WO2003068808A1 - Novel protein complexes and uses therefor - Google Patents

Novel protein complexes and uses therefor Download PDF

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Publication number
WO2003068808A1
WO2003068808A1 PCT/AU2003/000177 AU0300177W WO03068808A1 WO 2003068808 A1 WO2003068808 A1 WO 2003068808A1 AU 0300177 W AU0300177 W AU 0300177W WO 03068808 A1 WO03068808 A1 WO 03068808A1
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polypeptide
cd2ap
cortactin
cbl
endophilin
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PCT/AU2003/000177
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French (fr)
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Roger Daly
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Garvan Institute Of Medical Research
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Priority claimed from AUPS0514A external-priority patent/AUPS051402A0/en
Priority claimed from AU2002950698A external-priority patent/AU2002950698A0/en
Application filed by Garvan Institute Of Medical Research filed Critical Garvan Institute Of Medical Research
Priority to AU2003202655A priority Critical patent/AU2003202655A1/en
Publication of WO2003068808A1 publication Critical patent/WO2003068808A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates generally to the field of interactions between proteins and novel heterodimeric and heteromultimeric protein complexes, and, in particular, protein-protein interactions involving SH3 domain-containing proteins (i.e. proteins having a Src homology 3 domain) and proteins having one or more proline-rich regions (PRRs). More specifically, the present invention provides novel protein complexes comprising a cortactin protein and/or a CD2AP protein or a portion thereof and one or more other proteins. The present invention further provides methods for isolating the protein complexes or binding partner(s) of such protein complexes, and for producing the protein complexes of the invention in vitro.
  • SH3 domain-containing proteins i.e. proteins having a Src homology 3 domain
  • PRRs proline-rich regions
  • the present invention further provides prognostic and diagnostic methods for determining a predisposition for disease, or a disease state, said methods comprising detecting the presence or absence of a protein complex of the invention.
  • the present invention further provides methods for determining a modulator, specifically an agonist or antagonist of a protein complex of the invention.
  • the present invention further provides methods for determining a modulator of an interaction between a cortactin protein and/or a CD2AP protein or a portion thereof and one or more other proteins, particularly for determining modulators that are useful for the therapeutic or prophylactic treatment of a disease.
  • the present invention further provides kits for producing a protein complex of the invention or for identifying modulators of complex formation, optionally packaged with instructions for use.
  • the present invention further provides methods for the treatment of certain cellular proliferative disorders, said methods comprising modulating the level of a protein complex of the invention.
  • nucleotide and amino acid sequence information prepared using Patentln Version 3.1, presented herein after the claims.
  • Each nucleotide sequence is identified in the sequence listing by the numeric indicator ⁇ 210> followed by the sequence identifier (e.g. ⁇ 210>1, ⁇ 210>2, ⁇ 213> etc).
  • the length and type of sequence (DNA, protein (PRT), etc), and source organism for each nucleotide sequence are indicated by information provided in the numeric indicator fields ⁇ 211 >, ⁇ 212> and ⁇ 213>, respectively.
  • Nucleotide sequences referred to in the specification are defined by the term "SEQ ID NO:", followed by the sequence identifier (eg. SEQ ID NO: 1 refers to the sequence in the sequence listing designated as ⁇ 400>1).
  • nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.
  • derived from shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.
  • Regulation of growth and development of an individual and maintenance of a healthy state depend, in part, on the precise regulation of protein-protein interactions.
  • an interaction between an extracellular growth factor (e.g. epidermal growth factor) and its cell surface cognate receptor can trigger various intracellular protein- protein interactions that activate a signalling cascade from the cell surface to the cell nucleus, thereby regulating specific patterns of gene expression.
  • Protein-protein interactions are also involved in virtually all metabolic pathways that occur in a cell, including, for example, the formation of holoenzyme complexes, enzyme-substrate interactions, immune responses (e.g. recognition of self versus non-self, antigen- antibody complexes, T-cell mediated responses, etc), cell adhesion and cytoskeleton formation.
  • the identification of the proteins involved in such interactions can provide insight into the normal cellular metabolism in an organism. More practically, the identification and characterization of particular protein-protein interactions permits the identification of defects in such interactions that are associated with a diseased state.
  • the identification of such associations provides a target for diagnostic, prophylactic and therapeutic strategies that are highly specific.
  • the identification and characterization of protein-protein interactions provides a means to screen for drugs that modulate the interaction, such as antagonists or agonists of the interaction. Aberrant protein-protein interactions, or a defect in one or more of the binding partners to a protein-protein interaction, can have deleterious effects on a cell or produce a disease state.
  • RTKs receptor tyrosine kinases
  • Src homology 2 and Src homology 3 Src homology 3 domains.
  • SH2 and SH3 domains are necessary for interactions with protein substrates and determine intramolecular binding, protein localization and association with protein targets.
  • the SH3 domain was first described for Src, and is also present in a number of other tyrosine kinases, and proteins associated with organization of the cytoskeleton, including fodrin and cortactin.
  • the SH3 domain is thought to form a compact, globular, partly opened ⁇ -barrel structure, that is believed to assist in recruiting proteins having proline-rich regions (PRRs) to particular locations within the cell.
  • PRRs proline-rich regions
  • PRRs are thought to have a conserved amino acid consensus sequence, the presence or absence of this region alone is insufficient to determine whether or not a specific protein-protein interaction between an SH3 protein and another protein occurs in vitro or in vivo. This is because not all protein-protein interactions involving SH3 proteins necessarily involve proteins comprising PRRs. Moreover, PRRs also occur in proteins that do not bind to SH3 proteins. Cortactin
  • Cortactin is a multi-domain actin-binding protein that is probably tyrosine phosphorylated by a Src kinase, that has been shown to bind several other proteins, including proteins of the Arp2/3 complex (see below), brain cortactin binding protein-1 (CortBPI or Shank 2; Du et al., Mol. Cell. Biol. 18, 5838-5851, 1998; Weed and Parsons Oncogene 20, 6418-6434, 2001), brain ZO-1 protein (Katsube et al., J. Biol. Chem 273, 29672-29677, 1998), and dynamin-2 (McNiven et al., J. Cell Biol. 151, 187- 198, 2000).
  • cortactin contributes to the spatial organization of sites of actin polymerization, coupled to selected cell surface transmembrane receptor complexes. Accordingly, cortactin is thought to be involved in one or more signalling pathways that modify the adhesive properties of cells.
  • Cortactin is normally enriched within the lamellipodia of motile cells and in neuronal growth cones, particularly in the brain. Cortactin is localized with the actin-related protein (Arp) 2/3 complex, at sites within the lamellipodia where actin polymerizes. Cortactin stimulates nucleation activity of the Arp2/3 complex, and enhances actin polymerization induced by proteins associated with Wiskott-Aldrich Syndrome (WASPs), that are co-activators of the Arp2/3 complex (Weaver et al. Curr. Biol. 11, 37-374, 2001; Urono et al., Nature Cell Biol. 3, 259-266, 2001). Weaver et al.
  • WASPs Wiskott-Aldrich Syndrome
  • cortactin promotes the formation and stability of branched actin networks.
  • cortactin may regulate vesicle trafficking, for example, of endosomes (Kaksonen et al., J. Cell Sci. 113, 4421-4426, 2000), a role that is supported by its interaction with dynamin-2 (McNiven et al., J. Cell Biol. 151, 187-198, 2000).
  • cortactin also appears to regulate the organization and subcellular localization of transmembrane complexes, wherein CortBPI performs a scaffolding function in the organization of receptor complexes at post-synaptic sites of excitatory synapses, and ZO-1 interacts with the transmembrane proteins claudin and occludin at epithelial tight junctions (Weed and Parsons Oncogene 20, 6418-6434, 2001). Cortactin also binds to rpde ⁇ , in rat brain tissue.
  • the amino acid sequence of the cortactin polypeptide comprises the following domains: (i) an N-terminal stretch of acidic amino acid residues (NTA); (ii) Six to seven tandem repeats of an amino acid sequence that is related to that found in HS1, located C-terminal to the acidic domain; (iii) a sequence having the predicted structure of an ⁇ - helix located C-terminal to the Hs1/cortactin repeat; (iv) a region rich in serine, threonine and proline, located C-terminal to the ⁇ -helical domain; and (v) a C-terminal SH3 domain.
  • cortactin Over expression of cortactin also enhances cell motility and invasion in vitro (Patel et al., Oncogene 16, 3227-3232, 1998; Huang et al., J. Biol. Chem. 273, 25770-25776, 1998). Over expression of cortactin in breast cancer cells may also affect the invasive properties of the cancer cells (Kairouz and Daly, Breast Cancer Res 2, 197-202, 2000), and metastases in bone (Li et al., Cancer Res. 61, 6906-6911, 2001).
  • the EMS1 gene that encodes cortactin is commonly amplified in breast cancers and squamous cell carcinomas of the head and neck (Schuuring et al., Cancer Res.
  • Amplification of the EMS1 gene is also associated with a poor prognosis in node negative or ER-negative breast cancer (Hui et al, Oncogene 15, 1617-1623, 1997) and in head and neck cancers (Rodrigo et al., Clin. Cancer Res. 6, 3177-3182, 2000). Cortactin is localized to sites of invasion in the extracellular matrix in MDA-MB-231 breast cancer cells (Bowden et al., Oncogene 18, 4440-4449, 1999).
  • CD2AP CD2AP is a known adaptor molecule that associates with a variety of membrane proteins, to organize the cytoskeleton around a polarized site.
  • CD2AP is a Src substrate.
  • CD2AP is also known to be expressed at high levels in glomerular podocytes wherein it binds nephrin, and less so in renal tubular epithelial cells of the adult kidney, particularly in distal nephron segments, where it binds to polycystin-2 (Dustin et al., Cell 94, 667-677, 1998; Kirsch et al, Proc Natl Acad. Sci USA 96, 6211- 6216, 1999; Lehtonen et al., J. Biol.
  • CD2AP binds CD2 and functions in antigen recognition, by regulating receptor patterning and cytoskeleton polarization.
  • Cbl is a multi-adaptor protein that is involved in ligand-induced down regulation of receptor tyrosine kinases, wherein Cbl-mediated ubiquitination of active receptors is essential for receptor degradation and turnover, thereby leading to cessation of downstream signalling from the receptor (Soubeyran et al, Nature 416, 183-187, 2002).
  • the endophilins are a family of SH3 domain-containing proteins having homology to Grb2 (i.e. Grb2-like or "GL” proteins) designated endophilin I (human homolog SH3GL2) , endophilin II (human homolog SH3GL1), endophilin III (human homolog SH3GL3), endophilin B1 (human homolog SH3GLB1 and endophilin B2 (human homolog SH3GLB2).
  • endophilin I human homolog SH3GL2
  • endophilin II human homolog SH3GL1
  • endophilin III human homolog SH3GL3GL3GL3
  • endophilin B1 human homolog SH3GLB1
  • endophilin B2 human homolog SH3GLB2
  • the endophilins are implicated in clathrin-mediated endocytosis (Schmidt et al, Nature 401, 133-141, 1999; Ringstad et al, Neuron 24, 143-154, 1999; Simpson et al, Nature Cell. Biol. 1, 119-124-1999; Gad et al, Neuron 27, 301-327, 2000; Soubeyran et al, Nature 416, 183-187, 2002).
  • ASAP1 is a phosphatidylinositol 4,5-bisphosphate (PIP 2 )-dependent ADP-ribosylation factor-1 (ARF1) GTPase activating protein (GAP) (Brown et al, Mol. Cell. Biol: 18, 7038-7051, 1998; Andreev et al, Mol. Cell. Biol. 19, 2338-2350, 1999).
  • PIP 2 phosphatidylinositol 4,5-bisphosphate
  • ARF1 ADP-ribosylation factor-1 GAP
  • GAP GTPase activating protein
  • the ASAP1 protein is widely expressed as two variants, ASAPIa and ASAPIb, that differ by a 57 amino acid insert that is present in ASAPIa but not ASAPIb.
  • the amino acid sequences of both ASAPIa and ASAPIb comprise a GAP domain of about 70 amino acids in length that includes a zinc finger motif required for GAP activity, as well as multiple domains required for forming protein-protein interactions. These domains include a PH domain, three ankyrin repeats, a PRR for binding to SH3 domains, and an SH3 domain (Donaldson, Proc Natl Acad. Sci USA 97, 3792-3794, 2000).
  • ASAP1 can bind to the SH3 domains of Src and c-Crk, and is tyrosine phosphorylated in cells wherein Src is activated (Brown et al, Mol Cell. Biol. 18, 7038-7051, 1998).
  • ASAP1 is normally localized to focal adhesions, where it co-localizes with paxillin and vinculin (Randazzo et al, Proc Natl Acad. Sci USA 97, 4011-4016, 2000). Over expression of ASAP1 can modify cell spreading (Randazzo et al, Proc Natl Acad. Sci USA 97, 4011-4016, 2000).
  • N-WASP Neuronal Wiskott-Ald ch Syndrome Protein
  • N-WASP Neuronal Wiskott-Aldrich Syndrome Protein
  • FISH Protein FISH is a Src substrate adapter protein that is known to be tyrosine phosphorylated following disruption of the cytoskeleton using cytochalasin D (Lock et al., EMBO J. 17, 4346-4357, 1998). FISH comprises five SH3 domains.
  • Sam68 Protein Sam68 (Src-associated in mitosis) protein is tyrosine phosphorylated by c-Src, and can bind c-Src, Grb2, and RNA.
  • Sam68 is thought to be involved in cell cycle progression, because it is tyrosine phosphorylated in mitotic cells but not in asynchronous cells, and because its RNA binding activity decreases when it is phosphorylated.
  • EGF Epidermal Growth Factor
  • EGFR Epidermal Growth Factor Receptor
  • the EGF receptor regulates differentiation and growth in both normal and neoplastic cells. In normal cells exposed to EGF, the receptor is phosphorylated at tyrosine residues.
  • the signal protein Cbl Upon ligand-induced activation of EGFR, the signal protein Cbl (Langdon et al, J. Virol. 63, 5420-5424, 1989; Langdon et al, Proc. Natl Acad. Sci. USA 86, 1168-1172, 1989) binds to phosphorylated EGFR, promoting its ubiquitination.
  • Cbl is also tyrosine phosphorylated in this process and leads to translocation of a protein complex involving CIN85 and endophilin-A3 protein to the vicinity of the activated EGFR, which in turn enhances the clathrin-mediated endocytosis of the activated EGFR (Soubeyran et al., Nature 416, 183-187, 2002).
  • Cbl is also implicated in sorting internalized EGFR for lysosomal degradation, and appears to play a critical role in ligand-induced receptor down regulation (Soubeyran et al, Nature 416, 183-187, 2002).
  • Elevated levels of EGFR have been reported in many human tumors and cell lines, including breast cancer, adenocarcinoma and squamous lung cancer, gastrointestinal cancers (gastric, colon, pancreatic), renal cell cancer, bladder cancer, glioma, gynecological carcinomas, and prostate cancer.
  • the ectodomain of EGFR is also detectable in the urine of about 36% of squamous cell carcinoma patients and about 16% of patients having non-squamous carcinoma with higher levels in subjects having metastatic disease compared to those having a localized disease.
  • a mutant EGFR ie.
  • EGFRvlll occurs frequently in ovarian, breast, lung, glioblastoma, and medulloblastoma tumors.
  • the mechanism(s) regulating EGFR levels in normal cells or leading to elevated EGFR levels in neoplastic cells is(are) not known.
  • ERK-1, ERK-2, ERK-3 and ERK-4 function as proline-site-directed serine-threonine kinases in the phosphorylation of transcription factors such as p65TCF/Elk-1, c-jun and c-myc, and thus appear to play a crucial role in signal transduction by converting extracellular stimuli into transcriptional activation (Boulton et.
  • MAP kinases are believed to be directly involved in the regulation of genes that are responsible for cell proliferation.
  • MAP kinases are regulated by various levels of upstream regulatory proteins mediated in part by reversible protein phosphorylation involving a conserved signalling cascade initiated by ligand induced activation of receptor tyrosine kinases (RTKs) which sequentially activate a series of protein kinases.
  • RTKs receptor tyrosine kinases
  • an activated EGFR might signal via ras to the serine-threonine kinase raf to directly activate the MAP- kinase/ERK-kinase designated MEK.
  • MAP kinases are stimulated by phosphorylation on two regulatory threonine and tyrosine residues, respectively, which is catalyzed by activated MEK.
  • MAP kinases Upon activation, MAP kinases translocate to the nucleus and phosphorylate transcription factors.
  • the S6 kinase II (pp90 rsk) is another substrate of MAP kinases that is activated by ERKs, which may control protein translation.
  • MAP kinases can also be activated by TPA in a ras-dependent pathway to thereby stimulate PKC and signal to MEK via raf protein.
  • Oncogenes can lead to the hyperstimulation of one or more signal transduction pathways causing the cell nucleus to receive an inappropriate signal to proliferate.
  • Many oncogenes are members or targets of the family of the ras/MAP kinase pathways, such as, for example, ras, raf-1, myc, ski, myb, fos and jun (Blenis Proc. Natl. Acad Sci. USA., 90, 5889-5892, 1994; Cobb et al, J. Biol. Chem.
  • Increased MAP kinase expression is known for certain non-small cell lung carcinoma and breast cancer cell lines, and is associated with over expression of ERK-1 or ERK- 2 (Cobb , Melanie H., "The Role of MAP Kinase Pathway in Breast Cancer," National Technical Information Service, Accession No. AD-8301 655/7/XAB, 1995)
  • studies that illustrate the hyper-activation of MAP kinase do not provide any suggestions as to the cause of the hyper-activation or provide new routes of treating malignant neoplastic cell growth, such as primary breast carcinoma.
  • the inventors sought to identify the protein- protein interactions associated with normal or aberrant regulation, formation or activity of the cytoskeleton, including cell growth or motility, tumorigenesis, metastasis, tumor cell invasion, T cell-mediated immunity, autoimmunity, transplantation, allergy, or asthma.
  • the inventors identified several novel binding partners of cortactin, particularly proteins that bind to the cortactin SH3 domain.
  • the protein-protein interactions are useful as prognostic and diagnostic markers for diseases and disorders.
  • the inventors have also developed novel screens for compounds that modulate the protein-protein interactions of the invention, which compounds are useful in the treatment of a range of different diseases or disorders.
  • cortactin binds CD2AP indicates a role for cortactin in both antigen recognition and cell motility. Accordingly, blocking of the cortactin-CD2AP interaction provides a novel approach for treating a range of cancers and T cell mediated disorders, such as, for example, autoimmune diseases, transplant rejection, allergy, or asthma.
  • cortactin binds to the CD2 associated protein, CD2AP
  • the inventors subsequently focussed their attention on binding partners for CD2AP.
  • the inventors identified several novel binding partners of CD2AP, including endophilin, and subsequently identified tertiary and quaternary protein complexes comprising cortactin and/or CD2AP.
  • These higher- order protein complexes were also shown by the inventors to be involved in regulating EGFR levels in normal or neoplastic cells, such as, for example, by modulating EGFR endocytosis or EGFR recycling and, as a consequence, are important in EGF signalling in normal or neoplastic cells.
  • a protein complex comprising cortactin and CD2AP and endophilin and/or Cbl may positively regulate EGFR endocytosis.
  • endocytosis with reference to the EGFR is to be understood to include any down regulation of the steady state level of EGFR available for EGF signalling in a cell, such as, for example, as a consequence of Cbl-mediated ubiquitylation of EGFR that directs the receptor to the lysosome for destruction.
  • agonist compounds that enhance the level of formation of a protein complex comprising cortactin and CD2AP are particularly useful in preventing or treating elevated EGFR levels, such as, for example, in the prophylactic or therapeutic treatment of a cancer that is characterized by elevated EGFR.
  • the protein complexes described herein are useful for identifying a range of novel therapeutic agents for the prophylaxis or therapy of any condition characterized by elevated EGFR.
  • one aspect of the present invention provides an isolated or recombinant protein complex, such as, for example, an isolated heterodimer or hetero-multimer comprising: (i) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1 , FISH and Sam68; and (b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and (ii) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
  • a protein complex of the invention further comprises Epidermal growth factor receptor (EGFR).
  • EGFR Epidermal growth factor receptor
  • the binding partners of the invention may consist of two or three or four or five intact polypeptides, or two or three or four or five portions of the intact polypeptides capable of forming a complex, or a fusion protein comprising the binding partners of the complex.
  • cortactin-containing protein complexes and CD2AP-containing protein complexes such as, for example, a complex selected from the group consisting of: (i) a complex comprising CD2AP and cortactin; (ii) a complex comprising CD2AP and Cbl; (iii) a complex comprising CD2AP and endophilin; (iv) a complex comprising CD2AP and endophilin and cortactin; (v) a complex comprising CD2AP and Cbl and cortactin; (vi) a complex comprising CD2AP and endophilin and Cbl; (vii) a complex comprising CD2AP and endophilin and Cbl and cortactin; (viii) a complex comprising cortactin and ASAP1; (ix) a complex comprising cortactin and N-WASP; (x) a complex comprising cortactin and FISH; and (xi) a complex comprising cortactin
  • CD2AP shall be taken to mean any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence of a human or mouse CD2AP polypeptide set forth in SEQ ID NO: 1 or 2.
  • CD2AP shall also be taken to include a peptide, polypeptide or protein having the known biological activity of CD2AP, or the known binding specificity of CD2AP.
  • CD2AP shall also be taken to include a peptide, polypeptide, or protein having the known biological activity of CD2AP, or the known binding specificity of CD2AP, wherein said peptide, protein or polypeptide is further capable of binding to cortactin or a portion of cortactin and/or to an endophilin polypeptide or a portion of an endophilin polypeptide.
  • amino acid sequences of the human and mouse CD2AP polypeptides are exemplified herein, as SEQ ID Nos: 1 and 2, respectively.
  • the percentage identity to SEQ ID NO: 1 or 2 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
  • the CD2AP is human CD2AP.
  • cortactin shall be taken to mean any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence a human or mouse cortactin polypeptide set forth in SEQ ID NO: 3 or 4.
  • cortactin shall also be taken to include a peptide, polypeptide or protein having the known biological activity of cortactin, or the known binding specificity of cortactin, wherein said peptide, protein or polypeptide is further capable of binding to CD2AP or a portion of CD2AP.
  • the amino acid sequences of the human and mouse cortactin polypeptides are exemplified herein, as SEQ ID Nos: 3 and 4, respectively.
  • the percentage identity to SEQ ID NO: 3 or 4 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
  • the cortactin is human cortactin.
  • Cbl shall be taken to mean any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence of a human or mouse Cbl polypeptide set forth in SEQ ID NO: 5 or 6.
  • the term “Cbl” shall also be taken to include a peptide, polypeptide or protein having the known biological activity of Cbl, or the known binding specificity of Cbl, wherein said peptide, protein or polypeptide is further capable of binding to an endophilin polypeptide or a portion of an endophilin polypeptide.
  • amino acid sequences of the human and mouse Cbl polypeptides are exemplified herein, as SEQ ID Nos: 5 and 6, respectively.
  • the percentage identity to SEQ ID NO: 5 or 6 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
  • the Cbl is human Cbl.
  • endophilin shall be taken to mean any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence of a human endophilin polypeptide set forth in SEQ ID NO: 7 or 8 or 9.
  • the term “endophilin” shall also be taken to include a peptide, polypeptide or protein having the known biological activity of endophilin I, endophilin II or endophilin III, or the known binding specificity of said endophilin 1, endophilin II or endophilin III, wherein said peptide, protein or polypeptide is further capable of binding to CD2AP or a portion of CD2AP and preferably, in addition, to a Cbl polypeptide or a portion of a Cbl polypeptide.
  • amino acid sequences of the antigenically cross-reactive human endophilin I, II and III polypeptides are exemplified herein, as SEQ ID Nos: 7, 8 and 9, respectively.
  • the percentage identity to SEQ ID NO: 7 or 8 or 9 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
  • ASAP1 shall be taken to refer to any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence of a mouse ASAP1 polypeptide and capable of binding to a cortactin polypeptide.
  • the term “ASAP1” shall also be taken to include a peptide, polypeptide, or protein having the known biological activity of ASAP1, or the known binding specificity of ASAP1 , wherein said peptide, protein or polypeptide is further capable of binding to cortactin or a portion of cortactin.
  • amino acid sequences of the mouse ASAPIa and mouse ASAPIb polypeptides are provided herein, as SEQ ID Nos: 10 and 11, respectively.
  • the percentage identity to SEQ ID NO: 10 or 11 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
  • the ASAP1 protein is a human ortholog of a mouse ASAP1 protein exemplified in the sequence listing.
  • N-WASP shall be taken to refer to any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence of a human or rat N-WASP polypeptide and capable of binding to a cortactin polypeptide.
  • the term "N-WASP” shall also be taken to include a peptide, polypeptide, or protein having the known biological activity of N-WASP, or the known binding specificity of N-WASP, wherein said peptide, protein or polypeptide is further capable of binding to cortactin or a portion of cortactin.
  • the amino acid sequences of the human N-WASP and rat N-WASP polypeptides are provided herein, as SEQ ID Nos: 12 and 13, respectively.
  • the percentage identity to SEQ ID NO: 12 or 13 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
  • the N-WASP is human N-WASP.
  • FISH shall be taken to refer to any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence of a human or mouse FISH polypeptide and capable of binding to a cortactin polypeptide.
  • amino acid sequences of the human FISH and mouse FISH polypeptides are provided herein, as SEQ ID Nos: 14 and 15, respectively.
  • the percentage identity to SEQ ID NO: 14 or 15 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
  • the FISH is human FISH.
  • FISH shall also be taken to include a peptide, polypeptide, or protein having the known biological activity of FISH, or the known binding specificity of FISH, wherein said peptide, protein or polypeptide is further capable of binding to cortactin or a portion of cortactin.
  • the term "Sam68” shall be taken to refer to any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence of a human or rat Sam68 polypeptide and capable of binding to a cortactin polypeptide.
  • the amino acid sequences of the human Sam68 and rat Sam68 polypeptides are provided herein, as SEQ ID Nos: 16 and 17, respectively.
  • the percentage identity to SEQ ID NO: 16 or 17 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
  • the Sam68 is human Sam68.
  • Standard68 shall also be taken to include a peptide, polypeptide, or protein having the known biological activity of Sam68, or the known binding specificity of Sam68, wherein said peptide, protein or polypeptide is further capable of binding to cortactin or a portion of cortactin.
  • Another aspect of the present invention provides isolated peptides and kits comprising same for producing a protein complex comprising:
  • polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide, wherein said protein complex further optionally comprises EGFR.
  • peptides and/or kits of the present invention are also useful, or for identifying a modulator of the formation or stability of any one of said complexes.
  • Another aspect of the present invention provides an isolated antibody that binds to a protein complex comprising:
  • Another aspect of the present invention provides an anti-idiotypic antibody that binds to an antibody or ligand that binds to a protein complex comprising:
  • Another aspect of the present invention provides methods for isolating a cortactin- binding protein or CD2AP-binding protein from a suitable cellular source, such as, for example, an ER-negative breast cancer cell.
  • a cortactin polypeptide, or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1, FISH and Sam68 has been used as an affinity probe to bind said protein in a cellular extract from an ER-negative breast cancer cell or HeLa cell over expressing cortactin.
  • a CD2AP polypeptide, or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin has been used as an affinity probe to bind such a protein in a cellular extracts from an ER-negative breast cancer cell, or transfected HeLa cells over expressing cortactin.
  • an unbound protein or non-specifically bound protein is removed by washing, thereby isolating a CD2AP binding protein substantially free of conspecific proteins. It will be apparent to those skilled in the art that such affinity purification methods can also be adapted to the isolation of a protein complex comprising CD2AP or a portion of CD2AP and its cognate binding partner from ER-negative breast cancer cells or HeLa cells.
  • Another aspect of the present invention provides a method for isolating a protein complex comprising:
  • CD2AP, N-WASP, ASAP1, FISH and Sam68 ; and (b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and
  • nucleic acid comprising a sequence that encodes a cortactin polypeptide or a portion of a cortactin polypeptide and a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide, in operable connection with a suitable promoter sequence, is expressed in a suitable cell for a time and under conditions sufficient to produce a fusion polypeptide comprising the binding partners of the complex.
  • nucleic acid comprising a sequence that encodes a CD2AP polypeptide or a portion of a CD2AP polypeptide and a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide, in operable connection with a suitable promoter sequence, is expressed in a suitable cell for a time and under conditions sufficient for expression to occur.
  • the nucleic acid further comprises a nucleotide sequence encoding an EGFR polypeptide or a portion of said EGFR polypeptide sufficient to bind to a Cbl polypeptide or a portion of a Cbl polypeptide.
  • the EGFR polypeptide may indirectly bind to CD2AP or to a portion of CD2AP by virtue of the association between Cbl and CD2AP.
  • the EGFR indirectly binds to cortactin or a portion thereof that can form an association with CD2AP, or a portion of CD2AP that binds to cortactin and Cbl.
  • nucleic acid comprising a sequence encoding each binding partner is placed in operable connection with a promoter sequence and expressed in a suitable cell. If the protein partners are expressed in the same cell, they may freely associate in said cell to form the protein complex of the invention. If the protein partners are produced in different cells, the cells are lysed and the cellular lysates mixed under conditions sufficient to permit the association of the binding partners.
  • the protein-protein interactions of the invention are useful as prognostic and diagnostic markers for cancers in which EGFR is elevated.
  • Another aspect of the present invention provides prognostic and diagnostic methods for determining a predisposition for disease, or a disease state, said methods comprising detecting the presence or absence of a protein complex comprising:
  • polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide, wherein said protein complex further optionally comprises EGFR.
  • Preferred detection systems include any known assay for detecting a protein-protein interaction in a biological sample isolated from a human or mammalian subject, such as, for example, using one or more antibodies against the complex or each binding partner, or an epitope thereof.
  • a non-antibody ligand of the protein complex may be used, such as, for example, a small molecule (e.g. a chemical compound, agonist, antagonist, allosteric modulator, competitive inhibitor, or non- competitive inhibitor, of the complex that may or may not modulate complex formation or dissociation).
  • the antibody or small molecule may be used in any standard solid phase or solution phase assay format amenable to the detection of protein complexes or protein-protein interactions.
  • Optical or fluorescent detection such as, for example, using mass spectrometry, MALDI-TOF, biosensor technology, evanescent fiber optics, or fluorescence resonance energy transfer, is clearly encompassed by the present invention.
  • Assay systems suitable for use in high throughput screening of mass samples particularly a high throughput spectroscopy resonance method (e.g. MALDI-TOF, electrospray MS or nano- electrospray MS) or a detection system facilitating determination of real time association/dissociation constants, are particularly contemplated.
  • the biooogical sample or test sample or reference sample is obtained previously from the subject.
  • the prognostic or diagnostic method is performed ex vivo.
  • the prognostic methods described herein are particularly useful for detecting the occurrence of a disorder or disease associated with aberrant regulation, EGF- mediated signalling, aberrant formation or activity of the cytoskeleton, or a predisposition of an individual towards a disorder or disease associated with aberrant EGF-mediated signalling, or aberrant .regulation, formation or activity of the cytoskeleton.
  • Individuals having such conditions may exhibit, for example, aberrant cell growth or motility, the formation of tumors, tumor metastasis, or tumor cell invasion.
  • the present invention is particularly useful for diagnoses in relation to T cell- mediated immune system disorders, such as, for example, autoimmune diseases (Type I diabetes, multiple sclerosis, rheumatoid arthritis, psoriasis, Wiskott-Aldrich Syndrome), transplant rejection, allergy, asthma, aberrant T cell mediated immunity, aberrant antigen recognition or presentation, disorders of the central nervous system, nephritis, or kidney failure.
  • autoimmune diseases Type I diabetes, multiple sclerosis, rheumatoid arthritis, psoriasis, Wiskott-Aldrich Syndrome
  • a cancer selected from the group consisting of head and neck cancer, breast cancer, adenocarcinoma, squamous lung cancer, gastrointestinal cancer (eg. gastric, colon, or pancreatic cancer), renal cell cancer, bladder cancer, a gynecological carcinoma (eg. ovarian cancer), prostate cancer, squamous cell carcinoma, non-squamous carcinoma, glioblastoma, epithelial vulval carcinoma and medulloblastoma is particularly preferred.
  • a cancer selected from the group consisting of head and neck cancer, breast cancer, adenocarcinoma, squamous lung cancer, gastrointestinal cancer (eg. gastric, colon, or pancreatic cancer), renal cell cancer, bladder cancer, a gynecological carcinoma (eg. ovarian cancer), prostate cancer, squamous cell carcinoma, non-squamous carcinoma, glioblastoma, epithelial vulval carcinoma and medulloblastoma is particularly
  • Novel screens are also developed and used to identify compounds that modulate the protein-protein interactions of the invention, which compounds are useful in the treatment of a range of different cancers and other diseases.
  • a further aspect of the present invention provides methods for determining a modulator of a protein complex of the invention.
  • the methods of the present invention comprise determining the association or dissociation of the protein complex, or the structure of the complex, in the presence and absence of a candidate compound or a candidate antibody.
  • a modified association, dissociation, or structure, of the protein complex in the presence of a candidate compound or a candidate antibody indicates that the candidate is a modulator of the protein complex.
  • the association, dissociation, or structure of the complex may be determined by direct means, such as, for example, by determining real time association or dissociation constants in the presence and absence of the candidate, or modified binding of an antibody that recognizes a conformational epitope of the complex.
  • the association, dissociation, or structure of the complex may be determined by indirect means, such as, for example, using a protein recruitment system, n-hybrid screen, reverse n-hybrid screen, plate agar diffusion assay, ELISA, or other well known assay format for detecting protein-protein interactions.
  • indirect means generally use a reporter system to detect formation or dissociation of the protein complex.
  • the invention provides a method for determining a modulator of the formation or stability of a protein complex comprising: (i) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH and Sam68; and (b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and (ii) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
  • the modulators of the present invention are antagonists or inhibitors of a protein complex of the invention, particularly an inhibitor of complex formation or stability.
  • the modulators promote complex formation or stability.
  • the modulators identified using the methods described herein are useful for the therapeutic or prophylactic treatment of diseases associated with aberrant cytoskeletal regulation, formation or activity, such as, for example, aberrant cell growth or motility, tumorigenesis, tumor metastasis, tumor cell invasion, disorders of the central nervous system, nephritis, or kidney failure.
  • the modulators are preferably useful for the treatment of one or more symptoms associated with T cell-mediated immune system disorders, such as, for example, autoimmune diseases (Type I diabetes, multiple sclerosis, rheumatoid arthritis, psoriasis, Wiskott-Aldrich Syndrome), transplant rejection, allergy, asthma, aberrant T cell mediated immunity, aberrant antigen recognition or presentation.
  • the protein complexes of the invention represent novel targets for cancer therapy.
  • a cancer selected from the group consisting of head and neck cancer, breast cancer, adenocarcinoma, squamous lung cancer, gastrointestinal cancer (eg. gastric, colon, or pancreatic cancer), renal cell cancer, bladder cancer, a gynecological carcinoma (eg. ovarian cancer), prostate cancer, squamous cell carcinoma, non-squamous carcinoma, glioblastoma, epithelial vulval carcinoma and medulloblastoma is particularly contemplated by the present invention.
  • a cancer selected from the group consisting of head and neck cancer, breast cancer, adenocarcinoma, squamous lung cancer, gastrointestinal cancer (eg. gastric, colon, or pancreatic cancer), renal cell cancer, bladder cancer, a gynecological carcinoma (eg. ovarian cancer), prostate cancer, squamous cell carcinoma, non-squamous carcinoma, glioblast
  • a further aspect of the present invention provides a method of treatment comprising administering to a subject an amount of a modulatory compound identified in the screen of the present invention, including a modulator of an interaction selected from the group consisting of: (i) an interaction between CD2AP and a polypeptide selected from the group consisting of cortactin, Cbl, and endophilin, optionally further comprising EGFR; and (ii) an interaction between cortactin and a polypeptide selected from the group consisting of CD2AP, ASAP1, N-WASP, FISH and Sam68 optionally further comprising EGFR, wherein said contacting is performed under conditions sufficient to modulate the formation or stability of said interaction in a cell or tissue of said subject.
  • a modulatory compound identified in the screen of the present invention including a modulator of an interaction selected from the group consisting of: (i) an interaction between CD2AP and a polypeptide selected from the group consisting of cortactin, Cbl, and endophilin, optionally further comprising EG
  • the modulator modulates the activity, formation or stability of a protein complex selected from the group consisting of: (i) a complex comprising CD2AP and cortactin optionally further comprising EGFR; (ii) a complex comprising CD2AP and Cbl optionally further comprising EGFR; (iii) a complex comprising CD2AP and endophilin optionally further comprising EGFR; (iv) a complex comprising CD2AP and endophilin and cortactin optionally further comprising EGFR; (v) a complex comprising CD2AP and Cbl and cortactin optionally further comprising EGFR; (vi) a complex comprising CD2AP and endophilin and Cbl optionally further comprising EGFR; (vii) a complex comprising CD2AP and endophilin and Cbl and cortactin optionally further comprising EGFR; (viii) a complex comprising cortactin and ASAP1 optionally further comprising EGFR; (ix) a complex comprising cor
  • FIG. 1A is a schematic representation showing GST fusion proteins used to isolate cortactin binding partners from MDA-MB-231 cells.
  • the general structure of the cortactin polypeptide including the acidic N- terminal domain (horizontal hatched area), tandem repeats (arrow heads), helical domain (H, stippled area), region rich in serine, threonine and proline (P, diagonal hatching) and C- terminal SH3 domain (SH3, filled area).
  • the middle line indicates the structure of a peptide designated GST-HP comprising the helical domain of cortactin (H, stippled area) and region rich in serine, threonine and proline (P, diagonal hatching) fused to GST.
  • the lower line indicates the structure of a peptide designated GST-SH3 and consisting of only the SH3 domain of cortactin fused to GST.
  • Figure 1B is a photographic representation showing the binding of proteins in MDA- MB-231 cells to the peptides and polypeptides shown in Figure 1A. Peptides are indicated at the top of each lane.
  • Figure 2 is a representation of a MALDI-TOF MS fingerprint of bound and purified proteins, confirmed as CD2AP, ASAP1, N-WASP, FISH and Sam68.
  • the x-axis indicates the ion ratio m/z, and signal intensity for each peak is indicated on the abscissa.
  • Figure 3 is a photographic representation of a western blot showing specific binding of antibody against CD2AP in the protein fraction bound to the GST-SH3 fusion protein, but not in the protein fraction bound to the GST protein. Molecular weights are indicated at the left of the figure. The arrow indicates the position to which authentic CD2AP migrates on the gel.
  • Figure 4A is a photographic representation of a western blot (WB) of cortactin immune precipitates (IP) from MDA-MB-231 cells, using a CD2AP-specific antibody and cortactin-specific antibody. The positions of both CD2AP (above) and cortactin (below) are indicated by the arrows. The numbering refers to molecular weight (kDa).
  • FIG 4B is a photographic representation of a western blot (WB) of CD2AP immune precipitates (IP) from MDA-MB-231 cells, using a CD2AP-specific antibody and cortactin-specific antibody.
  • WB western blot
  • IP CD2AP immune precipitates
  • the positions of both cortactin(above) and CD2AP (below) are indicated by the arrows.
  • the numbering refers to molecular weight (kDa).
  • Figure 4C is a photographic representation showing expression of a truncation mutant protein of cortactin ( ⁇ SH3) in HEK 293 cells transfected with an expression vector encoding said truncation mutant protein, relative to the full-length cortactin polypeptide (Cortactin) and a negative control sample lacking a cortactin polypeptide.
  • CD2AP immune precipitates were probed in western blots, using antibodies against cortactin or CD2AP. The positions of both CD2AP (above) and cortactin (below) are indicated by the arrows.
  • Figure 5 is a photographic representation of a confocal micrograph showing coincident binding of antibodies against cortactin and CD2AP at regions of the cell cortex, structures resembling intracellular vesicles and cell-cell junctions.
  • Figure 6A is a schematic representation of the structure of CD2AP, showing the positions of the three SH3 domains, and three proline rich regions (PR), designated P1. P2, and P3.
  • PR proline rich regions
  • Figure 6B is a schematic representation showing the amino acid sequences of the three proline rich regions of CD2AP (i.e. SEQ ID Nos: 22-24) and a PRR consensus sequence (SEQ ID NO: 25).
  • Figure 6C is a photographic representation showing binding of GST fusion proteins comprising the three proline rich regions of CD2AP (i.e. SEQ ID Nos: 22-24), and a GST fusion comprising the a PRR consensus sequence (SEQ ID NO: 25), to the SH3 domain of cortactin in Far Western blots.
  • the left figure shows Coomassie-stained proteins.
  • the middle figure shows a far western blot using biotin-labelled GST.
  • the right figure shows a far western blot using the biotin-labelled GST-SH3 domain fusion protein. Numbering at the left of each panel shows molecular weight of the GST fusion proteins detected.
  • FIG. 7A is a copy of a photographic representation showing the induction of the association between CD2AP and cortactin by EGF.
  • CD2AP immunoprecipitates IP CD2AP
  • cortactin immunoprecipitates IP cortactin
  • SF serum- starved
  • EGF EGF-stimulated
  • Figure 7B is a copy of a photographic representation showing the requirement for the SH3 domain of cortactin in the EGF-inducible co-immunoprecipitation of cortactin and CD2AP in HEK 293 cells.
  • Cells were transfected with an expression vector (control), or an expression vector comprising nucleic acid encoding a full-length cortactin polypeptide (cortactin) or encoding a truncated cortactin polypeptide lacking the SH3 domain ( ⁇ SH3), and incubated in serum-free media (SF) or in the presence of EGF (EGF).
  • control an expression vector comprising nucleic acid encoding a full-length cortactin polypeptide
  • ⁇ SH3 truncated cortactin polypeptide lacking the SH3 domain
  • FIG 8A is a copy of a photographic representation showing the time course for recruitment of cortactin and Cbl to CD2AP following EGF stimulation of MDA-MB-231 cells.
  • CD2AP immunoprecipitates IP CD2AP
  • IP CD2AP CD2AP immunoprecipitates
  • FIG. 8B is a copy of a photographic representation showing the time course for recruitment of tyrosine phosphorylation of Cbl, cortactin and EGFR to CD2AP following EGF stimulation of MDA-MB-231 cells.
  • CD2AP immunoprecipitates IP CD2AP
  • IP CD2AP CD2AP immunoprecipitates
  • Data indicate the presence of several proteins phosphorylated at their tyrosine residues, having molecular weights of 180 kDa (EGFR), 130 kDa (unknown), 120 kDa (Cbl), and 80 kDa (cortactin). Data are consistent with the formation of a Cbl-CD2AP-endophilin complex that regulates receptor endocytosis, and demonstrates a novel role for cortactin and the formation of dynamic actin networks in this process.
  • Figure 9A is a copy of a photographic representation showing the time course for co- localization of CD2AP, cortactin and EGFR in HeLa cells transiently transfected with an expression construct comprising nucleic acid encoding a fusion protein consisting of EGFR and green fluorescent protein (GFP).
  • Cells were fixed either before EGF stimulation (time 0) or at 5 mins (5') or 15 mins (15') after EGF-stimulation, and analysed by indirect immunofluorescence using antibodies against cortactin or CD2AP.
  • EGFR was localized by fluorescence of the GFP moiety in the fusion protein.
  • Data indicate localization of EGFR primarily in the plasma membrane, with cortactin and CD2AP primarily in the punctate cytoplasmic structures, prior to EGF stimulation. After stimulation with EGF, EGFR moves into the membrane ruffles where it is associated with CD2AP and cortactin. The co-localization of EGFR with CD2AP and cortactin is transient and returns to basal levels within 15 mins after EGF stimulation.
  • Figure 9B is a copy of a photographic representation showing co-localization of CD2AP, cortactin and EGFR in membrane 5 mins (5') after EGF-stimulation of HeLa cells transiently transfected with an expression construct comprising nucleic acid encoding a fusion protein consisting to EGFR and green fluorescent protein (GFP).
  • Cells were analysed by indirect immunofluorescence using antibodies against cortactin or CD2AP.
  • EGFR was localized by fluorescence of the GFP moiety in the fusion protein.
  • Figure 10 is a copy of a photographic representation showing the effect of the over expression of cortactin or truncated cortactin on endocytic complex formation.
  • Cells were transfected with an "empty" expression vector (control), or an expression vector comprising nucleic acid encoding a full-length cortactin polypeptide (cortactin) or encoding a truncated cortactin polypeptide lacking the SH3 domain ( ⁇ SH3), and incubated in serum-free media (time 0) or in the presence of EGF (EGF) for 5 min (5') or 15 min (15').
  • control an expression vector comprising nucleic acid encoding a full-length cortactin polypeptide
  • ⁇ SH3 domain truncated cortactin polypeptide lacking the SH3 domain
  • CD2AP immunoprecipitates were analysed by western blotting using antibodies against CD2AP, endophilin, Cbl, and cortactin.
  • Over expression of full-length cortactin increased the basal association between cortactin and CD2AP and leads to enhanced and prolonged association between CD2AP and cortactin following EGF-stimulation.
  • Over-expression of truncated cortactin blocked recruitment of endogenous native cortactin into the complex following EGF stimulation of cells, as evidenced by the reduced level of cortactin in immunoprecipitates of cells over-expressing the mutant protein relative to control cells at 5 min post-EGF treatment.
  • FIG 11 is a copy of a photographic representation showing the kinetics of recruitment of cortactin and Cbl to CD2AP following EGF stimulation of MDA-MB-231 cells.
  • Cells were either serum-starved (lane marked 0) or treated with EGF for 1 min (1'), 2 min (2'), 5 min (5'), 15 min (15'), 30 min (30') or 60 min (60').
  • CD2AP-containing immune precipitates IP CD2AP
  • IP CD2AP CD2AP-containing immune precipitates
  • FIG. 12 is a copy of a photographic representation showing the association of Cbl with cortactin following EGF stimulation.
  • HeLa epithelial vulval carcinoma cells were transiently transfected with either an empty vector control (V), or alternatively, a cortactin-encoding expression vector (C). Cells were then serum-starved and left untreated (SF) or stimulated with EGF under standard conditions for 2 min (2' EGF).
  • IP cortactin cortactin-containing immune precipitates
  • P-Tyr phosphotyrosine
  • Cbl cortactin
  • kDa molecular weight
  • One aspect of the present invention provides an isolated or recombinant protein complex comprising:
  • CD2AP, N-WASP, ASAP1, FISH and Sam68 ; and (b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and
  • the protein complex of the invention comprises: (i) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide; and (ii) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a cortactin polypeptide or a portion of a cortactin polypeptide.
  • the protein complex of the invention comprises:
  • a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a Cbl polypeptide
  • a Cbl polypeptide or a portion of a Cbl polypeptide sufficient to bind to a CD2AP polypeptide or a portion of a CD2AP polypeptide.
  • an isolated or recombinant protein complex of the invention comprising cortactin and CD2AP further comprises a Cbl polypeptide and/or an endophilin polypeptide.
  • CD2AP may bind endophilin and/or Cbl in such an arrangement either directly or indirectly.
  • the protein complex of the invention comprises:
  • a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide; and (b) an endophilin polypeptide or a portion of an endophilin polypeptide; and (iii) an endophilin polypeptide or a portion of an endophilin polypeptide sufficient to bind to a CD2AP polypeptide or a portion of a CD2AP polypeptide.
  • the protein complex of the invention comprises: (i) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide; (ii) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide; and (b) a Cbl polypeptide or a portion of a Cbl polypeptide; and
  • the protein complex of the invention comprises:
  • a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide;
  • a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide; and (b) an endophilin polypeptide or a portion of an endophilin polypeptide;
  • an endophilin polypeptide or a portion of an endophilin polypeptide sufficient to bind to (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide; and (b) a Cbl polypeptide or a portion of a Cbl polypeptide; and
  • a Cbl polypeptide or a portion of a Cbl polypeptide sufficient to bind to an endophilin polypeptide or a portion of an endophilin polypeptide.
  • the protein complex of the invention comprises:
  • a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide;
  • a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide,
  • the protein complex of the invention comprises:
  • a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide;
  • a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide, (b) an endophilin polypeptide or a portion of an endophilin polypeptide, and (c) a Cbl polypeptide or a portion of a Cbl polypeptide;
  • an endophilin polypeptide or a portion of an endophilin polypeptide sufficient to bind to a CD2AP polypeptide or a portion of a CD2AP polypeptide; and
  • CD2AP polypeptide or a portion of a CD2AP polypeptide.
  • the protein complex of the invention comprises: (i) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide;
  • a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide, and (b) a Cbl polypeptide or a portion of a Cbl polypeptide;
  • an endophilin polypeptide or a portion of an endophilin polypeptide sufficient to bind to a Cbl polypeptide or a portion of a Cbl polypeptide.
  • a protein complex of the invention further comprises Epidermal growth factor receptor (EGFR), particularly in EGF-stimulated cells.
  • EGFR Epidermal growth factor receptor
  • a tertiary or quaternary protein complex comprising both cortactin and CD2AP and Cbl, may comprise EGFR in EGF-stimulated cells.
  • the protein complex is a heterodimer or hetero-multimeric protein.
  • a heterodimer or heterodimeric protein complex comprises two different peptide or polypeptide subunits.
  • the term "heteromultimer” shall be taken to mean a higher order protein complex comprising at least three peptide or polypeptide subunits, wherein at least two of said subunits are different.
  • a heterohexameric protein is known to comprise six peptide or polypeptide subunits, and, in the present context, may comprise three different homodimers, or six different monomers, or a dimer and a tetramer of different protein, etc. Accordingly, the present invention is not to be limited by the composition and size of the protein complex.
  • the protein subunits of the protein complex are held in physical relation by any means known to those skilled in the art.
  • This physical relation may involve the formation of an induced magnetic field or paramagnetic field, covalent bond formation such as a disulfide bridge formation between polypeptide subunits, an ionic interaction such as occur in an ionic lattice, a hydrogen bond or alternatively, a van der Waals interaction such as a dipole-dipole interaction, dipole-induced-dipole interaction, induced-dipole- induced-dipole interaction or a repulsive interaction or any combination of the above forces of attraction.
  • the peptide or polypeptide subunits may be held in physical relation by expressing them as a fusion polypeptide, optionally separated by a spacer to permit their folding. Accordingly, the physical relation between the peptide or polypeptide subunits may be a consequence of their binding capability and attraction toward one another, or alternatively, a consequence of their mode of production.
  • the peptide, polypeptide or protein partners are in direct physical relation.
  • direct physical relation is meant that the binding partners contact each other without any intervening protein moiety or non-protein moiety.
  • the protein complexes of the present invention can clearly include one or more additional protein moieties or non-protein moieties, such as, for example, a protein or non-protein moiety that enhances or stabilizes the physical relation between cortactin and/or CD2AP and the other binding partner(s).
  • any one or more protein or non- protein moieties selected from the group consisting of PIP 2 , Arp 2/3, CortBPI, Src, c- Src, c-Crk, rpde ⁇ , ZO-1, Nek, WIP, Grb2, SOS, nephrin, polycystin-2, dynamin-2, claudin, occludin, paxillin, vinculin, EGFR, Cbl, , and nucleic acid (RNA or DNA) is not to be excluded.
  • the present invention further encompasses a protein complex wherein one or more of the binding partners include a post-translational modification, such as, for example, a phosphorylated, fucosylated, myristoylated, farnesylated, or glycosylated residue.
  • a post-translational modification such as, for example, a phosphorylated, fucosylated, myristoylated, farnesylated, or glycosylated residue.
  • Such post-translational modifications may enhance complex formation or stabilize the complex once it is formed.
  • Phosphorylation of one or more tyrosine residues present on one or more of the binding partners, such as EGFR or Cbl is particularly contemplated herein.
  • Ubiqitination of one or more binding partners is also not to be excluded.
  • the binding partners of the protein complex are mammalian polypeptides or proteins, and more preferably of human or murine or rat origin. It is not strictly necessary for the binding partners to be derived from the same source, however this is preferred because the ability of the partners to associate or be maintained in physical non-covalent association with each other is generally enhanced if they are derived from the same organism.
  • cortactin or CD2AP or other polypeptide that can form the protein complex of the invention, such as, for example, the SH3 domain of cortactin or CD2AP or endophilin or ASAP1 or FISH, or a proline rich region (PRR) of CD2AP, Cbl, ASAP1, N-WASP, FISH or sam ⁇ .
  • PRR proline rich region
  • formation of the isolated or recombinant protein complex involves an SH3 domain of a polypeptide selected from the group consisting of cortactin, CD2AP, endophilin and ASAP1, more preferably an SH3 domain of cortactin, CD2AP or endophilin.
  • the "core" SH3 domain of a polypeptide involved in complex formation will comprise the consensus amino acid sequence WX ⁇ GXXXXXGXFP or WX1XGXXXXXGXFP or WXiXGXXXXXXGXFP or W ⁇ XGXXXXXXGXFP or WXiXGXXXXXXXXGXFP, wherein X ⁇ is W or Y and wherein X is any amino acid.
  • the SH3 domain of a polypeptide involved in complex formation will comprise the consensus amino acid sequence GWWXGXXXXXGXFP or GWWXGXXXXXXGXFP or GWWXGXXXXXXGXFP or GWWXGXXXXXXXGXFP, wherein X is any amino acid.
  • X is any amino acid.
  • Longer fragments or portions, comprising an additional 5 or 10 or 15 or 20 amino acid residues on the N- terminus and/or C-terminus are also encompassed within this definition of an SH3 domain, particularly wherein the extension is derived from the sequence of a naturally occurring protein described herein that flanks the SH3 domain in its native context.
  • Preferred SH3 domains of a cortactin polypeptide are functionally sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH, and Sam68.
  • the portion of cortactin suitable for protein complex formation is a portion of the SH3 domain of cortactin of at least about 5 amino acids in length, more preferably at least about 10 amino acids in length, even more preferably at least about 15 amino acids in length and still more preferably at least about 20 or 30 or 40 or 50 amino acids in length.
  • a portion of the SH3 domain of cortactin may comprise at least about 5 or 10 or 15 or 20 or 25 or 30 or 35 or 40 or 45 or 50 contiguous amino acid residues of a sequence having at least about 80% identity to SEQ ID NO: 3 or SEQ ID NO: 4.
  • the SH3 domain is from human cortactin (SEQ ID NO: 3) or mouse cortactin (SEQ ID NO: 4) or a portion thereof, and in a particularly preferred embodiment, comprises the SH3 domain of human cortactin.
  • Exemplary SH3 domains of cortactin are set forth in SEQ ID Nos: 18 and 19.
  • the percentage identity to SEQ ID NO: 18 or 19 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
  • SH3 domains of CD2AP comprise an amino acid sequence selected from the group consisting of: (i) amino acid residues from about position 97 to about position 111 of SEQ ID NO: 1 ; (ii) amino acid residues from about position 144 to about position 157 of SEQ ID NO: 2; (iii) amino acid residues from about position 261 to about position 274 of SEQ ID NO: 1; and amino acid residues from about position 306 to about position 319 of SEQ ID NO: 2.
  • an SH3 domain of CD2AP associates with a PRR of Cbl.
  • Preferred SH3 domains of ASAP1 comprise the amino acid sequence from about position 1121 to about position 1138 of SEQ ID NO: 10 or an equivalent portion of the murine sequence set forth in SEQ ID NO: 11. Again, longer fragments or portions, comprising an additional 5 or 10 or 15 or 20 amino acid residues on the N-terminus and/or C-terminus are also encompassed within this definition of an SH3 domain of ASAP1, such as, for example additional residues derived from the sequences set forth in the sequence listing (ie., SEQ ID Nos: 10 or 11). Peptides having about 80% identity to such SH3 domains of ASAP1 are also encompassed.
  • an endophilin polypeptide that is sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide or to said Cbl polypeptide or a portion thereof.
  • Preferred SH3 domains of endophilin are derived from amino acid residues from about position 327 to about position 340 of SEQ ID NO: 7 or from about position 328 to about position 341 of SEQ ID NO: 8 or from about position 322 to about position 335 of SEQ ID NO: 9.
  • conventional binding assays for determining the binding between two proteins may be used to assay the suitability of such portions.
  • the portion of endophilin that is sufficient to bind CD2AP or a portion of CD2AP comprises the SH3 domain of said endophilin.
  • an SH3 domain of endophilin associates with Cbl.
  • the term "SH3 domain of endophilin” shall be taken to refer to an amino acid sequence having at least about 80% amino acid sequence identity to the amino acid sequence of an SH3 domain of a human endophilin polypeptide, and preferably, capable of binding to a polypeptide selected from the group consisting of CD2AP and Cbl.
  • the amino acid sequences of exemplary peptides comprising a human endophilin SH3 domain are provided herein as SEQ ID Nos: 20 and 21.
  • the percentage identity to SEQ ID NO: 20 or 21 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
  • Peptides comprising additional sequences flanking the cores SH3 sequences presented in SEQ ID Nos: 18-21 are also encompassed by the present invention, particularly functionally equivalent peptides such as, for example, those peptides having additional flanking sequences derived from the native cortactin and/or native endophilin polypeptides. Functionally equivalent fragments of SEQ ID Nos: 18-21 are also encompassed by the present invention.
  • an SH3 domain of CD2AP or endophilin binds to Cbl in a higher order complex of the present invention.
  • proline rich region of a polypeptide selected from the group consisting of CD2AP, Cbl, ASAP1, N-WASP, FISH and Sam68, and more preferably, a PRR of CD2AP or Cbl.
  • PRR proline rich region
  • proline-rich region is meant that the amino acid sequence comprises two or three or more consecutive or adjacent proline residues and the consensus sequence PXXP wherein X is any amino acid and P is a proline residue.
  • the value of X is selected from the group consisting of lysine, proline, valine, leucine and glutamine.
  • the consecutive or adjacent proline residues are adjacent to at least one or two basic residues such as lysine or arginine or separated therefrom by one or two amino acid residues.
  • a proline-rich region is present as a tandemly-repeated sequence or duplication separated by up to ten amino acid residues in the naturally occurring polypeptide from which it is derived.
  • the portion of CD2AP suitable for protein complex formation is a portion of the PRR of CD2AP of at least about 5 amino acids in length, more preferably at least about 10 amino acids in length, even more preferably at least about 15 amino acids in length and still more preferably at least about 20 or 30 or 40 or 50 amino acids in length.
  • a portion of the PRR of CD2AP may comprise at least about 5 or 10 or 15 or 20 or 25 or 30 or 35 or 40 or 45 or 50 contiguous amino acid residues of a sequence having at least about 80% identity to the PRR of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the percentage identity is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
  • Preferred PRRs of a Cbl polypeptide are functionally sufficient to bind to an endophilin polypeptide.
  • Preferred PRRs of a polypeptide selected from the group consisting of CD2AP, ASAP1 , N-WASP, FISH and Sam68 are functionally sufficient to bind to a cortactin polypeptide.
  • Particularly preferred peptides having a PRR will comprise an amino acid sequence selected from the group consisting of:
  • the isolated or recombinant protein complex of the invention is selected from the group consisting of:
  • a complex comprising CD2AP and cortactin (i) a complex comprising CD2AP and cortactin; (ii) a complex comprising CD2AP and Cbl; (iii) a complex comprising CD2AP and endophilin; (iv) a complex comprising CD2AP and endophilin and cortactin; (v) a complex comprising CD2AP and Cbl and cortactin; (vi) a complex comprising CD2AP and endophilin and Cbl;
  • a PRR of CD2AP associates with an SH3 domain of cortactin or endophilin.
  • Another aspect of the present invention provides isolated peptides and kits comprising same for producing a protein complex comprising:
  • the peptide or polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO:24.
  • SEQ ID Nos: 1-17 relate to full-length polypeptide binding partners of a protein complex described herein
  • SEQ ID Nos: 18-21 relate to exemplary SH3 domain peptide sequences
  • SEQ ID Nos: 22-24 relate to exemplary peptides comprising CD2AP PRRs.
  • the kit comprises a first polypeptide consisting of cortactin or a portion thereof and a second peptide or polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH, Sam68, a portion of CD2AP, a portion of N- WASP, a portion of ASAP1, a portion of FISH and a portion of Sam68, wherein said portion of said second polypeptide is sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide.
  • the kit further comprises one or more antibodies or ligands that bind to the first polypeptide, to the second polypeptide, or to the complex formed between said first and said second polypeptide.
  • antibody refers to intact monoclonal or polyclonal antibodies, immunoglobulin (IgG, IgM, IgE) fractions, humanized antibodies, or recombinant single chain antibodies, as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which are capable of binding a linear or conformational epitope of at least one binding partner of the protein complex, or to a conformational epitope of the assembled protein complex.
  • Humanized antibodies are antibodies in which amino acids have been replaced in the non-antigen binding regions in order to more closely resemble a human antibody, while still retaining the original binding ability.
  • Antibodies referred to herein are obtained from a commercial source, or alternatively, produced by conventional means. Commercial sources will be well known to those skilled in the art.
  • an intact polypeptide, or a portion thereof containing a short amino acid sequence of interest is used as the immunizing antigen or immunogen.
  • the immunogen is derived from a natural source, produced by recombinant expression means or by in vitro translation of RNA, or synthesized chemically such as by Fmoc chemistry.
  • Immunogens consisting of short peptides a preferably conjugated to a carrier protein, such as, for example bovine serum albumin (BSA), thyroglobulin, or keyhole limpet hemocyanin (KLH), prior to immunization.
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • the coupled peptide is then used to immunize the animal.
  • Various host animals e.g.
  • adjuvants include, for example, Freund's complete or incomplete adjuvant, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are preferred.
  • Monoclonal antibodies are prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture, such as, for example, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al Nature 256, 495-497, 1975; Kozbor et al, J. Immunol. Methods 81, 31-42, 1985; Cote et al, Proc. Natl. Acad. Sci. USA 80, 2026- 2030, 1983; Cole e a/., Mol. Cell Biol. 62, 109-120, 1984).
  • Techniques developed for the production of chimeric antibodies are also employed. Such techniques involve splicing a mouse antibody gene to a human antibody gene to produce a molecule having the desired antigen specificity and biological activity (Morrison et al, Proc. Natl. Acad. Sci. USA 81, 6851-6855, 1984; Neuberger et al, Nature 312, 604-608, 1984; Takeda et al, Nature 314, 452-454, 1985). Alternatively, techniques described for the production of single chain antibodies are adapted, using methods known in the art, to produce single chain antibodies having the desired specificity.
  • Antibodies are also produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed by Oriandi et al, Proc. Natl. Acad. Sci. USA 86, 3833-3837, 1989; Winter et al, Nature 349, 293-299, 1991).
  • Antibody fragments such as, for example, F(ab') 2 fragments
  • F(ab') 2 fragments are produced by pepsin digestion of an intact antibody molecule.
  • Fab fragments are generated by reducing the disulfide bridges of F(ab') 2 fragments.
  • Fab expression libraries are constructed, to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al, Science 254, 1275-1281 , 1989).
  • the antibodies or ligands may assist in the subsequent isolation or detection of the complex formed between the first and second polypeptides. Binding of the antibody or ligand to a region of the first or second polypeptide that is not involved in complex formation is preferred for this purpose, the only requirement being that, in use, the ligand does not disrupt the complex formed between the first polypeptide and the second polypeptide. Binding of the antibody or ligand to a region other than the SH3 domain of cortactin is particularly preferred.
  • the ligand is a small molecule or alternatively, a binding partner for one of the first polypeptide or second polypeptide.
  • Preferred ligands for use in accordance with this embodiment are selected from the group consisting of PIP 2 , Arp 2/3, CortBPI, Src, c-Src, c-Crk, rpde ⁇ , ZO-1, Nek, WIP, Grb2, SOS, nephrin, polycystin-2, dynamin-2, claudin, occludin, paxillin, vinculin, and nucleic acid (RNA or DNA).
  • the antibody or ligand may be labeled using a suitable reporter molecule, such as, for example, a fluorophore, chromophore, or radioisotope.
  • kits are packaged with instructions for use.
  • the first polypeptide and second polypeptide are contacted for a time and under conditions sufficient for complex formation to occur, and when provided, the antibody or ligand is used to detect or isolate the complex formed.
  • the kit comprises a first compartment comprising cortactin or a portion thereof and a second compartment comprising an antibody or ligand that binds to a peptide or polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH, Sam68, a portion of CD2AP, a portion of N-WASP, a portion of ASAP1, a portion of FISH and a portion of Sam68, wherein said portion of said second polypeptide is sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide, or an antibody that binds to a complex formed between the content of the first compartment and the content of the second compartment.
  • a peptide or polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH, Sam68, a portion of CD2AP, a portion of N-WASP, a portion of ASAP1, a portion of FISH and a portion of Sam68
  • the ligand is a small molecule or alternatively, a binding partner for the protein of the second compartment.
  • Preferred ligands for use in accordance with this embodiment are selected from the group consisting of PIP 2 , Arp 2/3, Src, c-Src, c-Crk, Nek, WIP, Grb2, SOS, nephrin, polycystin-2, claudin, occludin, paxillin, vinculin, and nucleic acid (RNA or DNA).
  • the antibody of ligand may be labeled using a suitable reporter molecule, such as, for example, a fluorophore, chromophore, or radioisotope.
  • the kit is packaged with instructions for use.
  • the contents of the first compartment or the contents of the second compartment are contacted with an isolated protein selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH, Sam68, a portion of CD2AP, a portion of N-WASP, a portion of ASAP1, a portion of FISH and a portion of Sam68, or a mixture or extract of proteins comprising said protein, for a time and under conditions sufficient for binding to occur.
  • an isolated protein selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH, Sam68, a portion of CD2AP, a portion of N-WASP, a portion of ASAP1, a portion of FISH and a portion of Sam68, or a mixture or extract of proteins comprising said protein, for a time and under conditions sufficient for binding to occur.
  • a protein complex of the invention, or an antigen-antibody complex or protein-ligand complex is formed.
  • the complex is then reacted with the content of the remaining compartment of
  • the kit comprises a first compartment comprising a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH, Sam68, a portion of CD2AP, a portion of N-WASP, a portion of ASAP1, a portion of FISH and a portion of Sam68, wherein said portion of said second polypeptide is sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide and a second compartment comprising an antibody or ligand that binds to a polypeptide selected from the group consisting of cortactin, CD2AP, N-WASP, ASAP1 , FISH and Sam68, or an antibody that binds to a complex formed between cortactin and a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH and Sam68.
  • the antibody or ligand binds to cortactin or a portion thereof.
  • Preferred ligands for use in accordance with this embodiment are selected from the group consisting of Arp 2/3, CortBPI, Src, c-Src, rpde ⁇ , and Zo-1.
  • antibody or ligand may be labeled using a suitable reporter molecule, such as, for example, a fluorophore, chromophore, or radioisotope. In the case of antibodies and small molecules, these may also be detected using antibodies in accordance with procedures known to those skilled in the art.
  • the kit is packaged with instructions for use.
  • the contents of the first compartment or the contents of the second compartment are contacted with isolated cortactin or a portion of cortactin or a mixture or extract of proteins comprising same for a time and under conditions sufficient for binding to occur.
  • a protein complex of the invention, or an antigen-antibody complex or protein-ligand complex is formed.
  • the complex is then reacted with the content of the remaining compartment of the kit, under conditions sufficient to produce the protein complex of the invention bound to antibody or ligand.
  • the complex is then detected by virtue of the antibody or ligand tag according to standard procedures. Again, the ligand should be selected such that it does not disrupt the protein complex of the present invention.
  • the kit comprises a first polypeptide consisting of CD2AP or a portion thereof sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin, and a second polypeptide consisting of a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion thereof sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide.
  • kits are used to produce protein complexes comprising: (i) CD2AP and cortactin; (ii) CD2AP and endophilin; (iii) CD2AP and Cbl; (iv) CD2AP and endophilin and cortactin; (v) CD2AP and cortactin and Cbl; and (vi) CD2AP and cortactin and Cbl and endophilin.
  • the kit may also include one or more antibodies or ligands that bind to the first polypeptide or the second polypeptide, or to any one or more of the protein complexes supra comprising CD2AP, such as, for example, an antibody or ligand that specifically recognizes an assembled protein complex or the conformation of said protein complex, rather than the individual polypeptide components per se.
  • one or more antibodies or ligands that bind to the first polypeptide or the second polypeptide, or to any one or more of the protein complexes supra comprising CD2AP, such as, for example, an antibody or ligand that specifically recognizes an assembled protein complex or the conformation of said protein complex, rather than the individual polypeptide components per se.
  • the kit comprises:
  • a first compartment comprising a CD2AP polypeptide or a portion thereof sufficient to form a protein complex selected from the group consisting of: (i) CD2AP and cortactin; (ii) CD2AP and endophilin; (iii) CD2AP and Cbl; (iv)
  • CD2AP and endophilin and cortactin (v) CD2AP and cortactin and Cbl; and (vi) CD2AP and cortactin and Cbl and endophilin; and
  • the kit comprises a first compartment comprising an endophilin polypeptide or a portion thereof sufficient to bind CD2AP and a second compartment comprising an antibody or ligand that binds to CD2AP.
  • the kit comprises a first compartment comprising a cortactin polypeptide or a portion thereof sufficient to bind CD2AP and a second compartment comprising an antibody or ligand that binds to CD2AP.
  • the kit comprises a first compartment comprising a Cbl polypeptide or a portion thereof sufficient to bind CD2AP and a second compartment comprising an antibody or ligand that binds to CD2AP.
  • the kit comprises:
  • a first compartment comprising an isolated or recombinant protein complex selected from the group consisting of: (i) CD2AP and cortactin; (ii) CD2AP and endophilin; (iii) CD2AP and Cbl; (iv) CD2AP and endophilin and cortactin; (v) CD2AP and cortactin and Cbl; and (vi) CD2AP and cortactin and Cbl and endophilin; and
  • a second compartment comprising an (i) antibody or ligand that binds to a polypeptide selected from the group consisting of CD2AP, cortactin, Cbl and endophilin; or (ii) an antibody oMigand that binds to one or more protein complexes (a).
  • Moonoclonal or polyclonal antibodies that bind to CD2AP, cortactin, endophilin I, endophilin II, endophilin III, Cbl, or EGFR are obtained from a commercial source, such as for example, UBI, Lake Placid, New York, USA, or Santa Cruz Biotechnology, Inc, CA 95060, USA. Other commercial sources will be well known to those skilled in the art.
  • the antibodies or ligands may assist in the subsequent isolation or detection of the complex formed between the first and second polypeptides. Binding of the antibody or ligand to a region of the first or second polypeptide that is not involved in complex formation is preferred for this purpose, the only requirement being that, in use, the ligand does not disrupt the complex formed between the first polypeptide and the second polypeptide. Binding of the antibody or ligand to a region other than the SH3 domain or PRR of CD2AP is particularly preferred.
  • the ligand is a small molecule or alternatively, a binding partner for one of the protein complexes contemplated herein.
  • Particularly preferred ligands for use in accordance with this embodiment are selected from the group consisting of EGFR, Arp 2/3, CortBPI , Src, c-Src, rpde ⁇ , ZO-1 , Nek, WIP, SOS, nephrin, polycystin-2, dynamin-2, claudin, occludin, and nucleic acid (RNA or DNA).
  • the antibody or ligand may be labelled using a suitable reporter molecule, such as, for example, a fluorophore, chromophore, or radioisotope.
  • a suitable reporter molecule such as, for example, a fluorophore, chromophore, or radioisotope.
  • these may also be detected using antibodies in accordance with procedures known to those skilled in the art.
  • the kit is packaged with instructions for use.
  • kits of the invention are useful for producing and/or detecting the protein complexes of the invention in vitro or in vivo.
  • one or more of the non-antibody/ligand components of the kits is added to a cellular source for a time and under conditions sufficient for complex formation to occur.
  • the antibody components are particularly useful for isolating the complex(es) thus formed.
  • any one of the kit components is labelled with a protein tag to facilitate subsequent isolation or purification of the protein complex.
  • the polypeptide components are contacted for a time and under conditions sufficient for complex formation to occur. Additional proteins may be provided from cellular or non-cellular sources to produce protein complexes other than those specifically referred to herein.
  • the antibody or ligand is used to detect or isolate the complex formed. The ligand should be selected such that it does not disrupt the protein complex formed.
  • Another aspect of the present invention provides an isolated antibody that binds to a protein complex selected from the group consisting of:
  • a complex comprising CD2AP and cortactin (i) a complex comprising CD2AP and cortactin; (ii) a complex comprising CD2AP and Cbl; (iii) a complex comprising CD2AP and endophilin; (iv) a complex comprising CD2AP and endophilin and cortactin; (v) a complex comprising CD2AP and Cbl and cortactin; (vi) a complex comprising CD2AP and endophilin and Cbl; (vii) a complex comprising CD2AP and endophilin and Cbl and cortactin; (viii) a complex comprising cortactin and ASAP1; (ix) a complex comprising cortactin and N-WASP; (x) a complex comprising cortactin and FISH; and (xi) a complex comprising cortactin and Sam68, wherein said antibody binds to said complex in the presence or absence of EGFR, and wherein said antibody does not bind to any individual
  • the antibody recognizes a conformational epitope of the protein complex.
  • Another aspect of the present invention provides an anti-idiotypic antibody that binds to an antibody or ligand that binds to a protein complex selected from the group consisting of:
  • Another aspect of the present invention provides methods for isolating a cortactin- binding protein or CD2AP-binding protein or a complex comprising same from a suitable cellular source.
  • binding partners are separately isolated, from the same or a different cellular source that ectopically expresses or endogenously expresses at least one of the said binding partners.
  • the isolated binding partners are then combined in an amount and under conditions sufficient to facilitate their physical relation. Such conditions can be readily determined by those skilled in protein chemistry. Selection of buffer pH, ionic strength, and temperature, sufficient to maintain the binding partners in solution are generally preferred.
  • One or more protease inhibitors can also be included to prevent proteolytic digestion or degradation of the isolated polypeptides.
  • the protein complex per se may be isolated from a cellular source that expresses both binding partners endogenously or ectopically, including expression in response to EGF stimulation (i.e. EGF-modulated expression or EGF-induced expression or EGF-regulated expression).
  • binding partners are expressed as a fusion protein or as distinct polypeptides.
  • Preferred cellular sources of the isolated polypeptide binding partners, or the protein complex include any mammalian cell, and preferably, a mammalian cell that is known to express CD2AP, cortactin, endophilin, Cbl, EGFR, ASAP1, N-WASP, FISH, or Sam68 or can be engineered to express any one or more of said protein(s).
  • Exemplary cells for such a purpose include cancer cells (e.g.
  • carcinoma cells breast cancer cells such as ER-negative breast cancer cells, or squamous epithelial carcinoma cells
  • epithelial cells cells of the central nervous system, kidney cells, T cells, NIH3T3 cells, murine 10T fibroblasts, MDA-MB-231 cells, MDCK cells, COS cells, CHO cells, HeLa cells, or HEK 293 cells.
  • other cells e.g. insect sf9 or sf21 cells, chick embryo cells and the like
  • the protein complex or a binding partner thereof is isolated from cell line that endogenously expresses one or both binding partners, such as, for example, a cancer cell selected from the group consisting of head and neck cancer, breast cancer, adenocarcinoma, squamous lung cancer, gastrointestinal cancer (eg. gastric, colon, or pancreatic cancer), renal cell cancer, bladder cancer, a gynecological carcinoma (eg. ovarian cancer), prostate cancer, squamous cell carcinoma, non- squamous carcinoma, glioblastoma and medulloblastoma. More preferably, the cell will be a head and neck cancer cell or a breast cancer cell, or cell line derived from such cancers.
  • a cancer cell selected from the group consisting of head and neck cancer, breast cancer, adenocarcinoma, squamous lung cancer, gastrointestinal cancer (eg. gastric, colon, or pancreatic cancer), renal cell cancer, bladder cancer, a gynecological
  • the cell will be an ER-negative breast cancer cell or ER-negative breast cancer cell line of HeLa cell line.
  • the term "cell line” includes any derivative of a stated cell line that expresses a binding partner of a protein complex of the present invention wherein said derivative also expresses said binding partner or can be engineered to express said binding partner.
  • the protein complex or a binding partner thereof is isolated from a carcinoma cell or carcinoma cell line, preferably a breast cancer cell or breast cancer cell line, and more preferably a MDA-MB-231 cell or HeLa cell or derivative cell line thereof.
  • Means for isolating the peptide, polypeptide, or protein binding partners, or the protein complex include any means of protein isolation known to the skilled protein chemist, such as, for example, size exclusion chromatography, ion-exchange (anion or cation exchange) chromatography, reverse phase chromatography, or affinity chromatography. Both high pressure (e.g. HPLC, FLPC, MALDI) and low pressure systems can be used.
  • high pressure e.g. HPLC, FLPC, MALDI
  • low pressure systems can be used.
  • SU6656 is a competitive inhibitor of Src and Src-related tyrosine kinase proteins that may be used as an affinity tag for isolating any Src ligand.
  • Antibodies against the SH3 domain of an SH3 protein such as, for example, those antibodies described herein or in US Patent Nos. 6,342, 593 or 6,326,158 are particularly useful for isolating CD2AP or a binding partner of CD2AP that has an SH3 domain (i.e. cortactin or endophilin).
  • Antibodies against the PRR of CD2AP are also useful for isolating cortactin or endophilin.
  • antibodies against any polypeptide selected from the group consisting of CD2AP, Cortactin, Cbl, endophilin, ASAP1 , N-WASP, FISH, Sam 68, a portion of CD2AP, a portion of cortactin, a portion of Cbl, a portion of endophilin, a portion of ASAP1, a portion of N-WASP, a portion of FISH, and a portion of Sam 68 can be used.
  • Naturally-occurring or recombinant protein is purified free of conspecific proteins by providing a matrix comprising antibody coupled to activated chromatographic resin (eg. CNBr-activated Sepharose, Pharmacia), blocking the resin and washing to remove unbound antibody and blocking agent, contacting the resin with a protein extract comprising a peptide or polypeptide to which the antibody binds under conditions sufficient to allow binding of said peptide or polypeptide (e.g., high ionic strength buffers in the presence of detergent), and eluting said peptide or polypeptide under conditions that disrupt the antibody antigen binding (eg, a buffer of pH 2-3 or a high concentration of a chaotrope, such as urea or thiocyanate ion).
  • activated chromatographic resin eg. CNBr-activated Sepharose, Pharmacia
  • a protein extract comprising a peptide or polypeptide to which the antibody binds under conditions sufficient to allow binding of said peptide or polypeptide (e.
  • small molecules, or proteins capable of binding to one of the binding partners can also be used to isolate one or both binding partners, or the protein complex per se, by affinity means. Conditions to permit such isolation can be readily determined by those skilled in protein chemistry. Selection of buffer pH, ionic strength, and temperature, sufficient to maintain the binding partners in solution are generally preferred.
  • one or more protease inhibitors e.g. papain, PMSF, leupeptin
  • one or more protease inhibitors are included to prevent proteolytic digestion or degradation of the isolated polypeptides.
  • naturally-occurring or recombinant protein is purified free of conspecific proteins by providing a matrix comprising a small molecule or protein binding partner coupled to activated chromatographic resin (eg.
  • the isolated protein or complex is provided substantially free of conspecific proteins, meaning that it is at least about 1-5% pure as determined by an analysis of proteins by SDS/PAGE. More preferably, the protein is at least about 10%, even more preferably at least about 20% pure, even more preferably at least about 25% pure, even more preferably at least about 30% pure, and even more preferably at least about 50% pure, and still more preferably substantially pure.
  • Another aspect of the present invention provides methods for producing a protein complex described herein by recombinant means.
  • a protein-encoding nucleotide sequence is placed in operable connection with a promoter or other regulatory sequence capable of regulating expression in a cell-free system or cellular system.
  • nucleic acid comprising a sequence that encodes a cortactin polypeptide or a portion of a cortactin polypeptide and a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide, in operable connection with a suitable promoter sequence, is expressed in a suitable cell for a time and under conditions sufficient to produce a fusion polypeptide comprising the binding partners of the complex.
  • nucleic acid comprising a sequence that encodes a CD2AP polypeptide or a portion of a CD2AP polypeptide and a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide, in operable connection with a suitable promoter sequence, is expressed in a suitable cell for a time and under conditions sufficient for expression to occur.
  • the nucleic acid may also comprise a nucleotide sequence encoding a Cbl polypeptide or a portion of said Cbl polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide.
  • the nucleic acid may also comprise a nucleotide sequence encoding an endophilin polypeptide or a portion of said endophilin polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide.
  • any endophilin polypeptide, Cbl polypeptide, portion of endophilin, or any portion of Cbl to be sufficient to form a complex between Cbl and endophilin, a portion of Cbl and endophilin, a portion of Cbl and a portion of endophilin, or Cbl and a portion of endophilin, in addition to a capability of forming an association with CD2AP or a portion of CD2AP.
  • Nucleic acid encoding the binding partners is readily derived from the amino acid sequences set forth herein, or alternatively, publicly available.
  • the open reading frames are covalently linked in the same reading frame, such as, for example, using standard cloning procedures as described by Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, ISBN 047150338, 1992), which is herein inco ⁇ orated by reference.
  • a spacer is placed between the open reading frames of the binding partners to facilitate their physical relation.
  • Preferred spacers comprise protein- encoding nucleotide sequences of at least about 15-30 nucleotides in length, preferably sequences encoding amino acids rich in proline.
  • the spacer is designed such that it does not interrupt the open reading frames of the partners.
  • promoter includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e., upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner.
  • promoter is also used to describe a recombinant, synthetic or fusion molecule, or derivative which confers, activates or enhances the expression of a nucleic acid molecule to which it is operably connected, and which encodes the polypeptide or peptide fragment.
  • Preferred promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or to alter the spatial expression and/or temporal expression of the said nucleic acid molecule.
  • Placing a nucleic acid molecule under the regulatory control of, i.e., "in operable connection with”, a promoter sequence means positioning said molecule such that expression is controlled by the promoter sequence. Promoters are generally positioned 5' (upstream) to the coding sequence that they control. To construct heterologous promoter/structural gene combinations, it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e., the gene from which the promoter is derived. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.
  • the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
  • the prerequisite for producing intact polypeptides and peptides in bacteria such as E. coli is the use of a strong promoter with an effective ribosome binding site.
  • Typical promoters suitable for expression in bacterial cells such as £. coli include, but are not limited to, the lacz promoter, temperature-sensitive ⁇ or ⁇ R promoters, T7 promoter or the IPTG-inducible tac promoter.
  • a number of other vector systems for expressing the nucleic acid molecule of the invention in E. coli are well-known in the art and are described, for example, in Ausubel et al (In: Current Protocols in Molecular Biology.
  • Typical promoters suitable for expression in viruses of eukaryotic cells and eukaryotic cells include the SV40 late promoter, SV40 early promoter and cytomegalovirus (CMV) promoter, CMV IE (cytomegalovirus immediate early) promoter amongst others.
  • CMV cytomegalovirus
  • Preferred vectors for expression in mammalian cells eg.
  • COS CHO, 10T cells, 293T cells
  • pcDNA vector suite supplied by Invitrogen, in particular pcDNA 3.1 myc-His-tag comprising the CMV promoter and encoding a C-terminal 6xHis and MYC tag; and the retrovirus vector pSR ⁇ tkneo (Muller et al, Mol. Cell. Biol, 11, 1785, 1991).
  • the vector pcDNA 3.1 myc-His (Invitrogen) is particularly preferred for expressing a secreted form of a protein in 293T cells, wherein the expressed peptide or protein can be purified free of conspecific proteins, using standard affinity techniques that employ a Nickel column to bind the protein via the His tag.
  • Means for introducing the isolated nucleic acid molecule or a gene construct comprising same into a cell for expression are well-known to those skilled in the art. The technique used for a given organism depends on the known successful techniques. Means for introducing recombinant DNA into animal cells include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongst others.
  • nucleic acid comprising a sequence encoding each binding partner is placed in operable connection with a promoter sequence and expressed in a suitable cell. If the protein partners are expressed in the same cell, they may freely associate in said cell to form the protein complex of the invention. If the protein partners are produced in different cells, the cells are lysed and the cellular lysates mixed under conditions sufficient to permit the association of the binding partners.
  • the nucleotide sequences encoding the binding partners may be contained in the same or different nucleic acid molecules, and as a consequence, the use of single or multiple gene constructs to express the binding partners is clearly encompassed by the invention.
  • the requirements for expressing fusion polypeptides as described herein above are also relevant in this context, except that there is no need for a spacer.
  • different promoters will be used to express each binding partner, such as, for example, to prevent squelching or competition between promoters or regulatory sequences for cellular transcription factors.
  • Another aspect of the present invention provides prognostic and diagnostic methods for determining a predisposition for disease, or a disease state, said methods comprising detecting a protein complex comprising: (i) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH and Sam68; and (b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and (ii) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
  • the protein complex that is detected is a complex comprising cortactin and CD2AP, optionally further comprising EGFR; more preferably a complex comprising cortactin and CD2AP and endophilin, optionally further comprising EGFR; still more preferably a complex comprising cortactin and CD2AP and Cbl, optionally further comprising EGFR; and still more preferably comprising cortactin and CD2AP and endophilin and Cbl, optionally further comprising EGFR.
  • Preferred detection systems contemplated herein include any known assay for detecting a protein-protein interaction in a biological sample isolated from a human or mammalian subject, such as, for example, using one or more antibodies against the complex or each binding partner, or an epitope thereof.
  • a non-antibody ligand of the protein complex may be used, such as, for example, a small molecule (e.g. a chemical compound, agonist, antagonist, allosteric modulator, competitive inhibitor, or non-competitive inhibitor, of the complex that may or may not modulate complex formation or dissociation).
  • the use of antibody-based assay systems is particularly preferred.
  • the antibody or small molecule may be used in any standard solid phase or solution phase assay format amenable to the detection of protein complexes or protein-protein interactions.
  • Antibodies that specifically bind to the protein complex are used for the diagnosis of conditions or diseases characterized by the presence of said protein complex, or in prognostic assays to monitor disease progression in the presence of absence of treatment. Diagnostic assays for include methods which utilize the antibody and a label to detect the protein complex in human body fluids or extracts of cells or tissues.
  • the antibodies are used with or without modification, and may be labeled, either covalently or non-covalently, with a reporter molecule.
  • reporter molecules which are known in the art may be used, several of which are described above.
  • cortactin-comprising protein complexes of the present invention are associated with cancers induced by over expression of cortactin (eg.
  • breast cancer head and neck cancer
  • adenocarcinoma squamous lung cancer
  • gastrointestinal cancer gastric cancer
  • colon cancer pancreatic cancer
  • renal cell cancer bladder cancer
  • ovarian cancer prostate cancer
  • squamous cell carcinoma non-squamous carcinoma
  • glioblastoma medulloblastoma and ER-negative breast cancer
  • normal or standard values of the complex for a healthy individual are established by combining body fluids or cell extracts taken from normal or healthy subjects, preferably human subjects.
  • the amount of standard complex formation may be quantified by various methods, preferably by photometric means, or using antibodies in a quantitative immunoassay (e.g. ELISA), wherein the amount of protein complex is determined by comparison against known amounts of a standard peptide, such as, for example, a peptide comprising an SH3 domain of cortactin, CD2AP, ASAP1, or endophilin, or a PRR from CD2AP, Cbl, ASAP1 , N-WASP, FISH or Sam68.
  • a standard peptide such as, for example, a peptide comprising an SH3 domain of cortactin, CD2AP, ASAP1, or endophilin, or a PRR from CD2AP, Cbl, ASAP1 , N-WASP, FISH or Sam68.
  • Quantities of the protein complex expressed in subject samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease or establishing a prognosis.
  • a level of a protein complex in excess of the standard level of that protein complex detected in a healthy subject is diagnostic of disease, and indicates a poor prognosis for survival.
  • Optical or fluorescent detection such as, for example, using mass spectrometry, MALDI-TOF, biosensor technology, evanescent fiber optics, or fluorescence resonance energy transfer, is clearly encompassed by the present invention.
  • biosensor diagnostic devices the assay substrate and detector surface are integrated into a single device.
  • One general type of biosensor employs an electrode surface in combination with current or impedance measuring elements for detecting a change in current or impedance in response to the presence of a protein-protein binding event (e.g. U.S. Patent No. 5,567,301).
  • Gravimetric biosensors employ a piezoelectric crystal to generate a surface acoustic wave whose frequency, wavelength and/or resonance state are sensitive to surface mass on the crystal surface. The shift in acoustic wave properties is therefore indicative of a change in surface mass, such as, for example, as a consequence of protein-protein binding (e.g. U.S. Patent Nos. 5,478,756 and 4,789,804.
  • Biosensors based on surface plasmon resonance (SPR) effects have also been proposed, for example, in U.S. Patent Nos. 5,485,277 and 5,492,840, which exploit the shift in SPR surface reflection angle that occurs when protein binds to the SPR interface.
  • SPR surface plasmon resonance
  • Biosensors have a number of potential advantages over binding assay systems having separate reaction substrates and reader devices.
  • One important advantage is the ability to manufacture small-scale, but highly reproducible, biosensor units using microchip manufacturing methods, such as, for example, described in U.S. Patent Nos. 5,200,051 and 5,212,050.
  • Another advantage is the potentially large number of different analyte detection regions that can be integrated into a single biosensor unit, allowing sensitive detection of several analytes with a very small amount of body-fluid sample. Accordingly, the simultaneous detection of the individual binding partners that form the protein complex, or the simultaneous detection of one or more protein complexes of the present invention, is possible using a biosensor.
  • Evanescent biosensors are particularly preferred because they do not require separation of the protein complex from unbound material, and their use can be coupled to standard immunoassay formats, as originally described by Hirshfield in U.S. Patent No. 4,447,546.
  • evanescent biosensors rely upon light of a particular wavelength interacting with a fluorescent molecule, such as, for example, a fluorescent antibody or small molecule attached near the probe's surface, to emit fluorescence at another wavelength, on binding of the protein complex of the invention to the antibody or small molecule.
  • the biosensor is protected from sensitivity degradation caused by non-specific binding of proteins to the sensor surface, by exposing the sensor surface to a solution of non-interfering proteins, so that the non-interfering proteins bind to said sensor surface to prevent the subsequent binding of the interfering proteins.
  • Enhanced protection of surfaces from biological proteins is also possible by completely covering surfaces with protective coatings, such as, for example, amorphous copolymers of tetrafluoroethylene and bis-2,2-trifluoromethyl-4.5-difluoro-1,2-dioxole, dissolved in a solvent containing fluorinated alkanes, and applied by deposition as a thin protective coating (US. Patent No. 5,356,668 by Paton et al).
  • Assay systems suitable for use in high throughput screening of mass samples particularly a high throughput spectroscopy resonance method (e.g. MALDI-TOF, electrospray MS or nano-electrospray MS) or a detection system facilitating determination of real time association/dissociation constants, are particularly contemplated.
  • a high throughput spectroscopy resonance method e.g. MALDI-TOF, electrospray MS or nano-electrospray MS
  • detection system facilitating determination of real time association/dissociation constants are particularly contemplated.
  • a diagnosis or prognosis is made by separately determining the level(s) of expression of the binding partners, wherein a high level of expression of binding partners of a complex in a sample is indicative of disease, or, in the case of cancerous tissues, a poor prognosis for survival.
  • the level of expression of the binding partners is determined by standard protein-based detection systems, antibody-based methods, or nucleic acid-based methods.
  • Nucleic acid encoding a binding partner of the protein complex of the invention i.e., encoding a full-length protein or a portion thereof
  • a synthetic oligonucleotide, complementary RNA, DNA, or protein-nucleic acid (PNA) is used to detect and quantitate gene expression in biopsied tissues in which expression of the polypeptide is correlated with disease.
  • the diagnostic assay may be used to distinguish between absence, presence, and over expression of the binding partner, or to monitor expression following an initial diagnosis or during therapeutic intervention. As with protein detection systems, the detection of over expression of cortactin is preferred.
  • Co-localization of expression of several binding partners in a particular cell, tissue or organ is also indicative of disease.
  • hybridization with PCR probes capable of detecting the nucleic acid (RNA or genomic DNA) encoding a binding partner is used.
  • the specificity of the probe is determined by its nucleotide sequence and the stringency of the hybridization or amplification (maximal, high, intermediate, or low). Generally, highly specific probes are preferred for use under more stringent conditions.
  • the hybridization probes may be DNA or RNA and will preferably comprise a nucleotide sequence that encodes a polypeptide of a protein complex of the present invention, including a genomic gene sequence, or a promoter, enhancer element, or introns of the naturally occurring gene.
  • Hybridization probes may be labelled by a variety of reporter groups, for example, radionuclides such as 32 P or 35 S, or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • reporter groups for example, radionuclides such as 32 P or 35 S, or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • nucleic acid probes are labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions suitable for the formation of a hybridization complex. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the biopsied or extracted sample is significantly altered from that of a comparable control sample, the altered level of expression in the sample indicates a disease state.
  • assays are also used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
  • a normal or standard profile for expression of the partners is established, such as, for example, by combining body fluids or cell extracts taken from normal subjects with nucleic acid encoding the binding partners or a portion thereof, under conditions suitable for hybridization or amplification. Standard hybridization is then quantified by comparing the values obtained from normal subjects with the signal obtained using a known amount of a substantially purified nucleic acid. Standard values from normal samples are then compared with values from patient samples. Deviation between standard and subject values is diagnostic of the disease. Once a diagnosis is made by this or another method, hybridization assays are carried out to evaluate expression of the binding partners over time, or during a course of treatment.
  • RNA encoding a binding partner of an inventive protein complex indicates a predisposition for the development of the disease, or is otherwise diagnostic of the disease, preferably prior to the appearance of actual clinical symptoms.
  • high levels of these transcripts are indicative of a poor prognosis for survival.
  • Methods that are used to quantitate the expression include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and the use of standard curves onto which the experimental results are inte ⁇ olated (Melby et al, J. Immunol. Methods, 159, 235-244, 1993; Duplaa et al, Anal. Biochem. 212, 229-236, 1993).
  • an oligonucleotide derived from a nucleotide sequence encoding any one or more of the binding partners is used as a target in a microarray.
  • the microarray can be used to monitor the expression level of large numbers of genes simultaneously, to produce a transcript image, or to identify a genetic variant, mutant, or polymorphism for the protein complex of the invention.
  • the microarray is prepared and used according to the methods described by Chee et al, WO95/11995, Lockhart et al, Nat. Biotech. 14, 1675-1680, 1996, or Schena et al, Proc. Natl. Acad. Sci. USA 93, 10614-10619, 1996.
  • a microarray is preferably composed of a large number of unique, single-stranded nucleic acid sequences, such as, for example, synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support.
  • the oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length.
  • Preferred oligonucleotides have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides containing single nucleotide mismatches, to control for non-specific hybridization/amplification.
  • RNA or DNA from a biological sample is made into hybridization probes.
  • the mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA).
  • aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray so that the probe sequences hybridize to complementary oligonucleotides of the microarray. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of non-hybridized probes, a scanner is used to determine the levels and patterns of fluorescence.
  • the scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray.
  • the biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations.
  • a detection system is used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously.
  • the present invention is also used to diagnose the occurrence of a disorder or disease associated with aberrant regulation, formation or activity of the cytoskeleton, or a predisposition of an individual towards a disorder or disease associated with aberrant regulation, formation or activity of the cytoskeleton.
  • Individuals having a condition involving aberrant cytoskeletal regulation, formation or activity may exhibit, for example, aberrant cell growth or motility, the formation of tumors, tumor metastasis, or tumor cell invasion.
  • the present invention is particularly useful for diagnoses in relation to T cell-mediated immune system disorders, such as, for example, autoimmune diseases (Type I diabetes, multiple sclerosis, rheumatoid arthritis, psoriasis, Wiskott-Aldrich Syndrome), transplant rejection, allergy, asthma, aberrant T cell mediated immunity, aberrant antigen recognition or presentation, disorders of the central nervous system, nephritis, or kidney failure.
  • autoimmune diseases Type I diabetes, multiple sclerosis, rheumatoid arthritis, psoriasis, Wiskott-Aldrich Syndrome
  • transplant rejection allergy, asthma, aberrant T cell mediated immunity, aberrant antigen recognition or presentation, disorders of the central nervous system, nephritis, or kidney failure.
  • a further aspect of the present invention provides methods for determining a modulator of the activity, formation or stability of a protein complex comprising:
  • CD2AP, N-WASP, ASAP1 , FISH and Sam68 ; and (b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and
  • the modulator modulates the activity, formation or stability of a protein complex selected from the group consisting of: (i) a complex comprising CD2AP and cortactin; (ii) a complex comprising CD2AP and Cbl; (iii) a complex comprising CD2AP and endophilin; (iv) a complex comprising CD2AP and endophilin and cortactin; (v) a complex comprising CD2AP and Cbl and cortactin; (vi) a complex comprising CD2AP and endophilin and Cbl; (vii) a complex comprising CD2AP and endophilin and Cbl and cortactin; (viii) a complex comprising cortactin and ASAP1; (ix) a complex comprising cortactin and N-WASP; (x) a complex comprising cortactin and FISH; and (xi) a complex comprising cortactin and Sam68, wherein said protein complex optionally further comprises EGFR.
  • a protein complex selected from
  • the methods of the present invention comprise determining the association or dissociation of the protein complex, or the structure of the complex, in the presence and absence of a candidate compound or a candidate antibody.
  • a modified association, dissociation, or structure, of the protein complex in the presence of a candidate compound or a candidate antibody indicates that the candidate is a modulator of the protein complex.
  • association, dissociation, or structure of the complex may be determined by direct means, such as, for example, by determining real time association or dissociation constants in the presence and absence of the candidate, or modified binding of an antibody that recognizes a conformational epitope of the complex.
  • Biosensors used essentially as described herein above, in the presence or absence of the candidate compound or antibody, are particularly suited to such applications.
  • association, dissociation, or structure of the complex may be determined by indirect means, such as, for example, using a protein recruitment system, n-hybrid screen, reverse n-hybrid screen, plate agar diffusion assay, ELISA, or other well known assay format for detecting protein-protein interactions.
  • indirect means generally use a reporter system to detect formation or dissociation of the protein complex.
  • Standard solid-phase ELISA assay formats are particularly useful for identifying antagonists of the protein-protein interaction.
  • one of the binding partners e.g. cortactin or CD2AP or a portion thereof
  • a solid matrix such as, for example an array of polymeric pins or a glass support.
  • the immobilized binding partner is a fusion polypeptide comprising Glutathione-S-transferase (GST; e.g. a cortactin-GST fusion, CD2AP-GST fusion or SH3 domain-GST fusion), wherein the GST moiety facilitates immobilization of the protein to the solid phase support.
  • GST Glutathione-S-transferase
  • the second binding partner e.g. cortactin, FISH, N-WASP, ASAP1, Sam68, Cbl or endophilin
  • the antibodies are generally labelled with fluorescent molecules or conjugated to an enzyme (e.g.
  • a second labelled antibody can be used that binds to the first antibody.
  • the second binding partner is expressed as a fusion polypeptide with a FLAG or oligo-histidine peptide tag, or other suitable immunogenic peptide, wherein antibodies against the peptide tag are used to detect the binding partner.
  • oligo-HIS tagged protein complexes can be detected by their binding to nickel-NTA resin (Qiagen), or FLAG-labeled protein complexes detected by their binding to FLAG M2 Affinity Gel (Kodak). It will be apparent to the skilled person that the assay format described herein is amenable to high throughput screening of samples, such as, for example, using a microarray of bound peptides or fusion proteins.
  • the SH3 domain or PRR of CD2AP or the SH3 domain or PRR of a binding partner of CD2AP is immobilized on a solid support, such as by chemical synthesis thereon, or biotin-labelled and used in the liquid phase.
  • a two-hybrid assay is described in US Patent No. 6,316,223 to Payan et al, incorporated herein by reference.
  • the basic mechanism described by Payan et al. is similar to the yeast two hybrid system.
  • the binding partners are expressed as two distinct fusion proteins in a mammalian host cell.
  • a first fusion protein consists of a DNA binding domain which is fused to one of the binding partners
  • a second fusion protein consists of a transcriptional activation domain fused to the other binding partner.
  • the DNA binding domain binds to an operator sequence which controls expression of one or more reporter genes.
  • the transcriptional activation domain is recruited to the promoter through the functional interaction between binding partners. Subsequently, the transcriptional activation domain interacts with the basal transcription machinery of the cell, thereby activating expression of the reporter gene(s), the expression of which can be determined.
  • Candidate bioactive agents that modulate the protein-protein interaction between the binding partners are identified by their ability to modulate transcription of the reporter gene(s) when incubated with the host cell. Antagonists will prevent or reduce reporter gene expression, while agonists will enhance reporter gene expression. In the case of small molecule modulators, these are added directly to the cell medium and reporter gene expression determined.
  • peptide modulators are expressible from nucleic acid that is transfected into the host cell and reporter gene expression determined. In fact, whole peptide libraries can be screened in transfected cells.
  • reverse two hybrid screens such as, for example, described by Vidal et al, Proc. Natl Acad. Sci USA 93, 10315-10320, 1996, may be employed to identify antagonist molecules.
  • Reverse hybrid screens differ from forward screens supra in so far as they employ a counter-selectable reporter gene, such as for example, CYH2 or LYS2, to select against the protein-protein interaction.
  • Cell survival or growth is reduced or prevented in the presence of a non-toxic substrate of the counter- selectable reporter gene product, which is converted by said gene product to a toxic compound. Accordingly, cells in which the protein-protein interaction of the invention does not occur, such as in the presence of an antagonist of said interaction, survive in the presence of the substrate, because it will not be converted to the toxic product.
  • the cortactin SH3 domain or CD2AP PRR is expressed as a DNA binding domain fusion, such as with the DNA binding domain of GAL4, and the appropriate region of CD2AP or cortactin, respectively, is expressed as an appropriate transcription activation domain fusion polypeptide (e.g. with the GAL4 transcription activation domain).
  • the fusion polypeptides are expressed in yeast in operable connection with the URA3 counter-selectable reporter gene, wherein expression of URA3 requires a physical relation between the GAL4 DNA binding domain and transcriptional activation domain. This physical relation is achieved, for example, by placing reporter gene expression under the control of a promoter comprising nucleotide sequences to which GAL4 binds.
  • Cells in which the reporter gene is expressed do not grow in the presence of uracil and 5-fluoroorotic acid (5-FOA), because the 5-FOA is converted to a toxic compound.
  • Candidate peptide inhibitor(s) are expressed from libraries in such cells, wherein cells that grow in the presence of uracil and 5-FOA are retained for further analysis, such as, for example, analysis of the nucleic acid encoding the candidate peptide inhibitor(s).
  • Small molecules that antagonize the interaction are determined by incubating the cells in the presence of the small molecules and selecting cells that grow or survive in the presence of uracil and 5-FOA.
  • a protein recruitment system such as that described in U.S. Patent No. 5, 776, 689 to Karin et al, is used.
  • a protein-protein interaction is detected in a cell by the recruitment of an effector protein, which is not a transcription factor, to a specific cell compartment.
  • the effector protein Upon translocation of the effector protein to the cell compartment, the effector protein activates a reporter molecule present in that compartment, wherein activation of the reporter molecule is detectable, for example, by cell viability, indicating the presence of a protein-protein interaction.
  • the components of a protein recruitment system include a first expressible nucleic acid encoding a first fusion protein comprising the effector protein and one of the binding partners (e.g. CD2AP or a portion thereof), and a second expressible nucleic acid molecule encoding a second fusion protein comprising a cell compartment localization domain and the other binding partner (e.g. cortactin, endophilin or Cbl or a portion thereof).
  • a cell line or cell strain in which the activity of an endogenous effector protein is defective or absent e.g. a yeast cell or other non- mammalian cell
  • the reporter molecule is not expressed.
  • a complex is formed between the fusion polypeptides as a consequence of the interaction between the binding partners, thereby directing translocation of the complex to the appropriate cell compartment mediated by the cell compartment localization domain (e.g. plasma membrane localization domain, nuclear localization domain, mitochondrial membrane localization domain, and the like), where the effector protein then activates the reporter molecule.
  • the cell compartment localization domain e.g. plasma membrane localization domain, nuclear localization domain, mitochondrial membrane localization domain, and the like
  • the effector protein then activates the reporter molecule.
  • Such a protein recruitment system can be practiced in essentially any type of cell, including, for example, mammalian, avian, insect and bacterial cells, and using various effector protein/reporter molecule systems.
  • a yeast cell based assay is performed, in which the interaction between cortactin or CD2AP and one or more of their binding partners results in the recruitment of a guanine nucleotide exchange factor (GEF) to the plasma membrane, wherein GEF activates a reporter molecule, such as Ras, thereby resulting in the survival of cells that otherwise would not survive under the particular cell culture conditions.
  • GEF guanine nucleotide exchange factor
  • Suitable cells for this purpose include, for example, Saccharomyces cerevisiae cdc25- 2 cells, which grow at 36°C only when a functional GEF is expressed therein, Petitjean et al, Genetics 124, 797-806, 1990) Translocation of the GEF to the plasma membrane is facilitated by a plasma membrane localization domain.
  • Activation of Ras is detected, for example, by measuring cyclic AMP levels in the cells using commercially available assay kits and/or reagents.
  • duplicate incubations are carried out in the presence and absence of a test compound, or in the presence or absence of expression of a candidate antagonist peptide in the cell.
  • Reduced survival or growth of cells in the presence of a candidate compound or candidate peptide indicates that the peptide or compound is an antagonist of the interaction between cortactin or CD2AP, as appropriate, and one or more of their binding partners.
  • a "reverse" protein recruitment system is also contemplated, wherein modified survival or modified growth of the cells is contingent on the disruption of the protein-protein interaction by the candidate compound or candidate peptide.
  • NIH 3T3 cells that constitutively express activated Ras in the presence of GEF can be used, wherein the absence of cell transformation is indicative of disruption of the protein complex by a candidate compound or peptide.
  • NIH 3T3 cells that constitutively express activated Ras in the presence of GEF have a transformed phenotype (Aronheim et al., Cell. 78, 949-961, 1994)
  • small molecules are tested for their ability to dissociate the protein complex of the invention, by an adaptation of plate agar diffusion assay described by Vidal and Endoh, TIBS 17, 374-381, 1999, which is incorporated herein by reference.
  • a further embodiment of the invention provides a method for determining a modulator of an interaction between cortactin or a portion of cortactin and a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide, said method comprising:
  • a further embodiment of the invention provides a method for determining a modulator of an interaction between CD2AP or a portion of CD2AP and a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide, said method comprising:
  • determining the level of a protein complex selected from the group consisting of: (i) a complex comprising CD2AP and cortactin; (ii) a complex comprising CD2AP and endophilin; (iii) a complex comprising CD2AP and Cbl; (iv) a complex comprising CD2AP and endophilin and cortactin; (v) a complex comprising CD2AP and Cbl and cortactin; and (vi) a complex comprising CD2AP and endophilin and Cbl and cortactin in the absence of a candidate compound or candidate antibody; and (iv) determining the level of said protein complex in the presence of a candidate compound or in the presence of said candidate antibody wherein a difference in the level of said protein complex at (i) and (ii) indicates that the candidate compound or candidate antibody is a modulator of said interaction.
  • any one or more of the assay methods for antagonists as described herein above can be adapted for this purpose.
  • the level of the protein complex in the presence or absence of a candidate compound or antibody is related to antibody binding in the case of ELISAs, or to cell survival or growth, in the case of hybrid screens or protein recruitment assays.
  • ELISA-based assay formats are particularly suitable for this pu ⁇ ose, because they are readily quantifiable, by calibrating the detection system against known amounts of a protein standard to which the antibody binds. Such quantitation is well known to the skilled person.
  • the modulators of the present invention are agonists of complex formation or stability.
  • Preferred agonist compounds enhance EGFR endocytosis in a cell, or prevent or reduce an impairment of EGFR endocytosis in a cell, or prevent or reduce a reduced or limited EGFR endocytosis in a cell.
  • agonists of EGFR endocytosis in a cell will preferably enhance the formation or stability of a protein complex that is EGF-induced, such as, for example, a protein complex selected from the group consisting of: (i) a complex comprising cortactin and CD2AP and Cbl; and (ii) a complex comprising cortactin and CD2AP and Cbl and endophilin.
  • Exemplary agonist compounds include one or more polypeptides of the protein complex, such as, for example a CD2AP and/or Cbl polypeptide or a portion thereof sufficient to form a protein complex of the invention.
  • a mimetic of Cbl capable of binding to a protein complex cortactin and CD2AP is particularly contemplated.
  • a mimetic of CD2AP capable of binding to cortactin and/or Cbl can be used.
  • a mimetic compounds comprising an anti-idiotypic antibody as described herein above are also contemplated for use as agonists of the protein complex of the present invention or for treatment of a condition in which enhanced EGFR endocytosis is indicated.
  • an agonist comprises a nucleic acid encoding one or more of the binding partners of the protein complex, preferably in an expressible format and a form suitable for administering to a cell.
  • nucleic acids encoding a Cbl and/or CD2AP polypeptide or a portion thereof is particularly preferred.
  • the modulators of the present invention are antagonists or inhibitors of complex formation or stability, such as, for example, a compound that inhibits or disrupts SH3-mediated protein-protein interactions.
  • UCS15A to disrupt the stability or formation of a protein complex that does not bind to the EGFR and/or to disrupt the stability or formation of a protein complex that does not facilitate or enhance receptor endocytosis.
  • disruption of such complexes enhances the level of protein binding partners available for the formation of protein complexes (eg., the cortactin-CD2AP-Cbl protein complex) that bind to EGFR and/or enhance endocytosis of the receptor.
  • the modulators identified using the methods described herein are useful for the therapeutic or prophylactic treatment of diseases associated with aberrant cytoskeletal regulation, formation or activity, such as, for example, aberrant cell growth or motility, tumorigenesis, tumor metastasis, tumor cell invasion, disorders of the central nervous system, nephritis, or kidney failure.
  • the modulators are preferably useful for the treatment of one or more symptoms associated with T cell-mediated immune system disorders, such as, for example, autoimmune diseases (Type I diabetes, multiple sclerosis, rheumatoid arthritis, psoriasis, Wiskott-Aldrich Syndrome), transplant rejection, allergy, asthma, aberrant T cell mediated immunity, aberrant antigen recognition or presentation.
  • a cancer selected from the group consisting of head and neck cancer, breast cancer, adenocarcinoma, squamous lung cancer, gastrointestinal cancer (eg. gastric, colon, or pancreatic cancer), renal cell cancer, bladder cancer, a gynecological carcinoma (eg. ovarian cancer), prostate cancer, squamous cell carcinoma, non-squamous carcinoma, glioblastoma and medulloblastoma is particularly contemplated by the present invention, and preferably, treatment of a breast cancer, specifically an ER-negative breast cancer in which cortactin expression is elevated.
  • a cancer selected from the group consisting of head and neck cancer, breast cancer, adenocarcinoma, squamous lung cancer, gastrointestinal cancer (eg. gastric, colon, or pancreatic cancer), renal cell cancer, bladder cancer, a gynecological carcinoma (eg. ovarian cancer), prostate cancer, squamous cell carcinoma, non-squamous carcinoma,
  • a further aspect of the present invention provides a method of treatment comprising administering to a subject an amount of a modulator of the activity, formation or stability of a protein complex comprising:
  • this aspect of the invention relates to the modulation of EGFR endocytosis, and more particularly to enhancing EGFR endocytosis in a cell, or to preventing or reducing an impairment of EGFR endocytosis in a cell, or to preventing or reducing a reduced or limited EGFR endocytosis in a cell.
  • Conditions in which EGFR endocytosis is impaired or limited or reduced include a disease state or condition characterized by elevated EGFR levels, or elevated or sustained EGF-mediated signalling, such as, for example a cancer, infection by an oncogenic animal virus (eg., human papilloma virus such as HPV-16 or HPV-11 or HPV6; a poxvirus, a retrovirus).
  • an agonist of the formation or stability of a protein complex of the invention particularly an agonist of a protein complex of the invention the formation of which is EGF-induced, such as, for example, a protein complex selected from the group consisting of: (i) a complex comprising cortactin and CD2AP and Cbl; and
  • the present invention contemplates a method of enhancing EGFR endocytosis in a cancer cell characterized by elevated expression of EGFR and elevated expression of cortactin comprising administering to a subject an amount of a Cbl polypeptide or portion thereof and/or a CD2AP polypeptide or portion thereof for a time and under conditions sufficient for EGFR endocytosis to be enhanced in said cell.
  • EGFR endocytosis is preferably enhanced by the recruitment of cortactin and Cbl and
  • an amount of UCS15A sufficient to reduce the formation of protein complexes that do not enhance EGFR endocytosis but not sufficient to disrupt such recruitment of cortactin and CD2AP and Cbl to the EGFR is also administered to the cell.
  • nucleic acid encoding a Cbl polypeptide or portion thereof and/or a CD2AP polypeptide or portion thereof is used.
  • a mimetic compound such as, for example, an anti- idiotypic antibody that mimics a protein complex comprising CD2AP and cortactin and
  • Cbl and is capable of binding to the EGFR is administered.
  • the mimetic binds to EGFR to thereby enhance EGFR endocytosis.
  • a mimetic of Cbl capable of binding to a protein complex cortactin and CD2AP is also contemplated.
  • a mimetic of CD2AP capable of binding to cortactin and/or Cbl can be used.
  • the active ingredient, nucleic acid or protein or antibody or small molecule is formulated in a pharmaceutically acceptable carrier, diluent or excipient known to the skilled artisan.
  • a pharmaceutically acceptable carrier e.g., aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils, for instance.
  • Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers and the like (See, generally, Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., Pa., 1985).
  • the agent can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer, nebulizer or pressurized aerosol dispenser).
  • the agent can be administered via in vivo expression of the recombinant protein.
  • In vivo expression can be accomplished via somatic cell expression according to suitable methods (see, e.g. U.S. Pat. No. 5,399,346).
  • nucleic acid encoding the protein can be incorporated into a retroviral, adenoviral or other suitable vector (preferably, a replication deficient infectious vector) for delivery, or can be introduced into a transfected or transformed host cell capable of expressing the protein for delivery.
  • the cells can be implanted (alone or in a barrier device), injected or otherwise introduced in an amount effective to express the protein in a therapeutically effective amount.
  • EXAMPLE 1 Identification of Proteins binding to the SH3 domain of cortactin
  • cortactin links molecular scaffolds assembled around CD2AP to the Arp2/3 complex and hence to dynamic actin structures.
  • SH3-interacting proteins including CD2AP, ASAP1, FISH, N-WASP and Sam68, were identified by either MALDI-TOF MS or ⁇ LC-MS/MS.
  • the MALDI mass fingerprint for CD2AP is shown in Figure 2.
  • Peptide fragments of CD2AP resolved using MALDI-TOF are set forth in Table 1 (SEQ ID Nos: 26-49).
  • cortactin SH3 domain is essential for interaction of these proteins in vivo.
  • CD2AP contains three proline-rich regions harbouring potential SH3-binding sites
  • CD2AP contains two recruitment sites for the cortactin SH3 domain, wherein the relative close proximity of these recruitment sites enables them to function co-operatively in recruitment of cortactin to CD2AP.
  • EXAMPLE 5 EGF-inducible association between cortactin and CD2AP to investigate the in vivo association of cortactin and CD2AP.
  • a co- immunoprecipitation analysis was performed from lysates of serum-starved or EGF- treated MDA-MB-231 cells.
  • Western blotting of cortactin immunoprecipitates with a CD2AP-specific antibody, and the reciprocal analysis revealed EGF-inducible co- immunopreciptation of the two proteins, revealing that they associate at physiological levels in response to EGF (Figure 7A).
  • cortactin SH3 domain in its EGF-induced association with CD2AP was investigated by expressing a truncation mutant of cortactin lacking the SH3 domain ( ⁇ SH3) in HEK293 cells.
  • Cells were also transfected with an empty expression vector (control), or an expression vector comprising nucleic acid encoding a full-length cortactin polypeptide, and incubated in serum-free media (SF) or in the presence of EGF (EGF).
  • SF serum-free media
  • EGF EGF
  • WCL whole cell lysates
  • IP CD2AP CD2AP immunoprecipitates
  • CD2AP Binding of CD2AP to endophilin and Cbl
  • CD2AP immunoprecipitates were analysed by blotting with antibodies that are specific for cortactin, Cbl, endophilins I, II, and III, and for phosphotyrosine (UBI; Santa Cruz Biotechnology, Inc).
  • CD2AP has been localized to lamellipodia and punctate cytoplasmic structures (Kirsch et al, Proc. Natl Acad. Sci. USA 96, 6211-6216, 1999; Welsch et al, Am. J. Physiol. Renal Physiol. 4, 769-777, 2001).
  • the EGF-inducible association between cortactin and CD2AP and the appearance of the EGFR in the complex suggested that the proteins might co-localize.
  • the inventors therefore investigated the subcellular localization of cortactin, CD2AP and EGFR by confocal microscopy.
  • HeLa cells were transiently transfected with an expression construct comprising nucleic acid encoding a fusion protein consisting of EGFR and green fluorescent protein (GFP).
  • Cells were fixed either before EGF stimulation (time 0) or at 5 mins (5') or 15 mins (15') after EGF-stimulation, and analysed by indirect immunofluorescence using antibodies against cortactin or CD2AP.
  • EGFR was localized by fluorescence of the GFP moiety in the fusion protein.
  • Data in Figures 9A and 9B indicate localization of EGFR primarily in the plasma membrane, with cortactin and CD2AP primarily in punctate cytoplasmic structures, prior to EGF stimulation.
  • EGFR, cortactin and CD2AP are found in the membrane ruffles and a small subset of punctate cytoplasmic spots within 5 mins after EGF- stimulation. After 5 min EGF stimulation, the EGFR concentrates into prominent vesicular structures within the cytosol, presumably early endosomes. The co- localization of EGFR with CD2AP and cortactin is transient and returns to basal levels within 15 mins after EGF stimulation. These data are consistent with the formation of an EGF-induced complex containing both CD2AP and cortactin at membrane ruffles that regulates an early event in EGF receptor endocytosis.
  • EXAMPLE 7 The cortactin/CD2AP complex is implicated in regulation of EGFR endocytosis
  • a CD2AP-containing protein complex was involved in EGF receptor-mediated endocytosis
  • MDA-MB-231 cells were serum-starved and then treated with EGF for varying times up to 60 min.
  • the CD2AP-containing protein complexes were isolated by immune precipitation using antibodies against CD2AP, and subjected to Western blotting.
  • a negative control consisting of immune precipitates from serum-starved cells was also isolated by immune precipitation using antibodies against CD2AP, and subjected to Western blotting.
  • the antiphosphotyrosine antibodies were used to investigate the tyrosine phosphorylation status of proteins that immune precipitate with CD2AP, since the association of CD2AP with Cbl is dependent on tyrosine phosphorylation of the Cbl protein.
  • Data are presented in Figure 11.
  • anti-phosphotyrosine antibodies identified three proteins in CD2AP immune precipitates having molecular masses of approximately 180 kDa, 130 kDa and 120 kDa. A tight doublet of approximately 50 kDa was also detected. These tyrosine- phosphorylated proteins were detected between 1 min and 5 min following EGF stimulation ( Figure 11). A tyrosine phosphorylated protein of approximately 80 kDa was also detected between 2 min and 5 min following EGF stimulation of cells. The 180 kDa and 120 kDa bands were shown to correspond to EGFR and Cbl, respectively, as confirmed using antibodies against those proteins in Western blots ( Figure 11). Accordingly, Cbl protein in the CD2AP protein complexes was tyrosine phosphorylated.
  • CD2AP-containing protein complexes may exist in cells, ie., a protein complex consisting of CD2AP and cortactin, and a protein complex consisting of CD2AP and Cbl and EGFR.
  • a protein complex consisting of CD2AP and cortactin a protein complex consisting of CD2AP and Cbl and EGFR.
  • Cbl and EGFR a protein complex consisting of CD2AP and Cbl and EGFR.
  • the presence of Cbl and EGFR in cortactin-containing protein complexes was investigated.
  • HeLa cells were transiently transfected with either an empty vector control, or alternatively, a cortactin-encoding expression vector.
  • HeLa cells containing the empty vector control or expressing recombinant cortactin protein were either serum-starved and left untreated or stimulated with EGF under standard conditions for 2 min.
  • cortactin-containing protein complexes were isolated by immune precipitation using antibodies against cortactin, and subjected to Western blotting. Western blots were probed with antibodies against phosphotyrosine, Cbl and cortactin. Data are presented in Figure 12.
  • Peptide libraries are constructed in the fUSE5 gene III phage display system (Scott and Smith, Science 249, 386-390, 1990), and employing Escherichia coli host strains K91 Kan and MC1061 F as described by Pero et al, J. Biol. Chem 277, 11918-11926, 2002. Pools of transformants are plated on Pyrex dishes containing 2 YT/tetracycline/streptomycin medium (1 ml per dish). The dishes are incubated at 37°C overnight to allow and produce thousands of copies of each peptide phage particle library member.
  • nucleic acid encoding the SH3 domain of cortactin, or the SH3 domain of endophilin or the SH3 domain of CD2AP is inserted in the same reading frame as nucleic acid encoding GST present in plasmids essentially described by Janes et al, J. Biol. Chem. 272, 8490-8497, 1997 and Lowenstein et al, Cell 70, 431-442, 1992.
  • these proteins are expressed in a GST fusion bacterial expression system using the pGEX-2T expression vector (Amersham Biosciences, Inc.) and purified from isopropyl- ⁇ -D-thiogalactopyranoside-induced bacterial cultures as described previously (Smith and Johnson Gene (Amst.) 67, 31-40, 1988). Purity is determined by SDS-PAGE. Protein concentration is determined by Bradford based protein assay (Bio-Rad).
  • nucleic acid encoding a PRR of CD2AP or a PRR of Cbl or a PRR of endophilin is inserted in the same reading frame as nucleic acid encoding GST present in plasmids essentially described by Janes et al, J. Biol. Chem. 272, 8490- 8497, 1997 and Lowenstein et al, Cell 70, 431-442, 1992.
  • these PRR-GST fusion proteins are expressed in bacteria using the pGEX-2T expression vector (Amersham Biosciences, Inc.) and purified from isopropyl- ⁇ -D- thiogalactopyranoside-induced bacterial cultures as described previously (Smith and Johnson Gene (Amst.) 67, 31-40, 1988). Purity is determined by SDS-PAGE. Protein concentration is determined by Bradford basedprotein assay (Bio-Rad).
  • the peptide libraries are amplified so that there are about 1000 transforming units of each peptide phage representing 200 library equivalents.
  • the amplified phage are infected into K91/Kan cells, plated onto five 2YT agar dishes containing about 40 ⁇ g/ml tetracycline, incubated overnight at 30 °C, and harvested in 1x phosphate- buffered saline with a prokaryotic protease inhibitor mixture (1:1000)(Sigma) by sweeping the bacteria off the agar with a bent glass rod.
  • the bacterial cell suspension is centrifuged at about 8000 rpm for 10 min at 4°C, and the supernatant is filtered through a 0.22 ⁇ m polyethersulfone (PES) filter.
  • the filtrate is incubated for about 30 min on ice with 0.15 volume of polyethylene glycol/NaCI to precipitate the phage and centrifuged at 9000 rpm, 20 min, 4 °C.
  • the phage pellet is resuspended in 1x phosphate-buffered saline with a prokaryotic protease inhibitor mixture.
  • GST-binding peptide phage are subtracted from the library by preincubation of the library with 2 ⁇ g of GST immobilized on 20 ⁇ l of glutathione-Sepharose (Amersham Biosciences, Inc.).
  • the phage are screened by adding them to 12 ⁇ g of purified SH3-GST or PRR-GST fusion protein immobilized onto 20 ⁇ l glutathione-Sepharose and incubated for about 2 hr at 25 °C.
  • the unbound phage are removed by washing five times with 1x phosphate-buffered saline comprising a prokaryotic protease inhibitor mixture.
  • Phage that bind are eluted twice using a low pH buffer, such as pH 2.3 buffer, followed by two elutions using a high pH (eg., pH12) buffer.
  • K91/Kan cells are then infected with the pooled phage elutions, and a small aliquot is plated to determine the total amount of phage particles bound to the target GST fusion protein. The remaining phage are amplified overnight, harvested, screened, and eluted as described above.
  • half of the pre-cleared phage are added to fresh GST immobilized on glutathione beads to determine the amount of phage binding GST-glutathione-Sepharose compared to the binding of the phage to the GST fusion protein.
  • three rounds of screening are carried out before clones are sequenced or tested in an ELISA.
  • Isolated peptide phage-infected E. coli colonies are grown in 5 ml of LB/tetracycline broth.
  • the double-stranded phage replicative form DNA is isolated using the QIAprep Spin Miniprep kit (Qiagen).
  • Sequencing primers for sequencing a random insert in the fUSE5 vector is described by Scott and Smith, Science 249, 386-390, 1990). Sequencing reactions are carried out using BigDye Version 1 Dye Terminator kit (PerkinElmer Life Sciences).
  • the amino acid sequences of peptides displayed by peptide phage are deduced from the DNA sequence of the corresponding phage clones. Consensus sequence identification is performed by visual inspection and with the ClustalW alignment program.
  • synthetic peptides are produced using standard procedures for testing the ability of the peptides to modulate an interaction between the SH3 domain of cortactin, or the SH3 domain of endophilin or the SH3 domain of CD2AP, and a PRR of CD2AP or a PRR of Cbl or a PRR of endophilin.
  • a reverse phage ELISA (Valadon and Scharff, J. Immunol. Methods 197, 171-179, 1996; and Zwick et al, Curr. Opin. Biotechnol. 9, 427-436, 1998) is used to evaluate the ability of individual phage clones to bind to the SH3 domain or PRR present in the GST fusion protein.
  • protein targets at a concentration of about 5 ⁇ g/ml are added to a 96-well Maxisorp plate (Nunc) precoated with anti-GST polyclonal antibody at a concentration of about 1 ⁇ g/ml final concentration) (Amersham Biosciences, Inc.) and blocked using casein in Tris-buffered saline (Pierce).
  • the peptide phage are concentrated by precipitation using polyethylene glycol/NaCI, added to each well (1 x 10 7 to about 1 x 10 8 phage/well), and incubated for 2 h at room temperature.
  • Unbound phage are removed with 0.1 % Tween-Tris-buffered saline buffer, and phage are detected with horseradish peroxidase-conjugated anti-M13 monoclonal antibody (1 :1000) (Amersham Biosciences, Inc.) and 2,2'-azino-jb/s(3-ethylbenzothiazoline-6- sulfonic acid) (ABTS) substrate (Calbiochem). Plates are read at 405nm.
  • a competitive reverse phage ELISA is used, essentially as described by Barrett et al, Anal. Biochem. 204, 357-364, 1992 or Carcamo et al, Proc. Natl. Acad. Sci. )U. S. A.) 95, 11146-11151, 1998.
  • a competitive reverse phage ELISA purified GST fusion protein is added to a plate precoated with anti-GST polyclonal antibody and blocked with casein in Tris-buffered saline.
  • the peptide phage are concentrated by polyethylene glycol/NaCI precipitation,
  • the synthetic peptides are mixed at different concentrations (1, 10, and 100 ⁇ M) with peptide phage (1 x 10 6 transforming units/well) before adding to the GST fusion protein bound to the 96-well plate. Bound phage are detected as described above. Percent inhibition of binding achieved by the synthetic free peptides is calculated.
  • the relative ability of the free synthetic SH3-binding peptides or PRR-binding peptides to inhibit the formation of a protein complex described herein, particularly a complex comprising cortactin and CD2AP is determined by measuring the level of immunoprecipitated complex in the presence and absence of a synthetic free peptide comprising the SH3-binding or PRR-binding sequence.
  • whole cell extracts are made from MDA-MB-231 cells.
  • HeLa cells optionally transfected with cortactin-encoding nucleic acid, are grown until 70- 80% confluence in T-75 or T-150 flasks. After overnight starvation in serum-free media, the cells are stimulated with EGF for 2-5 min at 37 °C.
  • the cells are washed twice with phosphate-buffered saline, lysed with ice-cold lysis buffer (1% Triton X-100, 50 mM Hepes, 50 mM NaCl, 10% glycerol, 1.5mM MgCI2, 1 mM EGTA, 10 mM sodium pyrophosphate, 20 mM NaF, 10 ⁇ g/ml aprotinin, 10 ⁇ g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate) for 10 min on ice, and collected using a cell scraper.
  • ice-cold lysis buffer 1% Triton X-100, 50 mM Hepes, 50 mM NaCl, 10% glycerol, 1.5mM MgCI2, 1 mM EGTA, 10 mM sodium pyrophosphate, 20 mM NaF, 10 ⁇ g/ml a
  • the insoluble debris is separated by microfuging lysates for 5 min at 4 °C and 13,000 rpm ,and the supernatant is collected and frozen at -80 °C. Protein concentration of the lysate is determined by a Bradford- based protein assay (Bio-Rad).
  • the EGF-stimulated cell lysate (0.5-1 mg) is incubated with 20 ⁇ l of protein A-Sepharose to preclear the lysates.
  • the precleared lysate is incubated with increasing amounts of free synthetic peptide or no peptide (control) for 1 h at 4 °C and then immunoprecipitated with an amount (eg., 2 ⁇ g) of a polyclonal or monoclonal antibody against CD2AP or cortactin (UBI; Santa Cruz Biotechnology) overnight at 4°C.
  • the immune precipitated protein complexes are collected by incubation with 50 ⁇ l of protein A-Sepharose 4B beads (Zymed Laboratories Inc.) for 1 h at 4 °C.
  • the immune complexes are collected by centrifugation, washed five times in cold lysis buffer, and eluted with sample buffer at 100 °C for 5 min.
  • the immune complexes are then subjected to Western blot analysis on a 7.5% SDS-polyacrylamide gel using one or more of the following primary antibodies: horseradish peroxidase-conjugated anti-phosphotyrosine recombinant antibody (BD Transduction Laboratories); anti-cortactin polyclonal antibody (Santa Cruz Biotechnology) or anti-cortactin monoclonal antibody (UBI); anti-CD2AP polyclonal antibody (Santa Cruz Biotechnology); anti-Cbl polyclonal antibody (Santa Cruz Biotechnology); anti-endophilin polyclonal antibody (Santa Cruz Biotechnology); and anti-EGFR polyclonal antibody (Santa Cruz Biotechnology);.
  • primary antibodies horseradish peroxidase-conjugated anti-phosphotyrosine recombinant antibody (BD Transduction Laboratories); anti-cortactin polyclonal antibody (Santa Cruz Biotechnology) or anti-cortactin monoclonal antibody (UBI);
  • those peptides that modify the amount of a protein that is recruited to a cortactin immune precipitate or CD2AP immune precipitate such as, for example, by enhancing the level of recruitment of Cbl to cortactin and/or CD2AP, or that enhances the recruitment of a complex comprising cortactin and Cbl and CD2AP to the EGFR are retained.

Abstract

The present invention provides novel protein complexes involving SH3 domain-containing proteins (i.e. proteins having a Src homology 3 domain) and proteins having one or more proline-rich regions (PRRs), specifically novel protein complexes comprising a cortactin protein and/or a CD2AP protein or a portion thereof and one or more other proteins selected from the group consisting of Cbl, endophilin, ASAP1, N-WASP, FISH and Sam68, and methods and reagents for isolating, producing and determining the presence of the protein complexes, including prognostic and diagnostic methods for determining a predisposition for disease, or a disease state characterized by aberrant EGF receptor (EGFR) endocytosis. The present invention further provides methods for determining a modulator, specifically an agonist or antagonist of the formation or stability of a protein complex and uses therefor in the treatment of disorders characterized by aberrant EGFR endocytosis.

Description

Novel protein complexes and uses therefor
Field of the invention
The present invention relates generally to the field of interactions between proteins and novel heterodimeric and heteromultimeric protein complexes, and, in particular, protein-protein interactions involving SH3 domain-containing proteins (i.e. proteins having a Src homology 3 domain) and proteins having one or more proline-rich regions (PRRs). More specifically, the present invention provides novel protein complexes comprising a cortactin protein and/or a CD2AP protein or a portion thereof and one or more other proteins. The present invention further provides methods for isolating the protein complexes or binding partner(s) of such protein complexes, and for producing the protein complexes of the invention in vitro. The present invention further provides prognostic and diagnostic methods for determining a predisposition for disease, or a disease state, said methods comprising detecting the presence or absence of a protein complex of the invention. The present invention further provides methods for determining a modulator, specifically an agonist or antagonist of a protein complex of the invention. The present invention further provides methods for determining a modulator of an interaction between a cortactin protein and/or a CD2AP protein or a portion thereof and one or more other proteins, particularly for determining modulators that are useful for the therapeutic or prophylactic treatment of a disease. The present invention further provides kits for producing a protein complex of the invention or for identifying modulators of complex formation, optionally packaged with instructions for use. The present invention further provides methods for the treatment of certain cellular proliferative disorders, said methods comprising modulating the level of a protein complex of the invention.
Background to the invention 1. General
This specification contains nucleotide and amino acid sequence information prepared using Patentln Version 3.1, presented herein after the claims. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>1, <210>2, <213> etc). The length and type of sequence (DNA, protein (PRT), etc), and source organism for each nucleotide sequence, are indicated by information provided in the numeric indicator fields <211 >, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are defined by the term "SEQ ID NO:", followed by the sequence identifier (eg. SEQ ID NO: 1 refers to the sequence in the sequence listing designated as <400>1).
The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.
As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.
2. Description of the related art The effective diagnosis, prognosis, and treatment of certain cancers requires a clear understanding of signal transduction pathways that lead to cancer. This is because cancer is generally a disease of the intracellular signalling system. The signal transduction components involved in cancer development can be effective diagnostic or prognostic indicators of disease, as well as effective targets for treatment.
Regulation of growth and development of an individual and maintenance of a healthy state depend, in part, on the precise regulation of protein-protein interactions. For example, an interaction between an extracellular growth factor (e.g. epidermal growth factor) and its cell surface cognate receptor can trigger various intracellular protein- protein interactions that activate a signalling cascade from the cell surface to the cell nucleus, thereby regulating specific patterns of gene expression. Protein-protein interactions are also involved in virtually all metabolic pathways that occur in a cell, including, for example, the formation of holoenzyme complexes, enzyme-substrate interactions, immune responses (e.g. recognition of self versus non-self, antigen- antibody complexes, T-cell mediated responses, etc), cell adhesion and cytoskeleton formation.
Since protein-protein interactions are central to normal growth and development of the individual, the identification of the proteins involved in such interactions can provide insight into the normal cellular metabolism in an organism. More practically, the identification and characterization of particular protein-protein interactions permits the identification of defects in such interactions that are associated with a diseased state. The identification of such associations provides a target for diagnostic, prophylactic and therapeutic strategies that are highly specific. For example, the identification and characterization of protein-protein interactions provides a means to screen for drugs that modulate the interaction, such as antagonists or agonists of the interaction. Aberrant protein-protein interactions, or a defect in one or more of the binding partners to a protein-protein interaction, can have deleterious effects on a cell or produce a disease state. For example, the protein-protein interactions that facilitate signalling downstream of receptor tyrosine kinases (RTKs), such as, for example, Src cytoplasmic tyrosine kinase, are generally thought to be involved in the progression of certain cancers.
Many of the protein-protein interactions involving Src family proteins are mediated by specific functional protein domains, which are conserved in a number of proteins of different classes that are involved in distinct regulatory cascades. These domains are the so-called Src homology 2 and Src homology 3 (SH2 and SH3) domains. The SH2 domain and SH3 domains are necessary for interactions with protein substrates and determine intramolecular binding, protein localization and association with protein targets.
SH3 domain
The SH3 domain was first described for Src, and is also present in a number of other tyrosine kinases, and proteins associated with organization of the cytoskeleton, including fodrin and cortactin. The SH3 domain is thought to form a compact, globular, partly opened β-barrel structure, that is believed to assist in recruiting proteins having proline-rich regions (PRRs) to particular locations within the cell.
Notwithstanding that PRRs are thought to have a conserved amino acid consensus sequence, the presence or absence of this region alone is insufficient to determine whether or not a specific protein-protein interaction between an SH3 protein and another protein occurs in vitro or in vivo. This is because not all protein-protein interactions involving SH3 proteins necessarily involve proteins comprising PRRs. Moreover, PRRs also occur in proteins that do not bind to SH3 proteins. Cortactin
Cortactin is a multi-domain actin-binding protein that is probably tyrosine phosphorylated by a Src kinase, that has been shown to bind several other proteins, including proteins of the Arp2/3 complex (see below), brain cortactin binding protein-1 (CortBPI or Shank 2; Du et al., Mol. Cell. Biol. 18, 5838-5851, 1998; Weed and Parsons Oncogene 20, 6418-6434, 2001), brain ZO-1 protein (Katsube et al., J. Biol. Chem 273, 29672-29677, 1998), and dynamin-2 (McNiven et al., J. Cell Biol. 151, 187- 198, 2000). These interactions suggest that cortactin contributes to the spatial organization of sites of actin polymerization, coupled to selected cell surface transmembrane receptor complexes. Accordingly, cortactin is thought to be involved in one or more signalling pathways that modify the adhesive properties of cells.
Cortactin is normally enriched within the lamellipodia of motile cells and in neuronal growth cones, particularly in the brain. Cortactin is localized with the actin-related protein (Arp) 2/3 complex, at sites within the lamellipodia where actin polymerizes. Cortactin stimulates nucleation activity of the Arp2/3 complex, and enhances actin polymerization induced by proteins associated with Wiskott-Aldrich Syndrome (WASPs), that are co-activators of the Arp2/3 complex (Weaver et al. Curr. Biol. 11, 37-374, 2001; Urono et al., Nature Cell Biol. 3, 259-266, 2001). Weaver et al. (2001) also showed that cortactin promotes the formation and stability of branched actin networks. In addition to its role in forming cortical actin structures, cortactin may regulate vesicle trafficking, for example, of endosomes (Kaksonen et al., J. Cell Sci. 113, 4421-4426, 2000), a role that is supported by its interaction with dynamin-2 (McNiven et al., J. Cell Biol. 151, 187-198, 2000). Finally, cortactin also appears to regulate the organization and subcellular localization of transmembrane complexes, wherein CortBPI performs a scaffolding function in the organization of receptor complexes at post-synaptic sites of excitatory synapses, and ZO-1 interacts with the transmembrane proteins claudin and occludin at epithelial tight junctions (Weed and Parsons Oncogene 20, 6418-6434, 2001). Cortactin also binds to rpdeδ, in rat brain tissue.
The amino acid sequence of the cortactin polypeptide comprises the following domains: (i) an N-terminal stretch of acidic amino acid residues (NTA); (ii) Six to seven tandem repeats of an amino acid sequence that is related to that found in HS1, located C-terminal to the acidic domain; (iii) a sequence having the predicted structure of an α- helix located C-terminal to the Hs1/cortactin repeat; (iv) a region rich in serine, threonine and proline, located C-terminal to the α-helical domain; and (v) a C-terminal SH3 domain. It is known that the NTA region of cortactin mediates binding to the Arp2/3 complex, wherein the fourth tandem repeat four is necessary for cortactin to stably bind F-actin in vitro, (Weed et al., J. Cell Biol. 151, 29-40, 2000). On the other hand, the SH3 domain of cortactin is known to be involved in binding rpde6, CortBPI and ZO-1.
Over expression of cortactin also enhances cell motility and invasion in vitro (Patel et al., Oncogene 16, 3227-3232, 1998; Huang et al., J. Biol. Chem. 273, 25770-25776, 1998). Over expression of cortactin in breast cancer cells may also affect the invasive properties of the cancer cells (Kairouz and Daly, Breast Cancer Res 2, 197-202, 2000), and metastases in bone (Li et al., Cancer Res. 61, 6906-6911, 2001). The EMS1 gene that encodes cortactin is commonly amplified in breast cancers and squamous cell carcinomas of the head and neck (Schuuring et al., Cancer Res. 52, 5229-5234, 1992; Fantl et al, Cancer Surveys 18, 77-94, 1993; Williams et al, Arch. Otalaryngol. Head Neck Surg. 119, 1238-1243, 1993). Amplification of the EMS1 gene is also associated with a poor prognosis in node negative or ER-negative breast cancer (Hui et al, Oncogene 15, 1617-1623, 1997) and in head and neck cancers (Rodrigo et al., Clin. Cancer Res. 6, 3177-3182, 2000). Cortactin is localized to sites of invasion in the extracellular matrix in MDA-MB-231 breast cancer cells (Bowden et al., Oncogene 18, 4440-4449, 1999).
However, the specific nature of any interaction between cortactin and another protein that might be involved in disease progression has not been elucidated.
CD2AP CD2AP is a known adaptor molecule that associates with a variety of membrane proteins, to organize the cytoskeleton around a polarized site. CD2AP is a Src substrate. CD2AP is also known to be expressed at high levels in glomerular podocytes wherein it binds nephrin, and less so in renal tubular epithelial cells of the adult kidney, particularly in distal nephron segments, where it binds to polycystin-2 (Dustin et al., Cell 94, 667-677, 1998; Kirsch et al, Proc Natl Acad. Sci USA 96, 6211- 6216, 1999; Lehtonen et al., J. Biol. Chem 275, 32888-32893, 2000). Podocin, a component of the glomerular slit diaphragm, also binds CD2AP (Schwarz et al., J. Clin. Invest. 108, 1621-1629, 2001). In T cells, CD2AP binds CD2 and functions in antigen recognition, by regulating receptor patterning and cytoskeleton polarization.
Cbl adaptor protein
Cbl is a multi-adaptor protein that is involved in ligand-induced down regulation of receptor tyrosine kinases, wherein Cbl-mediated ubiquitination of active receptors is essential for receptor degradation and turnover, thereby leading to cessation of downstream signalling from the receptor (Soubeyran et al, Nature 416, 183-187, 2002).
Endophilin
The endophilins are a family of SH3 domain-containing proteins having homology to Grb2 (i.e. Grb2-like or "GL" proteins) designated endophilin I (human homolog SH3GL2) , endophilin II (human homolog SH3GL1), endophilin III (human homolog SH3GL3), endophilin B1 (human homolog SH3GLB1 and endophilin B2 (human homolog SH3GLB2). There are four isoforms of endophilin III and two isoforms of endophilin B1 generated by alternative splicing. The endophilins are implicated in clathrin-mediated endocytosis (Schmidt et al, Nature 401, 133-141, 1999; Ringstad et al, Neuron 24, 143-154, 1999; Simpson et al, Nature Cell. Biol. 1, 119-124-1999; Gad et al, Neuron 27, 301-327, 2000; Soubeyran et al, Nature 416, 183-187, 2002).
ASAP1 protein
ASAP1 is a phosphatidylinositol 4,5-bisphosphate (PIP2)-dependent ADP-ribosylation factor-1 (ARF1) GTPase activating protein (GAP) (Brown et al, Mol. Cell. Biol: 18, 7038-7051, 1998; Andreev et al, Mol. Cell. Biol. 19, 2338-2350, 1999).
The ASAP1 protein is widely expressed as two variants, ASAPIa and ASAPIb, that differ by a 57 amino acid insert that is present in ASAPIa but not ASAPIb. The amino acid sequences of both ASAPIa and ASAPIb comprise a GAP domain of about 70 amino acids in length that includes a zinc finger motif required for GAP activity, as well as multiple domains required for forming protein-protein interactions. These domains include a PH domain, three ankyrin repeats, a PRR for binding to SH3 domains, and an SH3 domain (Donaldson, Proc Natl Acad. Sci USA 97, 3792-3794, 2000).
ASAP1 can bind to the SH3 domains of Src and c-Crk, and is tyrosine phosphorylated in cells wherein Src is activated (Brown et al, Mol Cell. Biol. 18, 7038-7051, 1998). ASAP1 is normally localized to focal adhesions, where it co-localizes with paxillin and vinculin (Randazzo et al, Proc Natl Acad. Sci USA 97, 4011-4016, 2000). Over expression of ASAP1 can modify cell spreading (Randazzo et al, Proc Natl Acad. Sci USA 97, 4011-4016, 2000).
Neuronal Wiskott-Ald ch Syndrome Protein (N-WASP)
Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) is a substrate for PIP2 and Cdc42 (Fukuoka et al, Gene 196, 43-48, 1997; Miki et al, Nature 391, 93-96, '1998; Rohatgi et al, Cell 97, 221-223, 1999; Rohatgi et al, J. Cell Biol. 150, 1299-1309, 2000). N-WASP has also been shown to interact with Nek and WIP proteins.
FISH Protein FISH is a Src substrate adapter protein that is known to be tyrosine phosphorylated following disruption of the cytoskeleton using cytochalasin D (Lock et al., EMBO J. 17, 4346-4357, 1998). FISH comprises five SH3 domains.
Sam68 Protein Sam68 (Src-associated in mitosis) protein is tyrosine phosphorylated by c-Src, and can bind c-Src, Grb2, and RNA. Sam68 is thought to be involved in cell cycle progression, because it is tyrosine phosphorylated in mitotic cells but not in asynchronous cells, and because its RNA binding activity decreases when it is phosphorylated.
The Epidermal Growth Factor (EGF) Receptor (EGFR)
The EGF receptor (EGFR) regulates differentiation and growth in both normal and neoplastic cells. In normal cells exposed to EGF, the receptor is phosphorylated at tyrosine residues. Upon ligand-induced activation of EGFR, the signal protein Cbl (Langdon et al, J. Virol. 63, 5420-5424, 1989; Langdon et al, Proc. Natl Acad. Sci. USA 86, 1168-1172, 1989) binds to phosphorylated EGFR, promoting its ubiquitination. Cbl is also tyrosine phosphorylated in this process and leads to translocation of a protein complex involving CIN85 and endophilin-A3 protein to the vicinity of the activated EGFR, which in turn enhances the clathrin-mediated endocytosis of the activated EGFR (Soubeyran et al., Nature 416, 183-187, 2002). Cbl is also implicated in sorting internalized EGFR for lysosomal degradation, and appears to play a critical role in ligand-induced receptor down regulation (Soubeyran et al, Nature 416, 183-187, 2002).
Elevated levels of EGFR have been reported in many human tumors and cell lines, including breast cancer, adenocarcinoma and squamous lung cancer, gastrointestinal cancers (gastric, colon, pancreatic), renal cell cancer, bladder cancer, glioma, gynecological carcinomas, and prostate cancer. The ectodomain of EGFR is also detectable in the urine of about 36% of squamous cell carcinoma patients and about 16% of patients having non-squamous carcinoma with higher levels in subjects having metastatic disease compared to those having a localized disease. Additionally, a mutant EGFR (ie. EGFRvlll) occurs frequently in ovarian, breast, lung, glioblastoma, and medulloblastoma tumors. The mechanism(s) regulating EGFR levels in normal cells or leading to elevated EGFR levels in neoplastic cells is(are) not known.
The action of a variety of itogens, including EGF, is mediated by a signalling cascade leading to phosphorylation and activation of mitogen-activated protein (MAP) kinases or extracellular signal regulated kinases (ERKs). ERK-1, ERK-2, ERK-3 and ERK-4 function as proline-site-directed serine-threonine kinases in the phosphorylation of transcription factors such as p65TCF/Elk-1, c-jun and c-myc, and thus appear to play a crucial role in signal transduction by converting extracellular stimuli into transcriptional activation (Boulton et. al, Biochemistry 30, 278-286, 1991; Boulton et al, Science 249, 64-65, 1990; Boulton and Cobb, Cell Regulation 2, 357-371, 1991; Boulton et al, Cell 65, 663-675, 1991; Cobb et al, Cell Regulation 2, 965-978, 1991; and WO 91/19008 published Dec. 12, 1991). The MAP kinases are believed to be directly involved in the regulation of genes that are responsible for cell proliferation. MAP kinases are regulated by various levels of upstream regulatory proteins mediated in part by reversible protein phosphorylation involving a conserved signalling cascade initiated by ligand induced activation of receptor tyrosine kinases (RTKs) which sequentially activate a series of protein kinases. For example, an activated EGFR might signal via ras to the serine-threonine kinase raf to directly activate the MAP- kinase/ERK-kinase designated MEK. MAP kinases are stimulated by phosphorylation on two regulatory threonine and tyrosine residues, respectively, which is catalyzed by activated MEK. Upon activation, MAP kinases translocate to the nucleus and phosphorylate transcription factors. The S6 kinase II (pp90 rsk) is another substrate of MAP kinases that is activated by ERKs, which may control protein translation. MAP kinases can also be activated by TPA in a ras-dependent pathway to thereby stimulate PKC and signal to MEK via raf protein.
Considerable attention has been directed to how the expressed products of oncogenes alter the signal transduction pathway of cells, thereby leading to cancer. Oncogenes can lead to the hyperstimulation of one or more signal transduction pathways causing the cell nucleus to receive an inappropriate signal to proliferate. Many oncogenes are members or targets of the family of the ras/MAP kinase pathways, such as, for example, ras, raf-1, myc, ski, myb, fos and jun (Blenis Proc. Natl. Acad Sci. USA., 90, 5889-5892, 1994; Cobb et al, J. Biol. Chem. 270, 14843- 14846, 1995; Janes et al, Oncogene 9, 3601-3608, 1994). Currently, the focus on signal transduction pathway alterations as targets for effective therapies is primarily directed to the upstream regulators of MAP kinases, such as ras, raf-1, and MEK. However, strategies that inhibit or inactivate ras, raf-1 or MEK have generally not been effective, presumably because other pathways also operate to promote unregulated cell proliferation.
Increased MAP kinase expression is known for certain non-small cell lung carcinoma and breast cancer cell lines, and is associated with over expression of ERK-1 or ERK- 2 (Cobb , Melanie H., "The Role of MAP Kinase Pathway in Breast Cancer," National Technical Information Service, Accession No. AD-8301 655/7/XAB, 1995) However, studies that illustrate the hyper-activation of MAP kinase do not provide any suggestions as to the cause of the hyper-activation or provide new routes of treating malignant neoplastic cell growth, such as primary breast carcinoma.
Notwithstanding the importance of cellular signal cascades and tyrosine phosphorylation in normal cellular development and the progression of certain cancers, little is known of the specific protein-protein interactions involved in disease progression.
Summary of the invention In work leading up to the present invention, the inventors sought to identify the protein- protein interactions associated with normal or aberrant regulation, formation or activity of the cytoskeleton, including cell growth or motility, tumorigenesis, metastasis, tumor cell invasion, T cell-mediated immunity, autoimmunity, transplantation, allergy, or asthma.
The inventors identified several novel binding partners of cortactin, particularly proteins that bind to the cortactin SH3 domain. The protein-protein interactions are useful as prognostic and diagnostic markers for diseases and disorders. The inventors have also developed novel screens for compounds that modulate the protein-protein interactions of the invention, which compounds are useful in the treatment of a range of different diseases or disorders.
The finding by the present inventors that cortactin binds CD2AP indicates a role for cortactin in both antigen recognition and cell motility. Accordingly, blocking of the cortactin-CD2AP interaction provides a novel approach for treating a range of cancers and T cell mediated disorders, such as, for example, autoimmune diseases, transplant rejection, allergy, or asthma.
Based upon the novel finding by the inventors that cortactin binds to the CD2 associated protein, CD2AP, the inventors subsequently focussed their attention on binding partners for CD2AP. Surprisingly, the inventors identified several novel binding partners of CD2AP, including endophilin, and subsequently identified tertiary and quaternary protein complexes comprising cortactin and/or CD2AP. These higher- order protein complexes were also shown by the inventors to be involved in regulating EGFR levels in normal or neoplastic cells, such as, for example, by modulating EGFR endocytosis or EGFR recycling and, as a consequence, are important in EGF signalling in normal or neoplastic cells.
As exemplified herein, the present inventors have shown that a protein complex comprising cortactin and CD2AP and endophilin and/or Cbl may positively regulate EGFR endocytosis. As used herein, the term "endocytosis" with reference to the EGFR is to be understood to include any down regulation of the steady state level of EGFR available for EGF signalling in a cell, such as, for example, as a consequence of Cbl-mediated ubiquitylation of EGFR that directs the receptor to the lysosome for destruction.
Accordingly, agonist compounds that enhance the level of formation of a protein complex comprising cortactin and CD2AP are particularly useful in preventing or treating elevated EGFR levels, such as, for example, in the prophylactic or therapeutic treatment of a cancer that is characterized by elevated EGFR. In fact, the protein complexes described herein are useful for identifying a range of novel therapeutic agents for the prophylaxis or therapy of any condition characterized by elevated EGFR.
Accordingly, one aspect of the present invention provides an isolated or recombinant protein complex, such as, for example, an isolated heterodimer or hetero-multimer comprising: (i) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1 , FISH and Sam68; and (b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and (ii) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
(b) a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide.
Preferably, a protein complex of the invention further comprises Epidermal growth factor receptor (EGFR).
As will be apparent from the disclosure herein, the binding partners of the invention may consist of two or three or four or five intact polypeptides, or two or three or four or five portions of the intact polypeptides capable of forming a complex, or a fusion protein comprising the binding partners of the complex.
It will be apparent from the description that the present invention provides various cortactin-containing protein complexes and CD2AP-containing protein complexes, such as, for example, a complex selected from the group consisting of: (i) a complex comprising CD2AP and cortactin; (ii) a complex comprising CD2AP and Cbl; (iii) a complex comprising CD2AP and endophilin; (iv) a complex comprising CD2AP and endophilin and cortactin; (v) a complex comprising CD2AP and Cbl and cortactin; (vi) a complex comprising CD2AP and endophilin and Cbl; (vii) a complex comprising CD2AP and endophilin and Cbl and cortactin; (viii) a complex comprising cortactin and ASAP1; (ix) a complex comprising cortactin and N-WASP; (x) a complex comprising cortactin and FISH; and (xi) a complex comprising cortactin and Sam68. Preferably, the protein complex of the invention further comprises EGFR.
As used herein, the term "CD2AP" shall be taken to mean any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence of a human or mouse CD2AP polypeptide set forth in SEQ ID NO: 1 or 2. The term "CD2AP" shall also be taken to include a peptide, polypeptide or protein having the known biological activity of CD2AP, or the known binding specificity of CD2AP. The term "CD2AP" shall also be taken to include a peptide, polypeptide, or protein having the known biological activity of CD2AP, or the known binding specificity of CD2AP, wherein said peptide, protein or polypeptide is further capable of binding to cortactin or a portion of cortactin and/or to an endophilin polypeptide or a portion of an endophilin polypeptide. For the purposes of nomenclature, the amino acid sequences of the human and mouse CD2AP polypeptides are exemplified herein, as SEQ ID Nos: 1 and 2, respectively. Preferably, the percentage identity to SEQ ID NO: 1 or 2 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%. In a particularly preferred embodiment, the CD2AP is human CD2AP.
As used herein, the term "cortactin" shall be taken to mean any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence a human or mouse cortactin polypeptide set forth in SEQ ID NO: 3 or 4. The term "cortactin" shall also be taken to include a peptide, polypeptide or protein having the known biological activity of cortactin, or the known binding specificity of cortactin, wherein said peptide, protein or polypeptide is further capable of binding to CD2AP or a portion of CD2AP. For the purposes of nomenclature, the amino acid sequences of the human and mouse cortactin polypeptides are exemplified herein, as SEQ ID Nos: 3 and 4, respectively. Preferably, the percentage identity to SEQ ID NO: 3 or 4 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%. In a particularly preferred embodiment, the cortactin is human cortactin.
As used herein, the term "Cbl" shall be taken to mean any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence of a human or mouse Cbl polypeptide set forth in SEQ ID NO: 5 or 6. The term "Cbl" shall also be taken to include a peptide, polypeptide or protein having the known biological activity of Cbl, or the known binding specificity of Cbl, wherein said peptide, protein or polypeptide is further capable of binding to an endophilin polypeptide or a portion of an endophilin polypeptide. For the purposes of nomenclature, the amino acid sequences of the human and mouse Cbl polypeptides are exemplified herein, as SEQ ID Nos: 5 and 6, respectively. Preferably, the percentage identity to SEQ ID NO: 5 or 6 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%. In a particularly preferred embodiment, the Cbl is human Cbl.
As used herein, the term "endophilin" shall be taken to mean any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence of a human endophilin polypeptide set forth in SEQ ID NO: 7 or 8 or 9. The term "endophilin" shall also be taken to include a peptide, polypeptide or protein having the known biological activity of endophilin I, endophilin II or endophilin III, or the known binding specificity of said endophilin 1, endophilin II or endophilin III, wherein said peptide, protein or polypeptide is further capable of binding to CD2AP or a portion of CD2AP and preferably, in addition, to a Cbl polypeptide or a portion of a Cbl polypeptide. For the purposes of nomenclature, the amino acid sequences of the antigenically cross-reactive human endophilin I, II and III polypeptides are exemplified herein, as SEQ ID Nos: 7, 8 and 9, respectively. Preferably, the percentage identity to SEQ ID NO: 7 or 8 or 9 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
As used herein, the term "ASAP1" shall be taken to refer to any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence of a mouse ASAP1 polypeptide and capable of binding to a cortactin polypeptide. The term "ASAP1" shall also be taken to include a peptide, polypeptide, or protein having the known biological activity of ASAP1, or the known binding specificity of ASAP1 , wherein said peptide, protein or polypeptide is further capable of binding to cortactin or a portion of cortactin. For the purposes of nomenclature, the amino acid sequences of the mouse ASAPIa and mouse ASAPIb polypeptides are provided herein, as SEQ ID Nos: 10 and 11, respectively. Preferably, the percentage identity to SEQ ID NO: 10 or 11 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%. In a particularly preferred embodiment, the ASAP1 protein is a human ortholog of a mouse ASAP1 protein exemplified in the sequence listing.
As used herein, the term "N-WASP" shall be taken to refer to any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence of a human or rat N-WASP polypeptide and capable of binding to a cortactin polypeptide. The term "N-WASP" shall also be taken to include a peptide, polypeptide, or protein having the known biological activity of N-WASP, or the known binding specificity of N-WASP, wherein said peptide, protein or polypeptide is further capable of binding to cortactin or a portion of cortactin. For the purposes of nomenclature, the amino acid sequences of the human N-WASP and rat N-WASP polypeptides are provided herein, as SEQ ID Nos: 12 and 13, respectively. Preferably, the percentage identity to SEQ ID NO: 12 or 13 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%. In a particularly preferred embodiment, the N-WASP is human N-WASP.
As used herein, the term "FISH" shall be taken to refer to any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence of a human or mouse FISH polypeptide and capable of binding to a cortactin polypeptide. For the purposes of nomenclature, the amino acid sequences of the human FISH and mouse FISH polypeptides are provided herein, as SEQ ID Nos: 14 and 15, respectively. Preferably, the percentage identity to SEQ ID NO: 14 or 15 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%. In a particularly preferred embodiment, the FISH is human FISH. The term "FISH" shall also be taken to include a peptide, polypeptide, or protein having the known biological activity of FISH, or the known binding specificity of FISH, wherein said peptide, protein or polypeptide is further capable of binding to cortactin or a portion of cortactin.
As used herein, the term "Sam68" shall be taken to refer to any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence of a human or rat Sam68 polypeptide and capable of binding to a cortactin polypeptide. For the purposes of nomenclature, the amino acid sequences of the human Sam68 and rat Sam68 polypeptides are provided herein, as SEQ ID Nos: 16 and 17, respectively. Preferably, the percentage identity to SEQ ID NO: 16 or 17 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%. In a particularly preferred embodiment, the Sam68 is human Sam68. The term "Sam68" shall also be taken to include a peptide, polypeptide, or protein having the known biological activity of Sam68, or the known binding specificity of Sam68, wherein said peptide, protein or polypeptide is further capable of binding to cortactin or a portion of cortactin.
Another aspect of the present invention provides isolated peptides and kits comprising same for producing a protein complex comprising:
(i) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1 , FISH and Sam68; and (b) a polypeptide selected from the group consisting of CD2AP, N-
WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and (ii) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
(b) a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide, wherein said protein complex further optionally comprises EGFR.
The peptides and/or kits of the present invention are also useful, or for identifying a modulator of the formation or stability of any one of said complexes.
Another aspect of the present invention provides an isolated antibody that binds to a protein complex comprising:
(i) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1 , FISH and Sam68; and (b) a polypeptide selected from the group consisting of CD2AP, N-
WASP, ASAP1 , FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and (ii) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
(b) a polypeptide selected from the group consisting of cortactin,
Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide, wherein said protein complex further optionally comprises EGFR and wherein said antibody does not bind to any individual protein of said complex in the absence of another protein of said complex.
Another aspect of the present invention provides an anti-idiotypic antibody that binds to an antibody or ligand that binds to a protein complex comprising:
(i) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1 , FISH and Sam68; and
(b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1 , FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and (ii) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
(b) a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide, wherein said protein complex further optionally comprises EGFR and wherein said anti-idiotypic antibody does not bind to an antibody that binds to any individual protein of said complex in the absence of another protein of said complex.
Another aspect of the present invention provides methods for isolating a cortactin- binding protein or CD2AP-binding protein from a suitable cellular source, such as, for example, an ER-negative breast cancer cell. As exemplified herein, a cortactin polypeptide, or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1, FISH and Sam68, has been used as an affinity probe to bind said protein in a cellular extract from an ER-negative breast cancer cell or HeLa cell over expressing cortactin. Also exemplified herein, a CD2AP polypeptide, or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin, has been used as an affinity probe to bind such a protein in a cellular extracts from an ER-negative breast cancer cell, or transfected HeLa cells over expressing cortactin. During affinity purification, an unbound protein or non-specifically bound protein is removed by washing, thereby isolating a CD2AP binding protein substantially free of conspecific proteins. It will be apparent to those skilled in the art that such affinity purification methods can also be adapted to the isolation of a protein complex comprising CD2AP or a portion of CD2AP and its cognate binding partner from ER-negative breast cancer cells or HeLa cells.
Another aspect of the present invention provides a method for isolating a protein complex comprising:
(i) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of
CD2AP, N-WASP, ASAP1, FISH and Sam68; and (b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and
(ii) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
(b) a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide, wherein said protein complex further optionally comprises EGFR and wherein said method comprises contacting protein from an ER-negative breast cancer cell with an antibody or other ligand that binds to said protein complex under conditions sufficient for binding to occur and removing unbound protein, thereby isolating the protein complex substantially free of conspecific proteins.
Another aspect of the present invention provides methods for producing a protein complex described herein by recombinant means. In one embodiment of the invention, nucleic acid comprising a sequence that encodes a cortactin polypeptide or a portion of a cortactin polypeptide and a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide, in operable connection with a suitable promoter sequence, is expressed in a suitable cell for a time and under conditions sufficient to produce a fusion polypeptide comprising the binding partners of the complex. In another embodiment, nucleic acid comprising a sequence that encodes a CD2AP polypeptide or a portion of a CD2AP polypeptide and a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide, in operable connection with a suitable promoter sequence, is expressed in a suitable cell for a time and under conditions sufficient for expression to occur.
Preferably, the nucleic acid further comprises a nucleotide sequence encoding an EGFR polypeptide or a portion of said EGFR polypeptide sufficient to bind to a Cbl polypeptide or a portion of a Cbl polypeptide. In this respect, it is to be understood that the EGFR polypeptide may indirectly bind to CD2AP or to a portion of CD2AP by virtue of the association between Cbl and CD2AP. Similarly, by virtue of the association between cortactin and CD2AP, the EGFR indirectly binds to cortactin or a portion thereof that can form an association with CD2AP, or a portion of CD2AP that binds to cortactin and Cbl.
In another embodiment, nucleic acid comprising a sequence encoding each binding partner is placed in operable connection with a promoter sequence and expressed in a suitable cell. If the protein partners are expressed in the same cell, they may freely associate in said cell to form the protein complex of the invention. If the protein partners are produced in different cells, the cells are lysed and the cellular lysates mixed under conditions sufficient to permit the association of the binding partners.
The protein-protein interactions of the invention, particularly those involving cortactin and/or CD2AP in association with Cbl, are useful as prognostic and diagnostic markers for cancers in which EGFR is elevated.
Accordingly, another aspect of the present invention provides prognostic and diagnostic methods for determining a predisposition for disease, or a disease state, said methods comprising detecting the presence or absence of a protein complex comprising:
(i) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH and Sam68; and (b) a polypeptide selected from the group consisting of CD2AP, N-
WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and (ii) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polyp~eptide selected from the group consisting of cortactin, Cbl and endophilin; and
(b) a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide, wherein said protein complex further optionally comprises EGFR.
Preferred detection systems include any known assay for detecting a protein-protein interaction in a biological sample isolated from a human or mammalian subject, such as, for example, using one or more antibodies against the complex or each binding partner, or an epitope thereof. Alternatively, a non-antibody ligand of the protein complex may be used, such as, for example, a small molecule (e.g. a chemical compound, agonist, antagonist, allosteric modulator, competitive inhibitor, or non- competitive inhibitor, of the complex that may or may not modulate complex formation or dissociation). In accordance with these embodiments, the antibody or small molecule may be used in any standard solid phase or solution phase assay format amenable to the detection of protein complexes or protein-protein interactions. Optical or fluorescent detection, such as, for example, using mass spectrometry, MALDI-TOF, biosensor technology, evanescent fiber optics, or fluorescence resonance energy transfer, is clearly encompassed by the present invention. Assay systems suitable for use in high throughput screening of mass samples, particularly a high throughput spectroscopy resonance method (e.g. MALDI-TOF, electrospray MS or nano- electrospray MS) or a detection system facilitating determination of real time association/dissociation constants, are particularly contemplated.
In one embodiment of the diagnostic/prognostic methods described herein, the biooogical sample or test sample or reference sample, as the case may be, is obtained previously from the subject. In accordance with such an embodiment, the prognostic or diagnostic method is performed ex vivo.
The prognostic methods described herein are particularly useful for detecting the occurrence of a disorder or disease associated with aberrant regulation, EGF- mediated signalling, aberrant formation or activity of the cytoskeleton, or a predisposition of an individual towards a disorder or disease associated with aberrant EGF-mediated signalling, or aberrant .regulation, formation or activity of the cytoskeleton. Individuals having such conditions may exhibit, for example, aberrant cell growth or motility, the formation of tumors, tumor metastasis, or tumor cell invasion. The present invention is particularly useful for diagnoses in relation to T cell- mediated immune system disorders, such as, for example, autoimmune diseases (Type I diabetes, multiple sclerosis, rheumatoid arthritis, psoriasis, Wiskott-Aldrich Syndrome), transplant rejection, allergy, asthma, aberrant T cell mediated immunity, aberrant antigen recognition or presentation, disorders of the central nervous system, nephritis, or kidney failure.
The application of the methods described herein to the diagnosis or prognosis of a cancer selected from the group consisting of head and neck cancer, breast cancer, adenocarcinoma, squamous lung cancer, gastrointestinal cancer (eg. gastric, colon, or pancreatic cancer), renal cell cancer, bladder cancer, a gynecological carcinoma (eg. ovarian cancer), prostate cancer, squamous cell carcinoma, non-squamous carcinoma, glioblastoma, epithelial vulval carcinoma and medulloblastoma is particularly preferred.
Novel screens are also developed and used to identify compounds that modulate the protein-protein interactions of the invention, which compounds are useful in the treatment of a range of different cancers and other diseases.
Accordingly, a further aspect of the present invention provides methods for determining a modulator of a protein complex of the invention. In their general form, the methods of the present invention comprise determining the association or dissociation of the protein complex, or the structure of the complex, in the presence and absence of a candidate compound or a candidate antibody. In accordance with the embodiment described herein, a modified association, dissociation, or structure, of the protein complex in the presence of a candidate compound or a candidate antibody indicates that the candidate is a modulator of the protein complex. The association, dissociation, or structure of the complex may be determined by direct means, such as, for example, by determining real time association or dissociation constants in the presence and absence of the candidate, or modified binding of an antibody that recognizes a conformational epitope of the complex. Alternatively, the association, dissociation, or structure of the complex may be determined by indirect means, such as, for example, using a protein recruitment system, n-hybrid screen, reverse n-hybrid screen, plate agar diffusion assay, ELISA, or other well known assay format for detecting protein-protein interactions. Such indirect means generally use a reporter system to detect formation or dissociation of the protein complex.
In one embodiment, the invention provides a method for determining a modulator of the formation or stability of a protein complex comprising: (i) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH and Sam68; and (b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and (ii) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
(b) a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide, wherein said protein complex further optionally comprises EGFR, said method comprising:
(i) determining the level of said protein complex in the absence of a candidate compound or candidate antibody; and (ii) determining the level of said protein complex in the presence of a candidate compound or in the presence of said candidate antibody wherein a difference in the level of said protein complex at (i) and (ii) indicates that the candidate compound or candidate antibody is a modulator of said interaction.
In one preferred embodiment, the modulators of the present invention are antagonists or inhibitors of a protein complex of the invention, particularly an inhibitor of complex formation or stability. Alternatively, the modulators promote complex formation or stability.
The modulators identified using the methods described herein are useful for the therapeutic or prophylactic treatment of diseases associated with aberrant cytoskeletal regulation, formation or activity, such as, for example, aberrant cell growth or motility, tumorigenesis, tumor metastasis, tumor cell invasion, disorders of the central nervous system, nephritis, or kidney failure. The modulators are preferably useful for the treatment of one or more symptoms associated with T cell-mediated immune system disorders, such as, for example, autoimmune diseases (Type I diabetes, multiple sclerosis, rheumatoid arthritis, psoriasis, Wiskott-Aldrich Syndrome), transplant rejection, allergy, asthma, aberrant T cell mediated immunity, aberrant antigen recognition or presentation.
Moreover, the protein complexes of the invention, particularly CD2AP-comprising and/or cortactin-comprising complexes that also comprise a Cbl protein, represent novel targets for cancer therapy. The treatment of a cancer selected from the group consisting of head and neck cancer, breast cancer, adenocarcinoma, squamous lung cancer, gastrointestinal cancer (eg. gastric, colon, or pancreatic cancer), renal cell cancer, bladder cancer, a gynecological carcinoma (eg. ovarian cancer), prostate cancer, squamous cell carcinoma, non-squamous carcinoma, glioblastoma, epithelial vulval carcinoma and medulloblastoma is particularly contemplated by the present invention.
Accordingly, a further aspect of the present invention provides a method of treatment comprising administering to a subject an amount of a modulatory compound identified in the screen of the present invention, including a modulator of an interaction selected from the group consisting of: (i) an interaction between CD2AP and a polypeptide selected from the group consisting of cortactin, Cbl, and endophilin, optionally further comprising EGFR; and (ii) an interaction between cortactin and a polypeptide selected from the group consisting of CD2AP, ASAP1, N-WASP, FISH and Sam68 optionally further comprising EGFR, wherein said contacting is performed under conditions sufficient to modulate the formation or stability of said interaction in a cell or tissue of said subject.
Preferably, the modulator modulates the activity, formation or stability of a protein complex selected from the group consisting of: (i) a complex comprising CD2AP and cortactin optionally further comprising EGFR; (ii) a complex comprising CD2AP and Cbl optionally further comprising EGFR; (iii) a complex comprising CD2AP and endophilin optionally further comprising EGFR; (iv) a complex comprising CD2AP and endophilin and cortactin optionally further comprising EGFR; (v) a complex comprising CD2AP and Cbl and cortactin optionally further comprising EGFR; (vi) a complex comprising CD2AP and endophilin and Cbl optionally further comprising EGFR; (vii) a complex comprising CD2AP and endophilin and Cbl and cortactin optionally further comprising EGFR; (viii) a complex comprising cortactin and ASAP1 optionally further comprising EGFR; (ix) a complex comprising cortactin and N-WASP optionally further comprising EGFR; (x) a complex comprising cortactin and FISH optionally further comprising EGFR; and (xi) a complex comprising cortactin and Sam68 optionally further comprising EGFR.
Brief Description of the Drawings
Figure 1A is a schematic representation showing GST fusion proteins used to isolate cortactin binding partners from MDA-MB-231 cells. In the top line, the general structure of the cortactin polypeptide, including the acidic N- terminal domain (horizontal hatched area), tandem repeats (arrow heads), helical domain (H, stippled area), region rich in serine, threonine and proline (P, diagonal hatching) and C- terminal SH3 domain (SH3, filled area). The middle line indicates the structure of a peptide designated GST-HP comprising the helical domain of cortactin (H, stippled area) and region rich in serine, threonine and proline (P, diagonal hatching) fused to GST. The lower line indicates the structure of a peptide designated GST-SH3 and consisting of only the SH3 domain of cortactin fused to GST.
Figure 1B is a photographic representation showing the binding of proteins in MDA- MB-231 cells to the peptides and polypeptides shown in Figure 1A. Peptides are indicated at the top of each lane.
Figure 2 is a representation of a MALDI-TOF MS fingerprint of bound and purified proteins, confirmed as CD2AP, ASAP1, N-WASP, FISH and Sam68. The x-axis indicates the ion ratio m/z, and signal intensity for each peak is indicated on the abscissa.
Figure 3 is a photographic representation of a western blot showing specific binding of antibody against CD2AP in the protein fraction bound to the GST-SH3 fusion protein, but not in the protein fraction bound to the GST protein. Molecular weights are indicated at the left of the figure. The arrow indicates the position to which authentic CD2AP migrates on the gel. Figure 4A is a photographic representation of a western blot (WB) of cortactin immune precipitates (IP) from MDA-MB-231 cells, using a CD2AP-specific antibody and cortactin-specific antibody. The positions of both CD2AP (above) and cortactin (below) are indicated by the arrows. The numbering refers to molecular weight (kDa).
Figure 4B is a photographic representation of a western blot (WB) of CD2AP immune precipitates (IP) from MDA-MB-231 cells, using a CD2AP-specific antibody and cortactin-specific antibody. The positions of both cortactin(above) and CD2AP (below) are indicated by the arrows. The numbering refers to molecular weight (kDa).
Figure 4C is a photographic representation showing expression of a truncation mutant protein of cortactin (ΔSH3) in HEK 293 cells transfected with an expression vector encoding said truncation mutant protein, relative to the full-length cortactin polypeptide (Cortactin) and a negative control sample lacking a cortactin polypeptide. CD2AP immune precipitates were probed in western blots, using antibodies against cortactin or CD2AP. The positions of both CD2AP (above) and cortactin (below) are indicated by the arrows.
Figure 5 is a photographic representation of a confocal micrograph showing coincident binding of antibodies against cortactin and CD2AP at regions of the cell cortex, structures resembling intracellular vesicles and cell-cell junctions.
Figure 6A is a schematic representation of the structure of CD2AP, showing the positions of the three SH3 domains, and three proline rich regions (PR), designated P1. P2, and P3.
Figure 6B is a schematic representation showing the amino acid sequences of the three proline rich regions of CD2AP (i.e. SEQ ID Nos: 22-24) and a PRR consensus sequence (SEQ ID NO: 25).
Figure 6C is a photographic representation showing binding of GST fusion proteins comprising the three proline rich regions of CD2AP (i.e. SEQ ID Nos: 22-24), and a GST fusion comprising the a PRR consensus sequence (SEQ ID NO: 25), to the SH3 domain of cortactin in Far Western blots. The left figure shows Coomassie-stained proteins. The middle figure shows a far western blot using biotin-labelled GST. The right figure shows a far western blot using the biotin-labelled GST-SH3 domain fusion protein. Numbering at the left of each panel shows molecular weight of the GST fusion proteins detected.
Figure 7A is a copy of a photographic representation showing the induction of the association between CD2AP and cortactin by EGF. CD2AP immunoprecipitates (IP CD2AP) and cortactin immunoprecipitates (IP cortactin) were derived from serum- starved (SF) or EGF-stimulated (EGF) MDA-MB-231 cells and analysed by western blotting using antibodies against cortactin and CD2AP. Data indicate EGF-inducible co-immunoprecipitation of cortactin and CD2AP.
Figure 7B is a copy of a photographic representation showing the requirement for the SH3 domain of cortactin in the EGF-inducible co-immunoprecipitation of cortactin and CD2AP in HEK 293 cells. Cells were transfected with an expression vector (control), or an expression vector comprising nucleic acid encoding a full-length cortactin polypeptide (cortactin) or encoding a truncated cortactin polypeptide lacking the SH3 domain (ΔSH3), and incubated in serum-free media (SF) or in the presence of EGF (EGF). Whole cell lysates (WCL) and CD2AP immunoprecipitates (IP CD2AP) were analysed by western blotting using antibodies against cortactin and CD2AP. Data indicate EGF-inducible co-immunoprecipitation of cortactin and CD2AP that requires the SH3 domain of cortactin.
Figure 8A is a copy of a photographic representation showing the time course for recruitment of cortactin and Cbl to CD2AP following EGF stimulation of MDA-MB-231 cells. CD2AP immunoprecipitates (IP CD2AP) were derived from MDA-MB-231 cells either before EGF stimulation (time 0) or at 1 min (1'), 2 mins (2'), 5 mins (5'), 15 mins (15'), 30 mins (30') or 60 min (60') after EGF-stimulation, and analysed by western blotting using antibodies against cortactin, Cbl, endophilin and CD2AP. Data indicate a low basal level of cortactin-CD2AP complex that is enhanced following EGF- stimulation, reaching a peak at 5 min and returning to the basal level at 30 min after EGF stimulation (row marked cortactin). Association of Cbl with CD2AP is rapidly induced by EGF stimulation, reaching a peak at 1-5 min post EGF treatment, however decreases between 5-15 min and is not detectable after about 15 min (row marked Cbl). Association of endophilin with CD2AP appears to be constitutive and not induced by EGF stimulation (row marked endophilin).
Figure 8B is a copy of a photographic representation showing the time course for recruitment of tyrosine phosphorylation of Cbl, cortactin and EGFR to CD2AP following EGF stimulation of MDA-MB-231 cells. CD2AP immunoprecipitates (IP CD2AP) were derived from MDA-MB-231 cells either before EGF stimulation (time 0) or at 1 min (1'), 2 mins (2'), 5 mins (5'), 15 mins (15'), 30 mins (30') or 60 min (60') after EGF- stimulation, and analysed by western blotting using anti-phosphotyrosine antibodies (P-Tyr) or antibodies against EGFR (EGFR). Molecular weights of proteins are indicated on the left of the figure. Data indicate the presence of several proteins phosphorylated at their tyrosine residues, having molecular weights of 180 kDa (EGFR), 130 kDa (unknown), 120 kDa (Cbl), and 80 kDa (cortactin). Data are consistent with the formation of a Cbl-CD2AP-endophilin complex that regulates receptor endocytosis, and demonstrates a novel role for cortactin and the formation of dynamic actin networks in this process.
Figure 9A is a copy of a photographic representation showing the time course for co- localization of CD2AP, cortactin and EGFR in HeLa cells transiently transfected with an expression construct comprising nucleic acid encoding a fusion protein consisting of EGFR and green fluorescent protein (GFP). Cells were fixed either before EGF stimulation (time 0) or at 5 mins (5') or 15 mins (15') after EGF-stimulation, and analysed by indirect immunofluorescence using antibodies against cortactin or CD2AP. EGFR was localized by fluorescence of the GFP moiety in the fusion protein. Data indicate localization of EGFR primarily in the plasma membrane, with cortactin and CD2AP primarily in the punctate cytoplasmic structures, prior to EGF stimulation. After stimulation with EGF, EGFR moves into the membrane ruffles where it is associated with CD2AP and cortactin. The co-localization of EGFR with CD2AP and cortactin is transient and returns to basal levels within 15 mins after EGF stimulation. Figure 9B is a copy of a photographic representation showing co-localization of CD2AP, cortactin and EGFR in membrane 5 mins (5') after EGF-stimulation of HeLa cells transiently transfected with an expression construct comprising nucleic acid encoding a fusion protein consisting to EGFR and green fluorescent protein (GFP). Cells were analysed by indirect immunofluorescence using antibodies against cortactin or CD2AP. EGFR was localized by fluorescence of the GFP moiety in the fusion protein.
Figure 10 is a copy of a photographic representation showing the effect of the over expression of cortactin or truncated cortactin on endocytic complex formation.. Cells were transfected with an "empty" expression vector (control), or an expression vector comprising nucleic acid encoding a full-length cortactin polypeptide (cortactin) or encoding a truncated cortactin polypeptide lacking the SH3 domain (ΔSH3), and incubated in serum-free media (time 0) or in the presence of EGF (EGF) for 5 min (5') or 15 min (15'). CD2AP immunoprecipitates (IP CD2AP) were analysed by western blotting using antibodies against CD2AP, endophilin, Cbl, and cortactin.. Over expression of full-length cortactin increased the basal association between cortactin and CD2AP and leads to enhanced and prolonged association between CD2AP and cortactin following EGF-stimulation. Over-expression of truncated cortactin blocked recruitment of endogenous native cortactin into the complex following EGF stimulation of cells, as evidenced by the reduced level of cortactin in immunoprecipitates of cells over-expressing the mutant protein relative to control cells at 5 min post-EGF treatment.
Figure 11 is a copy of a photographic representation showing the kinetics of recruitment of cortactin and Cbl to CD2AP following EGF stimulation of MDA-MB-231 cells. Cells were either serum-starved (lane marked 0) or treated with EGF for 1 min (1'), 2 min (2'), 5 min (5'), 15 min (15'), 30 min (30') or 60 min (60'). CD2AP-containing immune precipitates (IP CD2AP) were then isolated and immunoblotted with specific antibodies against Cbl, endophilin, cortactin and CD2AP, as indicated at the right side of each of the lower four panels. Antibodies against EGFR and phosphotyrosine (P- Tyr) were also tested as indicated at the right of the top two panels. Numbers at the left of the top panel indicate molecular weight (kDa). Figure 12 is a copy of a photographic representation showing the association of Cbl with cortactin following EGF stimulation. HeLa epithelial vulval carcinoma cells were transiently transfected with either an empty vector control (V), or alternatively, a cortactin-encoding expression vector (C). Cells were then serum-starved and left untreated (SF) or stimulated with EGF under standard conditions for 2 min (2' EGF). Following washing, cortactin-containing immune precipitates (IP cortactin) were subjected to SDS/PAGE and Western blotting using antibodies against phosphotyrosine (P-Tyr), Cbl or cortactin as indicated at the right of each panel of the figure. Numbers at the right of the top panel indicate molecular weight (kDa). Data show the EGF-induced recruitment of Cbl to cortactin-containing complexes.
Description of the Preferred Embodiments
One aspect of the present invention provides an isolated or recombinant protein complex comprising:
(i) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of
CD2AP, N-WASP, ASAP1, FISH and Sam68; and (b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and
(ii) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
(b) a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide.
In one embodiment, the protein complex of the invention comprises: (i) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide; and (ii) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a cortactin polypeptide or a portion of a cortactin polypeptide.
In another preferred embodiment, the protein complex of the invention comprises:
(i) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a Cbl polypeptide; and (ii) a Cbl polypeptide or a portion of a Cbl polypeptide sufficient to bind to a CD2AP polypeptide or a portion of a CD2AP polypeptide.
Preferably, an isolated or recombinant protein complex of the invention comprising cortactin and CD2AP further comprises a Cbl polypeptide and/or an endophilin polypeptide. CD2AP may bind endophilin and/or Cbl in such an arrangement either directly or indirectly.
Accordingly, in another preferred embodiment, the protein complex of the invention comprises:
(i) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide;
(ii) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide; and (b) an endophilin polypeptide or a portion of an endophilin polypeptide; and (iii) an endophilin polypeptide or a portion of an endophilin polypeptide sufficient to bind to a CD2AP polypeptide or a portion of a CD2AP polypeptide.
In another preferred embodiment, the protein complex of the invention comprises: (i) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide; (ii) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide; and (b) a Cbl polypeptide or a portion of a Cbl polypeptide; and
(iii) a Cbl polypeptide or a portion of a Cbl polypeptide sufficient to bind to a CD2AP polypeptide or a portion of a CD2AP polypeptide.
In another preferred embodiment, the protein complex of the invention comprises:
(i) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide; (ii) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide; and (b) an endophilin polypeptide or a portion of an endophilin polypeptide; (iii) an endophilin polypeptide or a portion of an endophilin polypeptide sufficient to bind to (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide; and (b) a Cbl polypeptide or a portion of a Cbl polypeptide; and (iv) a Cbl polypeptide or a portion of a Cbl polypeptide sufficient to bind to an endophilin polypeptide or a portion of an endophilin polypeptide.
In another preferred embodiment, the protein complex of the invention comprises:
(i) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide; (ii) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide,
(b) an endophilin polypeptide or a portion of an endophilin polypeptide, and (c) a Cbl polypeptide or a portion of a Cbl polypeptide; (iii) an endophilin polypeptide or a portion of an endophilin polypeptide sufficient to bind to (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide and to (b) a Cbl polypeptide or a portion of a Cbl polypeptide; and (iv) a Cbl polypeptide or a portion of a Cbl polypeptide sufficient to bind to (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide, and (b) an endophilin polypeptide or a portion of an endophilin polypeptide.
In another preferred embodiment, the protein complex of the invention comprises:
(i) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide; (ii) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide, (b) an endophilin polypeptide or a portion of an endophilin polypeptide, and (c) a Cbl polypeptide or a portion of a Cbl polypeptide; (iii) an endophilin polypeptide or a portion of an endophilin polypeptide sufficient to bind to a CD2AP polypeptide or a portion of a CD2AP polypeptide; and (iv) a Cbl polypeptide or a portion of a Cbl polypeptide sufficient to bind to a
CD2AP polypeptide or a portion of a CD2AP polypeptide.
In yet another preferred embodiment, the protein complex of the invention comprises: (i) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide;
(ii) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide, and (b) a Cbl polypeptide or a portion of a Cbl polypeptide;
(iii) a Cbl polypeptide or a portion of a Cbl polypeptide sufficient to bind to (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide; and (b) an endophilin polypeptide or a portion of an endophilin polypeptide; and
(iv) an endophilin polypeptide or a portion of an endophilin polypeptide sufficient to bind to a Cbl polypeptide or a portion of a Cbl polypeptide.
Preferably, a protein complex of the invention further comprises Epidermal growth factor receptor (EGFR), particularly in EGF-stimulated cells. For example, a tertiary or quaternary protein complex comprising both cortactin and CD2AP and Cbl, may comprise EGFR in EGF-stimulated cells. The protein complex is a heterodimer or hetero-multimeric protein. Those skilled in the art will be aware that a heterodimer or heterodimeric protein complex comprises two different peptide or polypeptide subunits. As used herein, the term "heteromultimer" shall be taken to mean a higher order protein complex comprising at least three peptide or polypeptide subunits, wherein at least two of said subunits are different. For example, a heterohexameric protein is known to comprise six peptide or polypeptide subunits, and, in the present context, may comprise three different homodimers, or six different monomers, or a dimer and a tetramer of different protein, etc. Accordingly, the present invention is not to be limited by the composition and size of the protein complex.
The protein subunits of the protein complex are held in physical relation by any means known to those skilled in the art. This physical relation may involve the formation of an induced magnetic field or paramagnetic field, covalent bond formation such as a disulfide bridge formation between polypeptide subunits, an ionic interaction such as occur in an ionic lattice, a hydrogen bond or alternatively, a van der Waals interaction such as a dipole-dipole interaction, dipole-induced-dipole interaction, induced-dipole- induced-dipole interaction or a repulsive interaction or any combination of the above forces of attraction. Alternatively, the peptide or polypeptide subunits may be held in physical relation by expressing them as a fusion polypeptide, optionally separated by a spacer to permit their folding. Accordingly, the physical relation between the peptide or polypeptide subunits may be a consequence of their binding capability and attraction toward one another, or alternatively, a consequence of their mode of production.
Preferably, the peptide, polypeptide or protein partners are in direct physical relation. By "direct physical relation" is meant that the binding partners contact each other without any intervening protein moiety or non-protein moiety. However, the protein complexes of the present invention can clearly include one or more additional protein moieties or non-protein moieties, such as, for example, a protein or non-protein moiety that enhances or stabilizes the physical relation between cortactin and/or CD2AP and the other binding partner(s). The involvement of any one or more protein or non- protein moieties selected from the group consisting of PIP2, Arp 2/3, CortBPI, Src, c- Src, c-Crk, rpdeδ, ZO-1, Nek, WIP, Grb2, SOS, nephrin, polycystin-2, dynamin-2, claudin, occludin, paxillin, vinculin, EGFR, Cbl, , and nucleic acid (RNA or DNA) is not to be excluded.
The present invention further encompasses a protein complex wherein one or more of the binding partners include a post-translational modification, such as, for example, a phosphorylated, fucosylated, myristoylated, farnesylated, or glycosylated residue. Such post-translational modifications may enhance complex formation or stabilize the complex once it is formed. Phosphorylation of one or more tyrosine residues present on one or more of the binding partners, such as EGFR or Cbl, is particularly contemplated herein. Ubiqitination of one or more binding partners is also not to be excluded.
Preferably, the binding partners of the protein complex are mammalian polypeptides or proteins, and more preferably of human or murine or rat origin. It is not strictly necessary for the binding partners to be derived from the same source, however this is preferred because the ability of the partners to associate or be maintained in physical non-covalent association with each other is generally enhanced if they are derived from the same organism.
Those skilled in the art will be in a position to determine a suitable portion of cortactin or CD2AP or other polypeptide that can form the protein complex of the invention, such as, for example, the SH3 domain of cortactin or CD2AP or endophilin or ASAP1 or FISH, or a proline rich region (PRR) of CD2AP, Cbl, ASAP1, N-WASP, FISH or samδδ. This is achieved, for example, using conventional binding assays for determining the binding between two proteins without undue experimentation.
Preferably, formation of the isolated or recombinant protein complex involves an SH3 domain of a polypeptide selected from the group consisting of cortactin, CD2AP, endophilin and ASAP1, more preferably an SH3 domain of cortactin, CD2AP or endophilin. In the present context, the "core" SH3 domain of a polypeptide involved in complex formation will comprise the consensus amino acid sequence WX^GXXXXXGXFP or WX1XGXXXXXXGXFP or WXiXGXXXXXXXGXFP or W^XGXXXXXXXXGXFP or WXiXGXXXXXXXXXGXFP, wherein X^ is W or Y and wherein X is any amino acid. More preferably, the SH3 domain of a polypeptide involved in complex formation will comprise the consensus amino acid sequence GWWXGXXXXXXGXFP or GWWXGXXXXXXXGXFP or GWWXGXXXXXXXXGXFP or GWWXGXXXXXXXXXGXFP, wherein X is any amino acid. Longer fragments or portions, comprising an additional 5 or 10 or 15 or 20 amino acid residues on the N- terminus and/or C-terminus are also encompassed within this definition of an SH3 domain, particularly wherein the extension is derived from the sequence of a naturally occurring protein described herein that flanks the SH3 domain in its native context.
Preferred SH3 domains of a cortactin polypeptide are functionally sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH, and Sam68. Preferably, the portion of cortactin suitable for protein complex formation is a portion of the SH3 domain of cortactin of at least about 5 amino acids in length, more preferably at least about 10 amino acids in length, even more preferably at least about 15 amino acids in length and still more preferably at least about 20 or 30 or 40 or 50 amino acids in length. Accordingly, a portion of the SH3 domain of cortactin may comprise at least about 5 or 10 or 15 or 20 or 25 or 30 or 35 or 40 or 45 or 50 contiguous amino acid residues of a sequence having at least about 80% identity to SEQ ID NO: 3 or SEQ ID NO: 4. Preferably, the SH3 domain is from human cortactin (SEQ ID NO: 3) or mouse cortactin (SEQ ID NO: 4) or a portion thereof, and in a particularly preferred embodiment, comprises the SH3 domain of human cortactin. Exemplary SH3 domains of cortactin are set forth in SEQ ID Nos: 18 and 19. Preferably, the percentage identity to SEQ ID NO: 18 or 19 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
The three SH3 domains of CD2AP are presented schematically in Figure 6. Particularly preferred SH3 domains of CD2AP comprise an amino acid sequence selected from the group consisting of: (i) amino acid residues from about position 97 to about position 111 of SEQ ID NO: 1 ; (ii) amino acid residues from about position 144 to about position 157 of SEQ ID NO: 2; (iii) amino acid residues from about position 261 to about position 274 of SEQ ID NO: 1; and amino acid residues from about position 306 to about position 319 of SEQ ID NO: 2. Longer fragments or portions, comprising an additional 5 or 10 or 15 or 20 amino acid residues on the N-terminus and/or C-terminus are also encompassed within this definition of an SH3 domain of CD2AP, such as, for example additional residues derived from the sequences set forth in the sequence listing (ie., SEQ ID Nos: 1 or 2). Peptides having about 80% identity to such SH3 domains of CD2AP are also encompassed.
In one embodiment, an SH3 domain of CD2AP associates with a PRR of Cbl.
Preferred SH3 domains of ASAP1 comprise the amino acid sequence from about position 1121 to about position 1138 of SEQ ID NO: 10 or an equivalent portion of the murine sequence set forth in SEQ ID NO: 11. Again, longer fragments or portions, comprising an additional 5 or 10 or 15 or 20 amino acid residues on the N-terminus and/or C-terminus are also encompassed within this definition of an SH3 domain of ASAP1, such as, for example additional residues derived from the sequences set forth in the sequence listing (ie., SEQ ID Nos: 10 or 11). Peptides having about 80% identity to such SH3 domains of ASAP1 are also encompassed.
Similarly, the skilled artisan can readily determine a portion of an endophilin polypeptide that is sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide or to said Cbl polypeptide or a portion thereof. Preferred SH3 domains of endophilin are derived from amino acid residues from about position 327 to about position 340 of SEQ ID NO: 7 or from about position 328 to about position 341 of SEQ ID NO: 8 or from about position 322 to about position 335 of SEQ ID NO: 9. Again, conventional binding assays for determining the binding between two proteins may be used to assay the suitability of such portions. In a particularly preferred embodiment, the portion of endophilin that is sufficient to bind CD2AP or a portion of CD2AP comprises the SH3 domain of said endophilin. In another embodiment, an SH3 domain of endophilin associates with Cbl. As used herein, the term "SH3 domain of endophilin" shall be taken to refer to an amino acid sequence having at least about 80% amino acid sequence identity to the amino acid sequence of an SH3 domain of a human endophilin polypeptide, and preferably, capable of binding to a polypeptide selected from the group consisting of CD2AP and Cbl. For the purposes of nomenclature, the amino acid sequences of exemplary peptides comprising a human endophilin SH3 domain are provided herein as SEQ ID Nos: 20 and 21. Preferably, the percentage identity to SEQ ID NO: 20 or 21 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
Peptides comprising additional sequences flanking the cores SH3 sequences presented in SEQ ID Nos: 18-21 are also encompassed by the present invention, particularly functionally equivalent peptides such as, for example, those peptides having additional flanking sequences derived from the native cortactin and/or native endophilin polypeptides. Functionally equivalent fragments of SEQ ID Nos: 18-21 are also encompassed by the present invention.
Similarly, an SH3 domain of CD2AP or endophilin binds to Cbl in a higher order complex of the present invention.
It is also preferred that the formation of the isolated or recombinant protein complex of the invention involves a proline rich region (PRR) of a polypeptide selected from the group consisting of CD2AP, Cbl, ASAP1, N-WASP, FISH and Sam68, and more preferably, a PRR of CD2AP or Cbl. By "proline-rich region" is meant that the amino acid sequence comprises two or three or more consecutive or adjacent proline residues and the consensus sequence PXXP wherein X is any amino acid and P is a proline residue. Preferably, the value of X is selected from the group consisting of lysine, proline, valine, leucine and glutamine. Preferably, the consecutive or adjacent proline residues are adjacent to at least one or two basic residues such as lysine or arginine or separated therefrom by one or two amino acid residues. Optionally, a proline-rich region is present as a tandemly-repeated sequence or duplication separated by up to ten amino acid residues in the naturally occurring polypeptide from which it is derived.
Preferably, the portion of CD2AP suitable for protein complex formation is a portion of the PRR of CD2AP of at least about 5 amino acids in length, more preferably at least about 10 amino acids in length, even more preferably at least about 15 amino acids in length and still more preferably at least about 20 or 30 or 40 or 50 amino acids in length. Accordingly, a portion of the PRR of CD2AP may comprise at least about 5 or 10 or 15 or 20 or 25 or 30 or 35 or 40 or 45 or 50 contiguous amino acid residues of a sequence having at least about 80% identity to the PRR of SEQ ID NO: 1 or SEQ ID NO: 2. Preferably, the percentage identity is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
Preferred PRRs of a Cbl polypeptide are functionally sufficient to bind to an endophilin polypeptide. Preferred PRRs of a polypeptide selected from the group consisting of CD2AP, ASAP1 , N-WASP, FISH and Sam68 are functionally sufficient to bind to a cortactin polypeptide.
Particularly preferred peptides having a PRR will comprise an amino acid sequence selected from the group consisting of:
(i) amino acid residues 291-297 of SEQ ID NO: 1 ;
(ii) amino acid residues 341-348 of SEQ ID NO: 1;
(iii) amino acid residues 386-395 of SEQ ID NO: 2; (iv) amino acid residues 494-500 of SEQ ID NO: 5;
(v) amino acid residues 531-537 of SEQ ID NO: 5;
(vi) amino acid residues 540-551 of SEQ ID NO: 5;
(vii) amino acid residues 531-551 of SEQ ID NO: 5;
(viii) amino acid residues 512-518 of SEQ ID NO: 6; and (ix) amino acid residues 512-532 of SEQ ID NO: 6.
Peptides comprising additional sequences flanking the cores sequences (i) through
(ix) supra are also encompassed by the present invention, particularly functionally equivalent peptides such as, for example, those peptides having additional flanking sequences derived from the native CD2AP and/or native Cbl polypeptides.
In a particularly preferred embodiment, the isolated or recombinant protein complex of the invention is selected from the group consisting of:
(i) a complex comprising CD2AP and cortactin; (ii) a complex comprising CD2AP and Cbl; (iii) a complex comprising CD2AP and endophilin; (iv) a complex comprising CD2AP and endophilin and cortactin; (v) a complex comprising CD2AP and Cbl and cortactin; (vi) a complex comprising CD2AP and endophilin and Cbl;
(vii) a complex comprising CD2AP and endophilin and Cbl and cortactin; (viii) a complex comprising cortactin and ASAP1 ; (ix) a complex comprising cortactin and N-WASP; (x) a complex comprising cortactin and FISH; and (xi) a complex comprising cortactin and Sam68.
In one embodiment, a PRR of CD2AP associates with an SH3 domain of cortactin or endophilin.
Another aspect of the present invention provides isolated peptides and kits comprising same for producing a protein complex comprising:
(i) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH and Sam68; and (b) a polypeptide selected from the group consisting of CD2AP, N-
WASP, ASAP1 , FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and (ii) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
(b) a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide. optionally further comprising EGFR, or for identifying a modulator of the formation or stability of said complex. Preferably the peptide or polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO:24. In this respect, SEQ ID Nos: 1-17 relate to full-length polypeptide binding partners of a protein complex described herein, SEQ ID Nos: 18-21 relate to exemplary SH3 domain peptide sequences and SEQ ID Nos: 22-24 relate to exemplary peptides comprising CD2AP PRRs.
In one embodiment, the kit comprises a first polypeptide consisting of cortactin or a portion thereof and a second peptide or polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH, Sam68, a portion of CD2AP, a portion of N- WASP, a portion of ASAP1, a portion of FISH and a portion of Sam68, wherein said portion of said second polypeptide is sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide.
Preferably, the kit further comprises one or more antibodies or ligands that bind to the first polypeptide, to the second polypeptide, or to the complex formed between said first and said second polypeptide.
As used herein, the term "antibody" refers to intact monoclonal or polyclonal antibodies, immunoglobulin (IgG, IgM, IgE) fractions, humanized antibodies, or recombinant single chain antibodies, as well as fragments thereof, such as Fab, F(ab')2, and Fv, which are capable of binding a linear or conformational epitope of at least one binding partner of the protein complex, or to a conformational epitope of the assembled protein complex. Humanized antibodies are antibodies in which amino acids have been replaced in the non-antigen binding regions in order to more closely resemble a human antibody, while still retaining the original binding ability. Antibodies referred to herein are obtained from a commercial source, or alternatively, produced by conventional means. Commercial sources will be well known to those skilled in the art.
For the production of antibodies, an intact polypeptide, or a portion thereof containing a short amino acid sequence of interest (e.g. SH3 domain of cortactin), is used as the immunizing antigen or immunogen. The immunogen is derived from a natural source, produced by recombinant expression means or by in vitro translation of RNA, or synthesized chemically such as by Fmoc chemistry. Immunogens consisting of short peptides a preferably conjugated to a carrier protein, such as, for example bovine serum albumin (BSA), thyroglobulin, or keyhole limpet hemocyanin (KLH), prior to immunization. The coupled peptide is then used to immunize the animal. Various host animals (e.g. goats, rabbits, rats, mice, dogs, humans) are immunized by intramuscular, intraperitoneal, or intravenous injection, with immunogen, optionally in the presence of an adjuvant to enhance the immune response to the immunogen. Preferred adjuvants include, for example, Freund's complete or incomplete adjuvant, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are preferred.
Monoclonal antibodies are prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture, such as, for example, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al Nature 256, 495-497, 1975; Kozbor et al, J. Immunol. Methods 81, 31-42, 1985; Cote et al, Proc. Natl. Acad. Sci. USA 80, 2026- 2030, 1983; Cole e a/., Mol. Cell Biol. 62, 109-120, 1984).
Techniques developed for the production of chimeric antibodies are also employed. Such techniques involve splicing a mouse antibody gene to a human antibody gene to produce a molecule having the desired antigen specificity and biological activity (Morrison et al, Proc. Natl. Acad. Sci. USA 81, 6851-6855, 1984; Neuberger et al, Nature 312, 604-608, 1984; Takeda et al, Nature 314, 452-454, 1985). Alternatively, techniques described for the production of single chain antibodies are adapted, using methods known in the art, to produce single chain antibodies having the desired specificity.
Antibodies are also produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed by Oriandi et al, Proc. Natl. Acad. Sci. USA 86, 3833-3837, 1989; Winter et al, Nature 349, 293-299, 1991).
Antibody fragments, such as, for example, F(ab')2 fragments, are produced by pepsin digestion of an intact antibody molecule. Fab fragments are generated by reducing the disulfide bridges of F(ab')2 fragments. Alternatively, Fab expression libraries are constructed, to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al, Science 254, 1275-1281 , 1989).
The antibodies or ligands may assist in the subsequent isolation or detection of the complex formed between the first and second polypeptides. Binding of the antibody or ligand to a region of the first or second polypeptide that is not involved in complex formation is preferred for this purpose, the only requirement being that, in use, the ligand does not disrupt the complex formed between the first polypeptide and the second polypeptide. Binding of the antibody or ligand to a region other than the SH3 domain of cortactin is particularly preferred. Preferably, the ligand is a small molecule or alternatively, a binding partner for one of the first polypeptide or second polypeptide. Preferred ligands for use in accordance with this embodiment are selected from the group consisting of PIP2, Arp 2/3, CortBPI, Src, c-Src, c-Crk, rpdeδ, ZO-1, Nek, WIP, Grb2, SOS, nephrin, polycystin-2, dynamin-2, claudin, occludin, paxillin, vinculin, and nucleic acid (RNA or DNA). The antibody or ligand may be labeled using a suitable reporter molecule, such as, for example, a fluorophore, chromophore, or radioisotope. In the case of antibodies and small molecules, these may also be detected using antibodies in accordance with procedures known to those skilled in the art. Optionally, the kit is packaged with instructions for use. In use, the first polypeptide and second polypeptide are contacted for a time and under conditions sufficient for complex formation to occur, and when provided, the antibody or ligand is used to detect or isolate the complex formed.
In another embodiment, the kit comprises a first compartment comprising cortactin or a portion thereof and a second compartment comprising an antibody or ligand that binds to a peptide or polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH, Sam68, a portion of CD2AP, a portion of N-WASP, a portion of ASAP1, a portion of FISH and a portion of Sam68, wherein said portion of said second polypeptide is sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide, or an antibody that binds to a complex formed between the content of the first compartment and the content of the second compartment.
Preferably, the ligand is a small molecule or alternatively, a binding partner for the protein of the second compartment. Preferred ligands for use in accordance with this embodiment are selected from the group consisting of PIP2, Arp 2/3, Src, c-Src, c-Crk, Nek, WIP, Grb2, SOS, nephrin, polycystin-2, claudin, occludin, paxillin, vinculin, and nucleic acid (RNA or DNA). Again, the antibody of ligand may be labeled using a suitable reporter molecule, such as, for example, a fluorophore, chromophore, or radioisotope.
Optionally, the kit is packaged with instructions for use. In use, the contents of the first compartment or the contents of the second compartment are contacted with an isolated protein selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH, Sam68, a portion of CD2AP, a portion of N-WASP, a portion of ASAP1, a portion of FISH and a portion of Sam68, or a mixture or extract of proteins comprising said protein, for a time and under conditions sufficient for binding to occur. Thus, a protein complex of the invention, or an antigen-antibody complex or protein-ligand complex is formed. The complex is then reacted with the content of the remaining compartment of the kit, under conditions sufficient to produce the protein complex of the invention bound to antibody or ligand. The complex is then detected by virtue of the antibody or ligand tag according to standard procedures. Again, the ligand should be selected such that it does not disrupt the protein complex of the present invention.
In another embodiment, the kit comprises a first compartment comprising a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH, Sam68, a portion of CD2AP, a portion of N-WASP, a portion of ASAP1, a portion of FISH and a portion of Sam68, wherein said portion of said second polypeptide is sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide and a second compartment comprising an antibody or ligand that binds to a polypeptide selected from the group consisting of cortactin, CD2AP, N-WASP, ASAP1 , FISH and Sam68, or an antibody that binds to a complex formed between cortactin and a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH and Sam68.
Preferably, the antibody or ligand binds to cortactin or a portion thereof. Preferred ligands for use in accordance with this embodiment are selected from the group consisting of Arp 2/3, CortBPI, Src, c-Src, rpdeδ, and Zo-1. Again, antibody or ligand may be labeled using a suitable reporter molecule, such as, for example, a fluorophore, chromophore, or radioisotope. In the case of antibodies and small molecules, these may also be detected using antibodies in accordance with procedures known to those skilled in the art.
Optionally, the kit is packaged with instructions for use. In use, the contents of the first compartment or the contents of the second compartment are contacted with isolated cortactin or a portion of cortactin or a mixture or extract of proteins comprising same for a time and under conditions sufficient for binding to occur. Thus, a protein complex of the invention, or an antigen-antibody complex or protein-ligand complex is formed. The complex is then reacted with the content of the remaining compartment of the kit, under conditions sufficient to produce the protein complex of the invention bound to antibody or ligand. The complex is then detected by virtue of the antibody or ligand tag according to standard procedures. Again, the ligand should be selected such that it does not disrupt the protein complex of the present invention. In another embodiment, the kit comprises a first polypeptide consisting of CD2AP or a portion thereof sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin, and a second polypeptide consisting of a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion thereof sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide. Such kits are used to produce protein complexes comprising: (i) CD2AP and cortactin; (ii) CD2AP and endophilin; (iii) CD2AP and Cbl; (iv) CD2AP and endophilin and cortactin; (v) CD2AP and cortactin and Cbl; and (vi) CD2AP and cortactin and Cbl and endophilin.
The kit may also include one or more antibodies or ligands that bind to the first polypeptide or the second polypeptide, or to any one or more of the protein complexes supra comprising CD2AP, such as, for example, an antibody or ligand that specifically recognizes an assembled protein complex or the conformation of said protein complex, rather than the individual polypeptide components per se.
In another embodiment, the kit comprises:
(a) a first compartment comprising a CD2AP polypeptide or a portion thereof sufficient to form a protein complex selected from the group consisting of: (i) CD2AP and cortactin; (ii) CD2AP and endophilin; (iii) CD2AP and Cbl; (iv)
CD2AP and endophilin and cortactin; (v) CD2AP and cortactin and Cbl; and (vi) CD2AP and cortactin and Cbl and endophilin; and
(b) a second compartment comprising an antibody or ligand that binds to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin, or an antibody or ligand that binds to a protein complex selected from the group consisting of: (i) CD2AP and cortactin; (ii) CD2AP and endophilin; (iii) CD2AP and Cbl; (iv) CD2AP and endophilin and cortactin; (v) CD2AP and cortactin and Cbl; and (vi) CD2AP and cortactin and Cbl and endophilin, wherein said antibody or ligand that binds to a protein complex does not bind to Cbl in the absence of CD2AP. In another embodiment, the kit comprises a first compartment comprising an endophilin polypeptide or a portion thereof sufficient to bind CD2AP and a second compartment comprising an antibody or ligand that binds to CD2AP.
In another embodiment the kit comprises a first compartment comprising a cortactin polypeptide or a portion thereof sufficient to bind CD2AP and a second compartment comprising an antibody or ligand that binds to CD2AP.
In another embodiment, the kit comprises a first compartment comprising a Cbl polypeptide or a portion thereof sufficient to bind CD2AP and a second compartment comprising an antibody or ligand that binds to CD2AP.
In another embodiment, the kit comprises:
(a) a first compartment comprising an isolated or recombinant protein complex selected from the group consisting of: (i) CD2AP and cortactin; (ii) CD2AP and endophilin; (iii) CD2AP and Cbl; (iv) CD2AP and endophilin and cortactin; (v) CD2AP and cortactin and Cbl; and (vi) CD2AP and cortactin and Cbl and endophilin; and
(b) a second compartment comprising an (i) antibody or ligand that binds to a polypeptide selected from the group consisting of CD2AP, cortactin, Cbl and endophilin; or (ii) an antibody oMigand that binds to one or more protein complexes (a).
Moonoclonal or polyclonal antibodies that bind to CD2AP, cortactin, endophilin I, endophilin II, endophilin III, Cbl, or EGFR, are obtained from a commercial source, such as for example, UBI, Lake Placid, New York, USA, or Santa Cruz Biotechnology, Inc, CA 95060, USA. Other commercial sources will be well known to those skilled in the art.
The antibodies or ligands may assist in the subsequent isolation or detection of the complex formed between the first and second polypeptides. Binding of the antibody or ligand to a region of the first or second polypeptide that is not involved in complex formation is preferred for this purpose, the only requirement being that, in use, the ligand does not disrupt the complex formed between the first polypeptide and the second polypeptide. Binding of the antibody or ligand to a region other than the SH3 domain or PRR of CD2AP is particularly preferred.
Preferably, the ligand is a small molecule or alternatively, a binding partner for one of the protein complexes contemplated herein. Particularly preferred ligands for use in accordance with this embodiment are selected from the group consisting of EGFR, Arp 2/3, CortBPI , Src, c-Src, rpdeδ, ZO-1 , Nek, WIP, SOS, nephrin, polycystin-2, dynamin-2, claudin, occludin, and nucleic acid (RNA or DNA).
The antibody or ligand may be labelled using a suitable reporter molecule, such as, for example, a fluorophore, chromophore, or radioisotope. In the case of antibodies and small molecules, these may also be detected using antibodies in accordance with procedures known to those skilled in the art.
Optionally, the kit is packaged with instructions for use.
The kits of the invention are useful for producing and/or detecting the protein complexes of the invention in vitro or in vivo. For producing the protein complexes, one or more of the non-antibody/ligand components of the kits is added to a cellular source for a time and under conditions sufficient for complex formation to occur. The antibody components are particularly useful for isolating the complex(es) thus formed. Optionally any one of the kit components is labelled with a protein tag to facilitate subsequent isolation or purification of the protein complex.
In use, the polypeptide components are contacted for a time and under conditions sufficient for complex formation to occur. Additional proteins may be provided from cellular or non-cellular sources to produce protein complexes other than those specifically referred to herein. When provided, the antibody or ligand is used to detect or isolate the complex formed. The ligand should be selected such that it does not disrupt the protein complex formed. Another aspect of the present invention provides an isolated antibody that binds to a protein complex selected from the group consisting of:
(i) a complex comprising CD2AP and cortactin; (ii) a complex comprising CD2AP and Cbl; (iii) a complex comprising CD2AP and endophilin; (iv) a complex comprising CD2AP and endophilin and cortactin; (v) a complex comprising CD2AP and Cbl and cortactin; (vi) a complex comprising CD2AP and endophilin and Cbl; (vii) a complex comprising CD2AP and endophilin and Cbl and cortactin; (viii) a complex comprising cortactin and ASAP1; (ix) a complex comprising cortactin and N-WASP; (x) a complex comprising cortactin and FISH; and (xi) a complex comprising cortactin and Sam68, wherein said antibody binds to said complex in the presence or absence of EGFR, and wherein said antibody does not bind to any individual protein of said complex in the absence of another protein of said complex (ie., it is not merely an antibody that binds to a known protein rather than an antibody that binds to a protein complex of the present invention).
Preferably, the antibody recognizes a conformational epitope of the protein complex.
Another aspect of the present invention provides an anti-idiotypic antibody that binds to an antibody or ligand that binds to a protein complex selected from the group consisting of:
(i) a complex comprising CD2AP and cortactin;
(ii) a complex comprising CD2AP and Cbl;
(iii) a complex comprising CD2AP and endophilin;
(iv) a complex comprising CD2AP and endophilin and cortactin; (v) a complex comprising CD2AP and Cbl and cortactin;
(vi) a complex comprising CD2AP and endophilin and Cbl;
(vii) a complex comprising CD2AP and endophilin and Cbl and cortactin;
(viii) a complex comprising cortactin and ASAP1 ;
(ix) a complex comprising cortactin and N-WASP; (x) a complex comprising cortactin and FISH; and
(xi) a complex comprising cortactin and Sam68., wherein said anti-idiotypic antibody does not bind to an antibody or ligand that binds to an individual protein of said complex in the absence of another protein of said complex (ie., it is not merely an anti-idiotypic antibody against a known antibody or ligand that binds to an isolated protein rather than an anti-idiotypic antibody against an antibody or ligand that binds to a protein complex of the present invention).
Another aspect of the present invention provides methods for isolating a cortactin- binding protein or CD2AP-binding protein or a complex comprising same from a suitable cellular source.
To isolate the protein complex of the invention, one or both binding partners are separately isolated, from the same or a different cellular source that ectopically expresses or endogenously expresses at least one of the said binding partners. The isolated binding partners are then combined in an amount and under conditions sufficient to facilitate their physical relation. Such conditions can be readily determined by those skilled in protein chemistry. Selection of buffer pH, ionic strength, and temperature, sufficient to maintain the binding partners in solution are generally preferred. One or more protease inhibitors can also be included to prevent proteolytic digestion or degradation of the isolated polypeptides.
Alternatively, the protein complex per se may be isolated from a cellular source that expresses both binding partners endogenously or ectopically, including expression in response to EGF stimulation (i.e. EGF-modulated expression or EGF-induced expression or EGF-regulated expression).
It is also within the scope of this embodiment that the binding partners are expressed as a fusion protein or as distinct polypeptides.
Preferred cellular sources of the isolated polypeptide binding partners, or the protein complex, include any mammalian cell, and preferably, a mammalian cell that is known to express CD2AP, cortactin, endophilin, Cbl, EGFR, ASAP1, N-WASP, FISH, or Sam68 or can be engineered to express any one or more of said protein(s). Exemplary cells for such a purpose include cancer cells (e.g. carcinoma cells, breast cancer cells such as ER-negative breast cancer cells, or squamous epithelial carcinoma cells), epithelial cells, cells of the central nervous system, kidney cells, T cells, NIH3T3 cells, murine 10T fibroblasts, MDA-MB-231 cells, MDCK cells, COS cells, CHO cells, HeLa cells, or HEK 293 cells. The use of other cells (e.g. insect sf9 or sf21 cells, chick embryo cells and the like) is not excluded, particularly for isolation of a non-naturally occurring peptide, polypeptide or complex expressed by recombinant means.
Preferably, the protein complex or a binding partner thereof is isolated from cell line that endogenously expresses one or both binding partners, such as, for example, a cancer cell selected from the group consisting of head and neck cancer, breast cancer, adenocarcinoma, squamous lung cancer, gastrointestinal cancer (eg. gastric, colon, or pancreatic cancer), renal cell cancer, bladder cancer, a gynecological carcinoma (eg. ovarian cancer), prostate cancer, squamous cell carcinoma, non- squamous carcinoma, glioblastoma and medulloblastoma. More preferably, the cell will be a head and neck cancer cell or a breast cancer cell, or cell line derived from such cancers. Still more preferably, the cell will be an ER-negative breast cancer cell or ER-negative breast cancer cell line of HeLa cell line. As used herein, the term "cell line" includes any derivative of a stated cell line that expresses a binding partner of a protein complex of the present invention wherein said derivative also expresses said binding partner or can be engineered to express said binding partner.
In a particularly preferred embodiment, the protein complex or a binding partner thereof is isolated from a carcinoma cell or carcinoma cell line, preferably a breast cancer cell or breast cancer cell line, and more preferably a MDA-MB-231 cell or HeLa cell or derivative cell line thereof.
Means for isolating the peptide, polypeptide, or protein binding partners, or the protein complex, include any means of protein isolation known to the skilled protein chemist, such as, for example, size exclusion chromatography, ion-exchange (anion or cation exchange) chromatography, reverse phase chromatography, or affinity chromatography. Both high pressure (e.g. HPLC, FLPC, MALDI) and low pressure systems can be used.
Affinity methods using ligands or antibodies that bind to one or both of the binding partners to the protein-protein interaction are particularly preferred. For example, SU6656 is a competitive inhibitor of Src and Src-related tyrosine kinase proteins that may be used as an affinity tag for isolating any Src ligand. Antibodies against the SH3 domain of an SH3 protein, such as, for example, those antibodies described herein or in US Patent Nos. 6,342, 593 or 6,326,158 are particularly useful for isolating CD2AP or a binding partner of CD2AP that has an SH3 domain (i.e. cortactin or endophilin). Antibodies against the PRR of CD2AP are also useful for isolating cortactin or endophilin. In fact, antibodies against any polypeptide selected from the group consisting of CD2AP, Cortactin, Cbl, endophilin, ASAP1 , N-WASP, FISH, Sam 68, a portion of CD2AP, a portion of cortactin, a portion of Cbl, a portion of endophilin, a portion of ASAP1, a portion of N-WASP, a portion of FISH, and a portion of Sam 68 can be used.
For example, naturally-occurring or recombinant protein is purified free of conspecific proteins by providing a matrix comprising antibody coupled to activated chromatographic resin (eg. CNBr-activated Sepharose, Pharmacia), blocking the resin and washing to remove unbound antibody and blocking agent, contacting the resin with a protein extract comprising a peptide or polypeptide to which the antibody binds under conditions sufficient to allow binding of said peptide or polypeptide (e.g., high ionic strength buffers in the presence of detergent), and eluting said peptide or polypeptide under conditions that disrupt the antibody antigen binding (eg, a buffer of pH 2-3 or a high concentration of a chaotrope, such as urea or thiocyanate ion).
It will be apparent from the preceding description that small molecules, or proteins capable of binding to one of the binding partners, can also be used to isolate one or both binding partners, or the protein complex per se, by affinity means. Conditions to permit such isolation can be readily determined by those skilled in protein chemistry. Selection of buffer pH, ionic strength, and temperature, sufficient to maintain the binding partners in solution are generally preferred. Preferably, one or more protease inhibitors (e.g. papain, PMSF, leupeptin) are included to prevent proteolytic digestion or degradation of the isolated polypeptides. For example, naturally-occurring or recombinant protein is purified free of conspecific proteins by providing a matrix comprising a small molecule or protein binding partner coupled to activated chromatographic resin (eg. CNBr-activated Sepharose, Pharmacia), blocking the resin and washing to remove unbound material and blocking agent, contacting the resin with a protein extract comprising a peptide or polypeptide to which the antibody binds under conditions sufficient to allow binding of said peptide or polypeptide, and eluting said peptide or polypeptide under conditions that disrupt the binding.
Preferably, the isolated protein or complex is provided substantially free of conspecific proteins, meaning that it is at least about 1-5% pure as determined by an analysis of proteins by SDS/PAGE. More preferably, the protein is at least about 10%, even more preferably at least about 20% pure, even more preferably at least about 25% pure, even more preferably at least about 30% pure, and even more preferably at least about 50% pure, and still more preferably substantially pure.
Another aspect of the present invention provides methods for producing a protein complex described herein by recombinant means. For expressing peptides or polypeptides by recombinant means, a protein-encoding nucleotide sequence is placed in operable connection with a promoter or other regulatory sequence capable of regulating expression in a cell-free system or cellular system.
In one embodiment of the invention, nucleic acid comprising a sequence that encodes a cortactin polypeptide or a portion of a cortactin polypeptide and a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide, in operable connection with a suitable promoter sequence, is expressed in a suitable cell for a time and under conditions sufficient to produce a fusion polypeptide comprising the binding partners of the complex.
In an alternative embodiment of the invention, nucleic acid comprising a sequence that encodes a CD2AP polypeptide or a portion of a CD2AP polypeptide and a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide, in operable connection with a suitable promoter sequence, is expressed in a suitable cell for a time and under conditions sufficient for expression to occur. Wherein the recombinant expression of endophilin and CD2AP is performed, the nucleic acid may also comprise a nucleotide sequence encoding a Cbl polypeptide or a portion of said Cbl polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide. Similarly, wherein the recombinant expression of Cbl and CD2AP is performed, the nucleic acid" may also comprise a nucleotide sequence encoding an endophilin polypeptide or a portion of said endophilin polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide. In these embodiments, it is also within the scope of the present invention for any endophilin polypeptide, Cbl polypeptide, portion of endophilin, or any portion of Cbl to be sufficient to form a complex between Cbl and endophilin, a portion of Cbl and endophilin, a portion of Cbl and a portion of endophilin, or Cbl and a portion of endophilin, in addition to a capability of forming an association with CD2AP or a portion of CD2AP.
Nucleic acid encoding the binding partners is readily derived from the amino acid sequences set forth herein, or alternatively, publicly available. To produce a fusion polypeptide, the open reading frames are covalently linked in the same reading frame, such as, for example, using standard cloning procedures as described by Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, ISBN 047150338, 1992), which is herein incoφorated by reference.
Optionally, a spacer is placed between the open reading frames of the binding partners to facilitate their physical relation. Preferred spacers comprise protein- encoding nucleotide sequences of at least about 15-30 nucleotides in length, preferably sequences encoding amino acids rich in proline. The spacer is designed such that it does not interrupt the open reading frames of the partners.
Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e., upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. In the present context, the term "promoter" is also used to describe a recombinant, synthetic or fusion molecule, or derivative which confers, activates or enhances the expression of a nucleic acid molecule to which it is operably connected, and which encodes the polypeptide or peptide fragment. Preferred promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or to alter the spatial expression and/or temporal expression of the said nucleic acid molecule.
Placing a nucleic acid molecule under the regulatory control of, i.e., "in operable connection with", a promoter sequence means positioning said molecule such that expression is controlled by the promoter sequence. Promoters are generally positioned 5' (upstream) to the coding sequence that they control. To construct heterologous promoter/structural gene combinations, it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e., the gene from which the promoter is derived. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
The prerequisite for producing intact polypeptides and peptides in bacteria such as E. coli is the use of a strong promoter with an effective ribosome binding site. Typical promoters suitable for expression in bacterial cells such as £. coli include, but are not limited to, the lacz promoter, temperature-sensitive λ or λR promoters, T7 promoter or the IPTG-inducible tac promoter. A number of other vector systems for expressing the nucleic acid molecule of the invention in E. coli are well-known in the art and are described, for example, in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047150338, 1987) or Sambrook et al (In: Molecular cloning, A laboratory manual, second edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). Numerous plasmids with suitable promoter sequences for expression in bacteria and efficient ribosome binding sites have been described, such as for example, pKC30 (λ : Shimatake and Rosenberg, Nature 292, 128, 1981); pKK173-3 (tac: Amann and Brosius, Gene 40, 183, 1985), pET-3 (T7: Studier and Moffat, J. Mol. Biol. 189, 113, 1986); the pBAD/TOPO or pBAD/Thio-TOPO series of vectors containing an arabinose-inducible promoter (Invitrogen, Carlsbad, CA), the latter of which is designed to also produce fusion proteins with thioredoxin to enhance solubility of the expressed protein; the pFLEX series of expression vectors (Pfizer Inc., CT, USA); or the pQE series of expression vectors (Qiagen, CA), amongst others.
Typical promoters suitable for expression in viruses of eukaryotic cells and eukaryotic cells include the SV40 late promoter, SV40 early promoter and cytomegalovirus (CMV) promoter, CMV IE (cytomegalovirus immediate early) promoter amongst others. Preferred vectors for expression in mammalian cells (eg. 293, COS, CHO, 10T cells, 293T cells) include, but are not limited to, the pcDNA vector suite supplied by Invitrogen, in particular pcDNA 3.1 myc-His-tag comprising the CMV promoter and encoding a C-terminal 6xHis and MYC tag; and the retrovirus vector pSRαtkneo (Muller et al, Mol. Cell. Biol, 11, 1785, 1991). The vector pcDNA 3.1 myc-His (Invitrogen) is particularly preferred for expressing a secreted form of a protein in 293T cells, wherein the expressed peptide or protein can be purified free of conspecific proteins, using standard affinity techniques that employ a Nickel column to bind the protein via the His tag.
A wide range of additional host/vector systems suitable for expressing polypeptide binding partners or immunological derivatives thereof are available publicly, and described, for example, in Sambrook et al (In: Molecular cloning, A laboratory manual, second edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).
Means for introducing the isolated nucleic acid molecule or a gene construct comprising same into a cell for expression are well-known to those skilled in the art. The technique used for a given organism depends on the known successful techniques. Means for introducing recombinant DNA into animal cells include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongst others.
In another embodiment, nucleic acid comprising a sequence encoding each binding partner is placed in operable connection with a promoter sequence and expressed in a suitable cell. If the protein partners are expressed in the same cell, they may freely associate in said cell to form the protein complex of the invention. If the protein partners are produced in different cells, the cells are lysed and the cellular lysates mixed under conditions sufficient to permit the association of the binding partners.
In accordance with this embodiment, the nucleotide sequences encoding the binding partners may be contained in the same or different nucleic acid molecules, and as a consequence, the use of single or multiple gene constructs to express the binding partners is clearly encompassed by the invention. The requirements for expressing fusion polypeptides as described herein above are also relevant in this context, except that there is no need for a spacer. Generally, different promoters will be used to express each binding partner, such as, for example, to prevent squelching or competition between promoters or regulatory sequences for cellular transcription factors.
Another aspect of the present invention provides prognostic and diagnostic methods for determining a predisposition for disease, or a disease state, said methods comprising detecting a protein complex comprising: (i) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH and Sam68; and (b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and (ii) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
(b) a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide.
Preferably, the protein complex that is detected is a complex comprising cortactin and CD2AP, optionally further comprising EGFR; more preferably a complex comprising cortactin and CD2AP and endophilin, optionally further comprising EGFR; still more preferably a complex comprising cortactin and CD2AP and Cbl, optionally further comprising EGFR; and still more preferably comprising cortactin and CD2AP and endophilin and Cbl, optionally further comprising EGFR.
Preferred detection systems contemplated herein include any known assay for detecting a protein-protein interaction in a biological sample isolated from a human or mammalian subject, such as, for example, using one or more antibodies against the complex or each binding partner, or an epitope thereof. Alternatively, a non-antibody ligand of the protein complex may be used, such as, for example, a small molecule (e.g. a chemical compound, agonist, antagonist, allosteric modulator, competitive inhibitor, or non-competitive inhibitor, of the complex that may or may not modulate complex formation or dissociation).
The use of antibody-based assay systems is particularly preferred. In accordance with these embodiments, the antibody or small molecule may be used in any standard solid phase or solution phase assay format amenable to the detection of protein complexes or protein-protein interactions.
Antibodies that specifically bind to the protein complex are used for the diagnosis of conditions or diseases characterized by the presence of said protein complex, or in prognostic assays to monitor disease progression in the presence of absence of treatment. Diagnostic assays for include methods which utilize the antibody and a label to detect the protein complex in human body fluids or extracts of cells or tissues. The antibodies are used with or without modification, and may be labeled, either covalently or non-covalently, with a reporter molecule. A wide variety of reporter molecules which are known in the art may be used, several of which are described above.
A variety of protocols, including ELISA, RIA, and FACS, for measuring the protein complex are known in the art or described herein. Such methods provide a basis for diagnosing levels of the protein complex associated with disease. For example, the cortactin-comprising protein complexes of the present invention are associated with cancers induced by over expression of cortactin (eg. head and neck cancer, breast cancer, adenocarcinoma, squamous lung cancer, gastrointestinal cancer,, gastric cancer, colon cancer, pancreatic cancer, renal cell cancer, bladder cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, non-squamous carcinoma, glioblastoma, medulloblastoma and ER-negative breast cancer), and presence of one or more of those complexes provides a poor prognosis of survival from the disease. Normal or standard values of the complex for a healthy individual are established by combining body fluids or cell extracts taken from normal or healthy subjects, preferably human subjects.
The amount of standard complex formation may be quantified by various methods, preferably by photometric means, or using antibodies in a quantitative immunoassay (e.g. ELISA), wherein the amount of protein complex is determined by comparison against known amounts of a standard peptide, such as, for example, a peptide comprising an SH3 domain of cortactin, CD2AP, ASAP1, or endophilin, or a PRR from CD2AP, Cbl, ASAP1 , N-WASP, FISH or Sam68.
Quantities of the protein complex expressed in subject samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease or establishing a prognosis. In the case of cancerous tissues, a level of a protein complex in excess of the standard level of that protein complex detected in a healthy subject, is diagnostic of disease, and indicates a poor prognosis for survival. Optical or fluorescent detection, such as, for example, using mass spectrometry, MALDI-TOF, biosensor technology, evanescent fiber optics, or fluorescence resonance energy transfer, is clearly encompassed by the present invention.
In biosensor diagnostic devices, the assay substrate and detector surface are integrated into a single device. One general type of biosensor employs an electrode surface in combination with current or impedance measuring elements for detecting a change in current or impedance in response to the presence of a protein-protein binding event (e.g. U.S. Patent No. 5,567,301). Gravimetric biosensors employ a piezoelectric crystal to generate a surface acoustic wave whose frequency, wavelength and/or resonance state are sensitive to surface mass on the crystal surface. The shift in acoustic wave properties is therefore indicative of a change in surface mass, such as, for example, as a consequence of protein-protein binding (e.g. U.S. Patent Nos. 5,478,756 and 4,789,804. Biosensors based on surface plasmon resonance (SPR) effects have also been proposed, for example, in U.S. Patent Nos. 5,485,277 and 5,492,840, which exploit the shift in SPR surface reflection angle that occurs when protein binds to the SPR interface. Finally, a variety of biosensors that utilize changes in optical properties at a biosensor surface are known, (e.g., U.S. Patent No. 5,268,305).
Biosensors have a number of potential advantages over binding assay systems having separate reaction substrates and reader devices. One important advantage is the ability to manufacture small-scale, but highly reproducible, biosensor units using microchip manufacturing methods, such as, for example, described in U.S. Patent Nos. 5,200,051 and 5,212,050. Another advantage is the potentially large number of different analyte detection regions that can be integrated into a single biosensor unit, allowing sensitive detection of several analytes with a very small amount of body-fluid sample. Accordingly, the simultaneous detection of the individual binding partners that form the protein complex, or the simultaneous detection of one or more protein complexes of the present invention, is possible using a biosensor.
Evanescent biosensors are particularly preferred because they do not require separation of the protein complex from unbound material, and their use can be coupled to standard immunoassay formats, as originally described by Hirshfield in U.S. Patent No. 4,447,546. In general, evanescent biosensors rely upon light of a particular wavelength interacting with a fluorescent molecule, such as, for example, a fluorescent antibody or small molecule attached near the probe's surface, to emit fluorescence at another wavelength, on binding of the protein complex of the invention to the antibody or small molecule. The biosensor is protected from sensitivity degradation caused by non-specific binding of proteins to the sensor surface, by exposing the sensor surface to a solution of non-interfering proteins, so that the non-interfering proteins bind to said sensor surface to prevent the subsequent binding of the interfering proteins. Enhanced protection of surfaces from biological proteins is also possible by completely covering surfaces with protective coatings, such as, for example, amorphous copolymers of tetrafluoroethylene and bis-2,2-trifluoromethyl-4.5-difluoro-1,2-dioxole, dissolved in a solvent containing fluorinated alkanes, and applied by deposition as a thin protective coating (US. Patent No. 5,356,668 by Paton et al).
Assay systems suitable for use in high throughput screening of mass samples, particularly a high throughput spectroscopy resonance method (e.g. MALDI-TOF, electrospray MS or nano-electrospray MS) or a detection system facilitating determination of real time association/dissociation constants, are particularly contemplated.
In an alternative embodiment, a diagnosis or prognosis is made by separately determining the level(s) of expression of the binding partners, wherein a high level of expression of binding partners of a complex in a sample is indicative of disease, or, in the case of cancerous tissues, a poor prognosis for survival. In this case, the level of expression of the binding partners is determined by standard protein-based detection systems, antibody-based methods, or nucleic acid-based methods.
Nucleic acid encoding a binding partner of the protein complex of the invention (i.e., encoding a full-length protein or a portion thereof), such as, for example, a synthetic oligonucleotide, complementary RNA, DNA, or protein-nucleic acid (PNA) is used to detect and quantitate gene expression in biopsied tissues in which expression of the polypeptide is correlated with disease. The diagnostic assay may be used to distinguish between absence, presence, and over expression of the binding partner, or to monitor expression following an initial diagnosis or during therapeutic intervention. As with protein detection systems, the detection of over expression of cortactin is preferred.
Co-localization of expression of several binding partners in a particular cell, tissue or organ, such as, for example, using FISH or other expression detection system, is also indicative of disease.
In one embodiment, hybridization with PCR probes capable of detecting the nucleic acid (RNA or genomic DNA) encoding a binding partner is used. The specificity of the probe, is determined by its nucleotide sequence and the stringency of the hybridization or amplification (maximal, high, intermediate, or low). Generally, highly specific probes are preferred for use under more stringent conditions. The hybridization probes may be DNA or RNA and will preferably comprise a nucleotide sequence that encodes a polypeptide of a protein complex of the present invention, including a genomic gene sequence, or a promoter, enhancer element, or introns of the naturally occurring gene.
Means for producing specific hybridization probes are known in the art. Hybridization probes may be labelled by a variety of reporter groups, for example, radionuclides such as 32P or 35S, or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
In one embodiment, nucleic acid probes are labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions suitable for the formation of a hybridization complex. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the biopsied or extracted sample is significantly altered from that of a comparable control sample, the altered level of expression in the sample indicates a disease state. Such assays are also used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient. To provide a basis for the diagnosis of disease associated with expression of the binding partners, a normal or standard profile for expression of the partners is established, such as, for example, by combining body fluids or cell extracts taken from normal subjects with nucleic acid encoding the binding partners or a portion thereof, under conditions suitable for hybridization or amplification. Standard hybridization is then quantified by comparing the values obtained from normal subjects with the signal obtained using a known amount of a substantially purified nucleic acid. Standard values from normal samples are then compared with values from patient samples. Deviation between standard and subject values is diagnostic of the disease. Once a diagnosis is made by this or another method, hybridization assays are carried out to evaluate expression of the binding partners over time, or during a course of treatment.
With respect to cancer, the presence of relatively high amounts of RNA encoding a binding partner of an inventive protein complex in biopsied tissue from an individual indicates a predisposition for the development of the disease, or is otherwise diagnostic of the disease, preferably prior to the appearance of actual clinical symptoms. Alternatively, or in addition, high levels of these transcripts are indicative of a poor prognosis for survival.
Methods that are used to quantitate the expression include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and the use of standard curves onto which the experimental results are inteφolated (Melby et al, J. Immunol. Methods, 159, 235-244, 1993; Duplaa et al, Anal. Biochem. 212, 229-236, 1993).
In further embodiments, an oligonucleotide derived from a nucleotide sequence encoding any one or more of the binding partners is used as a target in a microarray. The microarray can be used to monitor the expression level of large numbers of genes simultaneously, to produce a transcript image, or to identify a genetic variant, mutant, or polymorphism for the protein complex of the invention. In one embodiment, the microarray is prepared and used according to the methods described by Chee et al, WO95/11995, Lockhart et al, Nat. Biotech. 14, 1675-1680, 1996, or Schena et al, Proc. Natl. Acad. Sci. USA 93, 10614-10619, 1996. A microarray is preferably composed of a large number of unique, single-stranded nucleic acid sequences, such as, for example, synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. Preferred oligonucleotides have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides containing single nucleotide mismatches, to control for non-specific hybridization/amplification.
To conduct sample analysis using a microarray, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray so that the probe sequences hybridize to complementary oligonucleotides of the microarray. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of non-hybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray. The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A detection system is used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously.
The present invention is also used to diagnose the occurrence of a disorder or disease associated with aberrant regulation, formation or activity of the cytoskeleton, or a predisposition of an individual towards a disorder or disease associated with aberrant regulation, formation or activity of the cytoskeleton. Individuals having a condition involving aberrant cytoskeletal regulation, formation or activity may exhibit, for example, aberrant cell growth or motility, the formation of tumors, tumor metastasis, or tumor cell invasion. The present invention is particularly useful for diagnoses in relation to T cell-mediated immune system disorders, such as, for example, autoimmune diseases (Type I diabetes, multiple sclerosis, rheumatoid arthritis, psoriasis, Wiskott-Aldrich Syndrome), transplant rejection, allergy, asthma, aberrant T cell mediated immunity, aberrant antigen recognition or presentation, disorders of the central nervous system, nephritis, or kidney failure. The application of the methods described herein to the diagnosis or prognosis of head and neck cancer, breast cancer, adenocarcinoma, squamous lung cancer, gastrointestinal cancer (eg. gastric, colon, or pancreatic cancer), renal cell cancer, bladder cancer, a gynecological carcinoma (eg. ovarian cancer), prostate cancer, squamous cell carcinoma, non- squamous carcinoma, glioblastoma or medulloblastoma is particularly preferred.
A further aspect of the present invention provides methods for determining a modulator of the activity, formation or stability of a protein complex comprising:
(i) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of
CD2AP, N-WASP, ASAP1 , FISH and Sam68; and (b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and
(ii) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
(b) a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide, wherein said protein complex further optionally comprises EGFR, said method comprising determining the level of said protein complex in the absence of a candidate compound or candidate antibody and determining the level of said protein complex in the presence of a candidate compound or in the presence of said candidate antibody wherein a difference in the level of said protein complex in the absence and presence of the candidate compound or candidate antibody indicates that the candidate compound or candidate antibody is a modulator of said interaction. Preferably, the modulator modulates the activity, formation or stability of a protein complex selected from the group consisting of: (i) a complex comprising CD2AP and cortactin; (ii) a complex comprising CD2AP and Cbl; (iii) a complex comprising CD2AP and endophilin; (iv) a complex comprising CD2AP and endophilin and cortactin; (v) a complex comprising CD2AP and Cbl and cortactin; (vi) a complex comprising CD2AP and endophilin and Cbl; (vii) a complex comprising CD2AP and endophilin and Cbl and cortactin; (viii) a complex comprising cortactin and ASAP1; (ix) a complex comprising cortactin and N-WASP; (x) a complex comprising cortactin and FISH; and (xi) a complex comprising cortactin and Sam68, wherein said protein complex optionally further comprises EGFR.
In their general form, the methods of the present invention comprise determining the association or dissociation of the protein complex, or the structure of the complex, in the presence and absence of a candidate compound or a candidate antibody. In accordance with the embodiment described herein, a modified association, dissociation, or structure, of the protein complex in the presence of a candidate compound or a candidate antibody indicates that the candidate is a modulator of the protein complex.
The association, dissociation, or structure of the complex may be determined by direct means, such as, for example, by determining real time association or dissociation constants in the presence and absence of the candidate, or modified binding of an antibody that recognizes a conformational epitope of the complex. Biosensors used essentially as described herein above, in the presence or absence of the candidate compound or antibody, are particularly suited to such applications.
Alternatively, the association, dissociation, or structure of the complex may be determined by indirect means, such as, for example, using a protein recruitment system, n-hybrid screen, reverse n-hybrid screen, plate agar diffusion assay, ELISA, or other well known assay format for detecting protein-protein interactions. Such indirect means generally use a reporter system to detect formation or dissociation of the protein complex. Standard solid-phase ELISA assay formats are particularly useful for identifying antagonists of the protein-protein interaction. In accordance with this embodiment, one of the binding partners (e.g. cortactin or CD2AP or a portion thereof) is immobilized on a solid matrix, such as, for example an array of polymeric pins or a glass support. Conveniently, the immobilized binding partner is a fusion polypeptide comprising Glutathione-S-transferase (GST; e.g. a cortactin-GST fusion, CD2AP-GST fusion or SH3 domain-GST fusion), wherein the GST moiety facilitates immobilization of the protein to the solid phase support. The second binding partner (e.g. cortactin, FISH, N-WASP, ASAP1, Sam68, Cbl or endophilin) in solution is brought into physical relation with the immobilized protein to form a protein complex, which complex is detected using antibodies directed against the second binding partner. The antibodies are generally labelled with fluorescent molecules or conjugated to an enzyme (e.g. horseradish peroxidase), or alternatively, a second labelled antibody can be used that binds to the first antibody. Conveniently, the second binding partner is expressed as a fusion polypeptide with a FLAG or oligo-histidine peptide tag, or other suitable immunogenic peptide, wherein antibodies against the peptide tag are used to detect the binding partner. Alternatively, oligo-HIS tagged protein complexes can be detected by their binding to nickel-NTA resin (Qiagen), or FLAG-labeled protein complexes detected by their binding to FLAG M2 Affinity Gel (Kodak). It will be apparent to the skilled person that the assay format described herein is amenable to high throughput screening of samples, such as, for example, using a microarray of bound peptides or fusion proteins.
In a modification of the standard ELISA-type assay format, the SH3 domain or PRR of CD2AP or the SH3 domain or PRR of a binding partner of CD2AP is immobilized on a solid support, such as by chemical synthesis thereon, or biotin-labelled and used in the liquid phase.
A two-hybrid assay is described in US Patent No. 6,316,223 to Payan et al, incorporated herein by reference. The basic mechanism described by Payan et al. is similar to the yeast two hybrid system. In the two-hybrid system, the binding partners are expressed as two distinct fusion proteins in a mammalian host cell. In adapting the standard two-hybrid screen to the present purpose, a first fusion protein consists of a DNA binding domain which is fused to one of the binding partners, and a second fusion protein consists of a transcriptional activation domain fused to the other binding partner. The DNA binding domain binds to an operator sequence which controls expression of one or more reporter genes. The transcriptional activation domain is recruited to the promoter through the functional interaction between binding partners. Subsequently, the transcriptional activation domain interacts with the basal transcription machinery of the cell, thereby activating expression of the reporter gene(s), the expression of which can be determined. Candidate bioactive agents that modulate the protein-protein interaction between the binding partners are identified by their ability to modulate transcription of the reporter gene(s) when incubated with the host cell. Antagonists will prevent or reduce reporter gene expression, while agonists will enhance reporter gene expression. In the case of small molecule modulators, these are added directly to the cell medium and reporter gene expression determined. On the other hand, peptide modulators are expressible from nucleic acid that is transfected into the host cell and reporter gene expression determined. In fact, whole peptide libraries can be screened in transfected cells.
Alternatively, reverse two hybrid screens, such as, for example, described by Vidal et al, Proc. Natl Acad. Sci USA 93, 10315-10320, 1996, may be employed to identify antagonist molecules. Reverse hybrid screens differ from forward screens supra in so far as they employ a counter-selectable reporter gene, such as for example, CYH2 or LYS2, to select against the protein-protein interaction. Cell survival or growth is reduced or prevented in the presence of a non-toxic substrate of the counter- selectable reporter gene product, which is converted by said gene product to a toxic compound. Accordingly, cells in which the protein-protein interaction of the invention does not occur, such as in the presence of an antagonist of said interaction, survive in the presence of the substrate, because it will not be converted to the toxic product. For example, the cortactin SH3 domain or CD2AP PRR is expressed as a DNA binding domain fusion, such as with the DNA binding domain of GAL4, and the appropriate region of CD2AP or cortactin, respectively, is expressed as an appropriate transcription activation domain fusion polypeptide (e.g. with the GAL4 transcription activation domain). The fusion polypeptides are expressed in yeast in operable connection with the URA3 counter-selectable reporter gene, wherein expression of URA3 requires a physical relation between the GAL4 DNA binding domain and transcriptional activation domain. This physical relation is achieved, for example, by placing reporter gene expression under the control of a promoter comprising nucleotide sequences to which GAL4 binds. Cells in which the reporter gene is expressed do not grow in the presence of uracil and 5-fluoroorotic acid (5-FOA), because the 5-FOA is converted to a toxic compound. Candidate peptide inhibitor(s) are expressed from libraries in such cells, wherein cells that grow in the presence of uracil and 5-FOA are retained for further analysis, such as, for example, analysis of the nucleic acid encoding the candidate peptide inhibitor(s). Small molecules that antagonize the interaction are determined by incubating the cells in the presence of the small molecules and selecting cells that grow or survive in the presence of uracil and 5-FOA.
Alternatively, a protein recruitment system, such as that described in U.S. Patent No. 5, 776, 689 to Karin et al, is used. In a standard protein recruitment system, a protein-protein interaction is detected in a cell by the recruitment of an effector protein, which is not a transcription factor, to a specific cell compartment. Upon translocation of the effector protein to the cell compartment, the effector protein activates a reporter molecule present in that compartment, wherein activation of the reporter molecule is detectable, for example, by cell viability, indicating the presence of a protein-protein interaction.
More specifically, the components of a protein recruitment system include a first expressible nucleic acid encoding a first fusion protein comprising the effector protein and one of the binding partners (e.g. CD2AP or a portion thereof), and a second expressible nucleic acid molecule encoding a second fusion protein comprising a cell compartment localization domain and the other binding partner (e.g. cortactin, endophilin or Cbl or a portion thereof). A cell line or cell strain in which the activity of an endogenous effector protein is defective or absent (e.g. a yeast cell or other non- mammalian cell), is also required, so that, in the absence of the protein-protein interaction, the reporter molecule is not expressed. A complex is formed between the fusion polypeptides as a consequence of the interaction between the binding partners, thereby directing translocation of the complex to the appropriate cell compartment mediated by the cell compartment localization domain (e.g. plasma membrane localization domain, nuclear localization domain, mitochondrial membrane localization domain, and the like), where the effector protein then activates the reporter molecule. Such a protein recruitment system can be practiced in essentially any type of cell, including, for example, mammalian, avian, insect and bacterial cells, and using various effector protein/reporter molecule systems.
For example, a yeast cell based assay is performed, in which the interaction between cortactin or CD2AP and one or more of their binding partners results in the recruitment of a guanine nucleotide exchange factor (GEF) to the plasma membrane, wherein GEF activates a reporter molecule, such as Ras, thereby resulting in the survival of cells that otherwise would not survive under the particular cell culture conditions. Suitable cells for this purpose include, for example, Saccharomyces cerevisiae cdc25- 2 cells, which grow at 36°C only when a functional GEF is expressed therein, Petitjean et al, Genetics 124, 797-806, 1990) Translocation of the GEF to the plasma membrane is facilitated by a plasma membrane localization domain. Activation of Ras is detected, for example, by measuring cyclic AMP levels in the cells using commercially available assay kits and/or reagents. To detect antagonists of the protein-protein interaction of the present invention, duplicate incubations are carried out in the presence and absence of a test compound, or in the presence or absence of expression of a candidate antagonist peptide in the cell. Reduced survival or growth of cells in the presence of a candidate compound or candidate peptide indicates that the peptide or compound is an antagonist of the interaction between cortactin or CD2AP, as appropriate, and one or more of their binding partners.
A "reverse" protein recruitment system is also contemplated, wherein modified survival or modified growth of the cells is contingent on the disruption of the protein-protein interaction by the candidate compound or candidate peptide. For example, NIH 3T3 cells that constitutively express activated Ras in the presence of GEF can be used, wherein the absence of cell transformation is indicative of disruption of the protein complex by a candidate compound or peptide. In contrast, NIH 3T3 cells that constitutively express activated Ras in the presence of GEF have a transformed phenotype (Aronheim et al., Cell. 78, 949-961, 1994)
In yet another embodiment, small molecules are tested for their ability to dissociate the protein complex of the invention, by an adaptation of plate agar diffusion assay described by Vidal and Endoh, TIBS 17, 374-381, 1999, which is incorporated herein by reference.
A further embodiment of the invention provides a method for determining a modulator of an interaction between cortactin or a portion of cortactin and a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide, said method comprising:
(i) determining the level of a protein complex comprising cortactin or a portion of cortactin and a polypeptide selected from the group consisting of CD2AP, N-
WASP, ASAP1 , FISH and Sam68 or a portion of said polypeptide in the absence of a candidate compound or candidate antibody; and (ii) determining the level of said protein complex in the presence of a candidate compound or in the presence of said candidate antibody wherein a difference in the level of said protein complex at (i) and (ii) indicates that the candidate compound or candidate antibody is a modulator of said interaction.
A further embodiment of the invention provides a method for determining a modulator of an interaction between CD2AP or a portion of CD2AP and a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide, said method comprising:
(iii) determining the level of a protein complex selected from the group consisting of: (i) a complex comprising CD2AP and cortactin; (ii) a complex comprising CD2AP and endophilin; (iii) a complex comprising CD2AP and Cbl; (iv) a complex comprising CD2AP and endophilin and cortactin; (v) a complex comprising CD2AP and Cbl and cortactin; and (vi) a complex comprising CD2AP and endophilin and Cbl and cortactin in the absence of a candidate compound or candidate antibody; and (iv) determining the level of said protein complex in the presence of a candidate compound or in the presence of said candidate antibody wherein a difference in the level of said protein complex at (i) and (ii) indicates that the candidate compound or candidate antibody is a modulator of said interaction.
These embodiments of the invention apply mutatis mutandis to the determination of protein complexes comprising a portion of any one or more of the protein binding partners of a protein complex described herein.
It will be understood by those skilled in the art that any one or more of the assay methods for antagonists as described herein above can be adapted for this purpose. This is because the level of the protein complex in the presence or absence of a candidate compound or antibody is related to antibody binding in the case of ELISAs, or to cell survival or growth, in the case of hybrid screens or protein recruitment assays. ELISA-based assay formats are particularly suitable for this puφose, because they are readily quantifiable, by calibrating the detection system against known amounts of a protein standard to which the antibody binds. Such quantitation is well known to the skilled person.
In a particularly preferred embodiment, the modulators of the present invention are agonists of complex formation or stability. Preferred agonist compounds enhance EGFR endocytosis in a cell, or prevent or reduce an impairment of EGFR endocytosis in a cell, or prevent or reduce a reduced or limited EGFR endocytosis in a cell. More particularly, agonists of EGFR endocytosis in a cell will preferably enhance the formation or stability of a protein complex that is EGF-induced, such as, for example, a protein complex selected from the group consisting of: (i) a complex comprising cortactin and CD2AP and Cbl; and (ii) a complex comprising cortactin and CD2AP and Cbl and endophilin.
Exemplary agonist compounds include one or more polypeptides of the protein complex, such as, for example a CD2AP and/or Cbl polypeptide or a portion thereof sufficient to form a protein complex of the invention. A mimetic of Cbl capable of binding to a protein complex cortactin and CD2AP is particularly contemplated. Alternatively, or in addition a mimetic of CD2AP capable of binding to cortactin and/or Cbl can be used. A mimetic compounds comprising an anti-idiotypic antibody as described herein above are also contemplated for use as agonists of the protein complex of the present invention or for treatment of a condition in which enhanced EGFR endocytosis is indicated.
In a related embodiment, an agonist comprises a nucleic acid encoding one or more of the binding partners of the protein complex, preferably in an expressible format and a form suitable for administering to a cell. In accordance with this embodiment, nucleic acids encoding a Cbl and/or CD2AP polypeptide or a portion thereof is particularly preferred.
In an alternative embodiment, the modulators of the present invention are antagonists or inhibitors of complex formation or stability, such as, for example, a compound that inhibits or disrupts SH3-mediated protein-protein interactions. The antagonist compound UCS15A isolated from Streptomyces sp. (Oneyama et al, Oncogene 21,
2037-2050, 2002) is particularly preferred for disrupting the formation of protein complexes formed by an association with an SH3 domain of cortactin or CD2AP or endophilin or ASAP1. In this respect, it is particularly preferred to use UCS15A to disrupt the stability or formation of a protein complex that does not bind to the EGFR and/or to disrupt the stability or formation of a protein complex that does not facilitate or enhance receptor endocytosis. Without being bound by any theory or mode of action, disruption of such complexes enhances the level of protein binding partners available for the formation of protein complexes (eg., the cortactin-CD2AP-Cbl protein complex) that bind to EGFR and/or enhance endocytosis of the receptor.
The modulators identified using the methods described herein are useful for the therapeutic or prophylactic treatment of diseases associated with aberrant cytoskeletal regulation, formation or activity, such as, for example, aberrant cell growth or motility, tumorigenesis, tumor metastasis, tumor cell invasion, disorders of the central nervous system, nephritis, or kidney failure. The modulators are preferably useful for the treatment of one or more symptoms associated with T cell-mediated immune system disorders, such as, for example, autoimmune diseases (Type I diabetes, multiple sclerosis, rheumatoid arthritis, psoriasis, Wiskott-Aldrich Syndrome), transplant rejection, allergy, asthma, aberrant T cell mediated immunity, aberrant antigen recognition or presentation. The treatment of a cancer selected from the group consisting of head and neck cancer, breast cancer, adenocarcinoma, squamous lung cancer, gastrointestinal cancer (eg. gastric, colon, or pancreatic cancer), renal cell cancer, bladder cancer, a gynecological carcinoma (eg. ovarian cancer), prostate cancer, squamous cell carcinoma, non-squamous carcinoma, glioblastoma and medulloblastoma is particularly contemplated by the present invention, and preferably, treatment of a breast cancer, specifically an ER-negative breast cancer in which cortactin expression is elevated.
Accordingly, a further aspect of the present invention provides a method of treatment comprising administering to a subject an amount of a modulator of the activity, formation or stability of a protein complex comprising:
(i) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH and Sam68; and (b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1, FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and (ii) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
(b) a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide.
Preferably, this aspect of the invention relates to the modulation of EGFR endocytosis, and more particularly to enhancing EGFR endocytosis in a cell, or to preventing or reducing an impairment of EGFR endocytosis in a cell, or to preventing or reducing a reduced or limited EGFR endocytosis in a cell. Conditions in which EGFR endocytosis is impaired or limited or reduced include a disease state or condition characterized by elevated EGFR levels, or elevated or sustained EGF-mediated signalling, such as, for example a cancer, infection by an oncogenic animal virus (eg., human papilloma virus such as HPV-16 or HPV-11 or HPV6; a poxvirus, a retrovirus).
To enhance EGFR endocytosis in a cell, it is particularly preferred to administer an agonist of the formation or stability of a protein complex of the invention, particularly an agonist of a protein complex of the invention the formation of which is EGF-induced, such as, for example, a protein complex selected from the group consisting of: (i) a complex comprising cortactin and CD2AP and Cbl; and
(ii) a complex comprising cortactin and CD2AP and Cbl and endophilin. Exemplary agonist compounds are described herein above.
In a particularly preferred embodiment, the present invention contemplates a method of enhancing EGFR endocytosis in a cancer cell characterized by elevated expression of EGFR and elevated expression of cortactin comprising administering to a subject an amount of a Cbl polypeptide or portion thereof and/or a CD2AP polypeptide or portion thereof for a time and under conditions sufficient for EGFR endocytosis to be enhanced in said cell. As will be apparent from the description provided herein, EGFR endocytosis is preferably enhanced by the recruitment of cortactin and Cbl and
CD2AP to the EGFR. Optionally, an amount of UCS15A sufficient to reduce the formation of protein complexes that do not enhance EGFR endocytosis but not sufficient to disrupt such recruitment of cortactin and CD2AP and Cbl to the EGFR is also administered to the cell.
In an alternative but related embodiment, nucleic acid encoding a Cbl polypeptide or portion thereof and/or a CD2AP polypeptide or portion thereof is used.
In an alternative embodiment, a mimetic compound, such as, for example, an anti- idiotypic antibody that mimics a protein complex comprising CD2AP and cortactin and
Cbl and is capable of binding to the EGFR is administered. In use, the mimetic binds to EGFR to thereby enhance EGFR endocytosis. A mimetic of Cbl capable of binding to a protein complex cortactin and CD2AP is also contemplated. Alternatively, or in addition a mimetic of CD2AP capable of binding to cortactin and/or Cbl can be used.
Preferably, the active ingredient, nucleic acid or protein or antibody or small molecule is formulated in a pharmaceutically acceptable carrier, diluent or excipient known to the skilled artisan. Formulations of the active ingerdient to be administered will vary according to the route of administration selected (e.g., solution, emulsion, capsule). An appropriate composition comprising the agent to be administered can be prepared in a physiologically acceptable vehicle or carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils, for instance. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers and the like (See, generally, Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., Pa., 1985). For inhalation, the agent can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer, nebulizer or pressurized aerosol dispenser).
Furthermore, where the active ingredient is a protein or peptide, the agent can be administered via in vivo expression of the recombinant protein. In vivo expression can be accomplished via somatic cell expression according to suitable methods (see, e.g. U.S. Pat. No. 5,399,346). In this embodiment, nucleic acid encoding the protein can be incorporated into a retroviral, adenoviral or other suitable vector (preferably, a replication deficient infectious vector) for delivery, or can be introduced into a transfected or transformed host cell capable of expressing the protein for delivery. In the latter embodiment, the cells can be implanted (alone or in a barrier device), injected or otherwise introduced in an amount effective to express the protein in a therapeutically effective amount.
The present invention is further described with reference to the following examples and the accompanying drawings.
EXAMPLE 1 Identification of Proteins binding to the SH3 domain of cortactin We have established in vitro assays for the binding of the cortactin SH3 region to either CD2AP in cell lysates or peptides corresponding to the CD2AP binding sites for the cortactin SH3 domain. These systems are used to screen for peptides, low molecular weight compounds etc that inhibit the interaction, and are useful in the treatment of the above-mentioned disorders.
We have utilized a proteomics-based approach to identify binding partners for the cortactin SH3 domain from the ER-negative breast cancer cell line MDA-MB-231. This has revealed that CD2AP, ASAP1 , FISH, N-WASP and Sam68 proteins associate with cortactin in vitro and in vivo, and co-localizes with cortactin in cortical actin-containing regions. These data suggest that cortactin links molecular scaffolds assembled around CD2AP to the Arp2/3 complex and hence to dynamic actin structures.
To identify proteins interacting with the cortactin SH3 region in ER-negative breast cancer cells, we performed affinity chromatography on 35S-methionine-labelled lysates from MDA-MB-231 cells. This cell line was chosen since it lacks ERs and exhibits a motile, invasive phenotype (Bae et al, Breast Cancer Res. Treat. 24, 241-2555, 1993). Affinity purification was performed using a matrix consisting of a GST-SH3 fusion protein (Figure 1A) coupled with glutathione Sepharose. We also investigated the protein-binding capability of the cortactin HP region (Figure 1A) by the same strategy. GST was included as a negative control. Following washing, bound proteins were separated by SDS-PAGE and detected by autoradiography.
Since the Drosophilia homologues of cortactin and ZO-1 interact via the SH3 domain (Katsube et al, 1998), the activity of the GST-SH3 fusion protein was confirmed in a parallel experiment using unlabelled cell lysates, where specific binding of ZO-1 could be detected by Western blotting.
Fourteen proteins were specifically bound by the SH3 domain and not by the GST control (Figure 1B). One protein of approximately 30 kDa bound the HP region.
The procedure was scaled-up, and bound proteins were detected by Coomassie Blue staining. Following gel excision and trypsin digestion, SH3-interacting proteins, including CD2AP, ASAP1, FISH, N-WASP and Sam68, were identified by either MALDI-TOF MS or μLC-MS/MS.
The MALDI mass fingerprint for CD2AP is shown in Figure 2. Peptide fragments of CD2AP resolved using MALDI-TOF, are set forth in Table 1 (SEQ ID Nos: 26-49).
The specific binding of CD2AP to the cortactin SH3 domain was confirmed by Western blotting of pull-downs from unlabelled cell lysates using specific antibodies (Figure 3).
TABLE 1
SEQ ID NO: Peptide (with amino acid positions)
26 468 TSKETDWNFDDIASSENLLHLTANRPK 495
27 276 TLFAYEGTNEDELTFKEGEIIHLISK 301
28 471 ETDWNFDDIASSENLLHLTANRPK 495
29 317 EGVFPDNFAVQINELDKDFPKPK 339
30 231 TSSSETEEKKPEKPLILQSLGPK 253
31 373 STLEQKPSKPAAPQVPPK 390
32 85 STYGLPAGGIQPHPQTK 102
33 31 KLQEEGWLEGELNGR 45
34 32 LQEEGWLEGELNGR 45
35 59 RETEFKDDSLPIK 71
36 254 TQSVEITKTDTEGK 267
37 412 RPEKPVPPPPPIAK 425
38 558 ANTTAFLTPLEIK 570
39 73 ERHGNVASLVQR 84
40 447 LDSEQLPLRPK 457
41 46 RGMFPDNFVK 55
42 292 EGEIIHLISK 301
43 154 LGLFPSNFVK 163
44 302 ETGEAGW R 310
45 75 HGNVASLVQR 84
46 47 GMFPDNFVK 55
47 231 TSSSETEEK 239
48 213 GIGFGDIFK 221 Example 2 Association of CD2AP with Cortactin in vivo Since both CD2AP and cortactin have been identified as substrates of c-Src family kinases with roles in organization of the cytoskeleton (Kirsch et al, 1999, Weed and Parsons, 2001), this suggested that the binding of the cortactin SH3 domain to CD2AP in vitro might reflect a functional relationship between the two proteins. To investigate their association in living cells, cortactin immune precipitates from MDA-MB-231 breast cancer cells were treated with a CD2AP-specific antibody and cortactin-specific antibody (Figure 4A). The reciprocal analysis using CD2AP immune precipitates (Figure 4B), was also performed. Results showed co-immunoprecipitation of the two proteins, indicating that these proteins associate at physiological expression levels.
The requirement for the cortactin SH3 domain in CD2AP association was investigated by expressing a truncation mutant of CD2AP lacking the SH3 domain (ΔSH3) in HEK 293 cells. Cells were also transfected with an expression vector for full-length cortactin as a control. The expressed cortactin, but not the ΔSH3 mutant, could readily be detected in CD2AP immunoprecipitates (Figure 4C), indicating that the cortactin SH3 domain is essential for interaction of these proteins in vivo.
EXAMPLE 3
Subcellular localization of cortactin and CD2AP In order to further investigate the functional role of cortactin/CD2AP complexes, the subcellular distribution of the two proteins was investigated by staining MDA-MB-231 cells with cortactin- or CD2AP- specific antibodies, separately or in combination. Analysis by confocal microscopy revealed co-incident staining at regions of the cell cortex, within a subset of cytoplasmic dots (possibly representing small vesicles), and at cell-cell contacts (Figure 5). These findings support the association of cortactin and CD2AP detected by co-immunoprecipitation analysis. Our data suggest a role for the protein interaction between cortactin and CD2AP in the regulation of dynamic cortical actin structures, vesicle trafficking and protein complexes involved in cell-cell adhesion. EXAMPLE 4
Mapping of the binding site for the cortactin SH3 domain on CD2AP
CD2AP contains three proline-rich regions harbouring potential SH3-binding sites
(Figures 6A and 6B; Kirsch et al, 1999). To determine whether the cortactin SH3 binds directly to CD2AP, a Western blot immunoassay was performed (Figure 6C).
The GST-SH3 fusion protein, and GST as a negative control, were labeled with biotin and used to blot CD2AP immunoprecipitates that had been resolved by SDS-PAGE and transferred to a PVDF membrane. Detection of bound biotin-labeled protein revealed that the cortactin SH3 region directly binds to CD2AP. No binding of GST was detected.
The optimal amino acid sequence of the PRR for CD2AP binding via the cortactin SH3 domain has been characterized (Sparks et al, Proc. Natl. Acad. Sci. USA 93, 1540- 1544, 1996). Examination of the CD2AP amino acid sequence did not reveal an exact match with this sequence. To determine the binding site in CD2AP for the cortactin SH3 domain, each of three proline rich regions of CD2AP (P1-P3 in Figure 6B; SEQ ID Nos: 21-24) were expressed as GST fusion proteins. A fusion protein corresponding to a deduced optimum sequence for binding to the cortactin SH3 domain (Popt in Figure 6B; SEQ ID NO: 25) was also generated as a positive control. These fusion proteins, and GST as a negative control, were subjected to a Western assay with biotin-labeled GST-SH3 or GST alone. Strong binding of the SH3 domain to Popt was detected, but significant binding to P1 (SEQ ID NO: 22) and P2 (SEQ ID NO: 23) also occurred (Figure 6C). Binding to P3 (SEQ ID NO: 24) was undetectable.
Analysis of these proline-rich regions reveals that both P1 and P2 contain a sequence similar to the deduced optimum sequence for binding to the cortactin SH3 domain, KPPPPAKA and KPTPPTK respectively (Figure 6B). Although P3 contains the sequence PKRPEKP, it is likely that the basic residues within the PXXP core, and the lack of a basic residue prior to it, prevent binding.
Consequently CD2AP contains two recruitment sites for the cortactin SH3 domain, wherein the relative close proximity of these recruitment sites enables them to function co-operatively in recruitment of cortactin to CD2AP. EXAMPLE 5 EGF-inducible association between cortactin and CD2AP To investigate the in vivo association of cortactin and CD2AP, a co- immunoprecipitation analysis was performed from lysates of serum-starved or EGF- treated MDA-MB-231 cells. Western blotting of cortactin immunoprecipitates with a CD2AP-specific antibody, and the reciprocal analysis, revealed EGF-inducible co- immunopreciptation of the two proteins, revealing that they associate at physiological levels in response to EGF (Figure 7A).
The requirement for the cortactin SH3 domain in its EGF-induced association with CD2AP was investigated by expressing a truncation mutant of cortactin lacking the SH3 domain (ΔSH3) in HEK293 cells. Cells were also transfected with an empty expression vector (control), or an expression vector comprising nucleic acid encoding a full-length cortactin polypeptide, and incubated in serum-free media (SF) or in the presence of EGF (EGF). Whole cell lysates (WCL) and CD2AP immunoprecipitates (IP CD2AP) were analysed by western blotting using antibodies against cortactin, and CD2AP. Data in Figure 7B indicate EGF-inducible co-immunoprecipitation of cortactin and CD2AP is enhanced by EGF-stimulation of HEK 293 cells and requires the SH3 domain of cortactin. As with the association of the endogenous proteins, co- immunoprecipitation was increased by short-term EGF treatment.
EXAMPLE 6
Binding of CD2AP to endophilin and Cbl To investigate whether or not CD2AP binds to endophilin or Cbl, and participate in
EGFR endocytosis, the different protein-protein interactions were investigated following EGF stimulation of MDA-MB-231 cells in the absence of EGF stimulation, and at various time points after EGF treatment. CD2AP immunoprecipitates were analysed by blotting with antibodies that are specific for cortactin, Cbl, endophilins I, II, and III, and for phosphotyrosine (UBI; Santa Cruz Biotechnology, Inc).
As shown in Figure 8A, western blotting with antibodies specific for cortactin reveals a low basal level of cortactin-CD2AP complex in serum-starved cells that is transiently enhanced following EGF-stimulation, reaching a peak at 5 min and returning to the basal level at 30 min after EGF stimulation. Association of Cbl with CD2AP is rapidly induced by EGF stimulation, reaching a peak at 1-5 min post EGF treatment, however decreases between 5-15 min and is not detectable after about 15 min. Association of endophilin with CD2AP appears to be constitutive and not induced by EGF stimulation.
To investigate whether or not CD2AP association with Cbl is dependent on Cbl tyrosine phosphorylation, we probed western blots with anti-phosphotyrosine antibodies (Figure 8B). Such western blotting revealed two proteins of about 120 kDa and 130 kDa, the mobility of the former being consistent with the mobility of Cbl under these conditions. A co-precipitating tyrosine phosphorylated protein of about 180 kDa in size exhibited similar recruitment kinetics to Cbl and was identified as EGFR using specific antibodies (Figure 8B). Another tyrosine phosphorylated protein of about 80 kDa in size, detected at 2 min and 5 min post-EGF treatment, is likely to be cortactin (Figure 8B). Accordingly, available data are consistent with EGF-induced formation of a Cbl-CD2AP-endophilin complex that regulates the endocytosis of EGFR, and also demonstrates a novel role for cortactin and hence the formation of dynamic actin networks in this process.
Depending on the cell type, CD2AP has been localized to lamellipodia and punctate cytoplasmic structures (Kirsch et al, Proc. Natl Acad. Sci. USA 96, 6211-6216, 1999; Welsch et al, Am. J. Physiol. Renal Physiol. 4, 769-777, 2001). The EGF-inducible association between cortactin and CD2AP and the appearance of the EGFR in the complex suggested that the proteins might co-localize. The inventors therefore investigated the subcellular localization of cortactin, CD2AP and EGFR by confocal microscopy. HeLa cells were transiently transfected with an expression construct comprising nucleic acid encoding a fusion protein consisting of EGFR and green fluorescent protein (GFP). Cells were fixed either before EGF stimulation (time 0) or at 5 mins (5') or 15 mins (15') after EGF-stimulation, and analysed by indirect immunofluorescence using antibodies against cortactin or CD2AP. EGFR was localized by fluorescence of the GFP moiety in the fusion protein. Data in Figures 9A and 9B indicate localization of EGFR primarily in the plasma membrane, with cortactin and CD2AP primarily in punctate cytoplasmic structures, prior to EGF stimulation. EGFR, cortactin and CD2AP are found in the membrane ruffles and a small subset of punctate cytoplasmic spots within 5 mins after EGF- stimulation. After 5 min EGF stimulation, the EGFR concentrates into prominent vesicular structures within the cytosol, presumably early endosomes. The co- localization of EGFR with CD2AP and cortactin is transient and returns to basal levels within 15 mins after EGF stimulation. These data are consistent with the formation of an EGF-induced complex containing both CD2AP and cortactin at membrane ruffles that regulates an early event in EGF receptor endocytosis.
Since the association between cortactin and CD2AP requires the cortactin SH3 domain, the effect of expressing a truncation mutant of cortactin lacking the SH3 domain on the formation of the Cbl-CD2AP-endophilin complex was investigated. HeLa cells were transfected with an "empty" expression vector (control), or an expression vector comprising nucleic acid encoding a full-length cortactin polypeptide (cortactin) or encoding a truncated cortactin polypeptide lacking the SH3 domain (ΔSH3), and incubated in serum-free media (time 0) or in the presence of EGF (EGF) for 5 min or 15 min. CD2AP immunoprecipitates were analysed by western blotting using antibodies against CD2AP, endophilin, Cbl, and cortactin.
Data in Figure 10 indicate that over expression of full-length cortactin increased the basal association between cortactin and CD2AP and leads to enhanced and prolonged association between CD2AP and cortactin following EGF-stimulation, consistent with a positive regulation of the receptor by cortactin. Over expression of truncated cortactin blocked recruitment of endogenous native cortactin into the complex following EGF stimulation of cells, as evidenced by the reduced basal level of cortactin in immunoprecipitates of cells over expressing the mutant protein relative to control cells at 5 min post-EGF treatment. These data suggest that the ΔSH3 mutant may act as a dominant negative mutant with respect to endogenous cortactin, in EGF- induced cells. EXAMPLE 7 The cortactin/CD2AP complex is implicated in regulation of EGFR endocytosis To investigate whether or not a CD2AP-containing protein complex was involved in EGF receptor-mediated endocytosis, we investigated the kinetics of formation of the various CD2AP-comprising protein complexes following EGF stimulation of MDA-MB- 231 cells. MDA-MB-231 cells were serum-starved and then treated with EGF for varying times up to 60 min. The CD2AP-containing protein complexes were isolated by immune precipitation using antibodies against CD2AP, and subjected to Western blotting. A negative control consisting of immune precipitates from serum-starved cells was also isolated by immune precipitation using antibodies against CD2AP, and subjected to Western blotting. Western blots were probed with antibodies against phosphotyrosine, EGFR, Cbl, endophilin, cortactin and CD2AP. The antiphosphotyrosine antibodies were used to investigate the tyrosine phosphorylation status of proteins that immune precipitate with CD2AP, since the association of CD2AP with Cbl is dependent on tyrosine phosphorylation of the Cbl protein. Data are presented in Figure 11.
In Western blots, anti-phosphotyrosine antibodies identified three proteins in CD2AP immune precipitates having molecular masses of approximately 180 kDa, 130 kDa and 120 kDa. A tight doublet of approximately 50 kDa was also detected. These tyrosine- phosphorylated proteins were detected between 1 min and 5 min following EGF stimulation (Figure 11). A tyrosine phosphorylated protein of approximately 80 kDa was also detected between 2 min and 5 min following EGF stimulation of cells. The 180 kDa and 120 kDa bands were shown to correspond to EGFR and Cbl, respectively, as confirmed using antibodies against those proteins in Western blots (Figure 11). Accordingly, Cbl protein in the CD2AP protein complexes was tyrosine phosphorylated.
Western blotting also showed that the association between CD2AP and endophilin was constitutive and unaffected by EGF-stimulation (Figure 11 , lanes marked "endophilin"). Accordingly, the data presented in Figure 11 indicate that CD2AP associates with both receptor-bound Cbl and endophilin, and that the interaction between CD2AP and Cbl is dependent on tyrosine phosphorylation of Cbl whereas the interaction between CD2AP and endophilin is not.
Western blotting of CD2AP immune precipitates using an antibody against cortactin detected a low basal level of association in serum-starved cells. Following EGF stimulation, there was a marked but transient increase in the recruitment of cortactin into CD2AP -containing protein complexes that peaked at 5 min and returned to a low/basal level about 30 min following EGF-stimulation of cells (Figure 11). Comparison of recruitment kinetics and gel mobility suggests that the 80 kDa tyrosine phosphorylated protein detected in CD2AP immune precipitates at 2 min and 5 min following EGF treatment is likely to be cortactin. These data are consistent with recruitment of cortactin to the endocytic complex, but association of cortactin with CD2AP is slightly delayed compared to the association of Cbl and the EGFR with CD2AP.
An alternative explanation for the data presented in Figure 11 is that two independent CD2AP-containing protein complexes may exist in cells, ie., a protein complex consisting of CD2AP and cortactin, and a protein complex consisting of CD2AP and Cbl and EGFR. To investigate this possibility, the presence of Cbl and EGFR in cortactin-containing protein complexes was investigated. HeLa cells were transiently transfected with either an empty vector control, or alternatively, a cortactin-encoding expression vector. HeLa cells containing the empty vector control or expressing recombinant cortactin protein were either serum-starved and left untreated or stimulated with EGF under standard conditions for 2 min. The cortactin-containing protein complexes were isolated by immune precipitation using antibodies against cortactin, and subjected to Western blotting. Western blots were probed with antibodies against phosphotyrosine, Cbl and cortactin. Data are presented in Figure 12.
Western blotting of cortactin immune precipitates with an antiphosphotyrosine antibody revealed an EGF-inducible protein having an approximate molecular mass of 120 kDa, which was subsequently identified as Cbl (Figure 12). A second EGF-induced protein having a molecular mass of about 180 kDa was also detected in cells that over expressed cortactin, which is likely to correspond to EGFR. Accordingly, data presented in Figure 12 indicate that Cbl is indeed recruited to cortactin following EGF stimulation of cells that express cortactin. Together with the data presented in Figure 4 showing that CD2AP and cortactin interact, these data confirm that cortactin is indeed recruited to a CD2AP/Cbl/EGFR complex following EGF stimulation of cells.
EXAMPLE 8
Isolation of peptide modulators using phage display
1. Construction of the Random and Peptide Phage Libraries
Peptide libraries are constructed in the fUSE5 gene III phage display system (Scott and Smith, Science 249, 386-390, 1990), and employing Escherichia coli host strains K91 Kan and MC1061 F as described by Pero et al, J. Biol. Chem 277, 11918-11926, 2002. Pools of transformants are plated on Pyrex dishes containing 2 YT/tetracycline/streptomycin medium (1 ml per dish). The dishes are incubated at 37°C overnight to allow and produce thousands of copies of each peptide phage particle library member.
2. Expression of SH3 domains and PRRs
To produce plasmids encoding glutathione S-transferase (GST) SH3 domain fusion proteins, nucleic acid encoding the SH3 domain of cortactin, or the SH3 domain of endophilin or the SH3 domain of CD2AP is inserted in the same reading frame as nucleic acid encoding GST present in plasmids essentially described by Janes et al, J. Biol. Chem. 272, 8490-8497, 1997 and Lowenstein et al, Cell 70, 431-442, 1992. Thus, these proteins are expressed in a GST fusion bacterial expression system using the pGEX-2T expression vector (Amersham Biosciences, Inc.) and purified from isopropyl-β-D-thiogalactopyranoside-induced bacterial cultures as described previously (Smith and Johnson Gene (Amst.) 67, 31-40, 1988). Purity is determined by SDS-PAGE. Protein concentration is determined by Bradford based protein assay (Bio-Rad).
Similarly, to produce plasmids encoding glutathione S-transferase (GST) PRR domain fusion proteins, nucleic acid encoding a PRR of CD2AP or a PRR of Cbl or a PRR of endophilin is inserted in the same reading frame as nucleic acid encoding GST present in plasmids essentially described by Janes et al, J. Biol. Chem. 272, 8490- 8497, 1997 and Lowenstein et al, Cell 70, 431-442, 1992. As with the SH3-GST fusions, these PRR-GST fusion proteins are expressed in bacteria using the pGEX-2T expression vector (Amersham Biosciences, Inc.) and purified from isopropyl-β-D- thiogalactopyranoside-induced bacterial cultures as described previously (Smith and Johnson Gene (Amst.) 67, 31-40, 1988). Purity is determined by SDS-PAGE. Protein concentration is determined by Bradford basedprotein assay (Bio-Rad).
3. Peptide Phage Library Screening
The peptide libraries are amplified so that there are about 1000 transforming units of each peptide phage representing 200 library equivalents. The amplified phage are infected into K91/Kan cells, plated onto five 2YT agar dishes containing about 40 μg/ml tetracycline, incubated overnight at 30 °C, and harvested in 1x phosphate- buffered saline with a prokaryotic protease inhibitor mixture (1:1000)(Sigma) by sweeping the bacteria off the agar with a bent glass rod. The bacterial cell suspension is centrifuged at about 8000 rpm for 10 min at 4°C, and the supernatant is filtered through a 0.22μm polyethersulfone (PES) filter. The filtrate is incubated for about 30 min on ice with 0.15 volume of polyethylene glycol/NaCI to precipitate the phage and centrifuged at 9000 rpm, 20 min, 4 °C. The phage pellet is resuspended in 1x phosphate-buffered saline with a prokaryotic protease inhibitor mixture.
GST-binding peptide phage are subtracted from the library by preincubation of the library with 2 μg of GST immobilized on 20 μl of glutathione-Sepharose (Amersham Biosciences, Inc.).
The phage are screened by adding them to 12 μg of purified SH3-GST or PRR-GST fusion protein immobilized onto 20 μl glutathione-Sepharose and incubated for about 2 hr at 25 °C. The unbound phage are removed by washing five times with 1x phosphate-buffered saline comprising a prokaryotic protease inhibitor mixture. Phage that bind are eluted twice using a low pH buffer, such as pH 2.3 buffer, followed by two elutions using a high pH (eg., pH12) buffer.
K91/Kan cells are then infected with the pooled phage elutions, and a small aliquot is plated to determine the total amount of phage particles bound to the target GST fusion protein. The remaining phage are amplified overnight, harvested, screened, and eluted as described above.
To determine the amount of enrichment after the second and third rounds of panning, half of the pre-cleared phage are added to fresh GST immobilized on glutathione beads to determine the amount of phage binding GST-glutathione-Sepharose compared to the binding of the phage to the GST fusion protein.
Preferably, three rounds of screening are carried out before clones are sequenced or tested in an ELISA.
4. DNA and Amino Acid Sequence Determination of Harvested Phage
Isolated peptide phage-infected E. coli colonies are grown in 5 ml of LB/tetracycline broth. The double-stranded phage replicative form DNA is isolated using the QIAprep Spin Miniprep kit (Qiagen). Sequencing primers for sequencing a random insert in the fUSE5 vector is described by Scott and Smith, Science 249, 386-390, 1990). Sequencing reactions are carried out using BigDye Version 1 Dye Terminator kit (PerkinElmer Life Sciences). The amino acid sequences of peptides displayed by peptide phage are deduced from the DNA sequence of the corresponding phage clones. Consensus sequence identification is performed by visual inspection and with the ClustalW alignment program.
5. Production of free synthetic peptides
Based upon the amino acid sequences of phage clones, synthetic peptides are produced using standard procedures for testing the ability of the peptides to modulate an interaction between the SH3 domain of cortactin, or the SH3 domain of endophilin or the SH3 domain of CD2AP, and a PRR of CD2AP or a PRR of Cbl or a PRR of endophilin.
6. Reverse Phage ELISA
A reverse phage ELISA (Valadon and Scharff, J. Immunol. Methods 197, 171-179, 1996; and Zwick et al, Curr. Opin. Biotechnol. 9, 427-436, 1998) is used to evaluate the ability of individual phage clones to bind to the SH3 domain or PRR present in the GST fusion protein. Briefly, protein targets at a concentration of about 5 μg/ml are added to a 96-well Maxisorp plate (Nunc) precoated with anti-GST polyclonal antibody at a concentration of about 1 μg/ml final concentration) (Amersham Biosciences, Inc.) and blocked using casein in Tris-buffered saline (Pierce). The peptide phage are concentrated by precipitation using polyethylene glycol/NaCI, added to each well (1 x 107 to about 1 x 108 phage/well), and incubated for 2 h at room temperature. Unbound phage are removed with 0.1 % Tween-Tris-buffered saline buffer, and phage are detected with horseradish peroxidase-conjugated anti-M13 monoclonal antibody (1 :1000) (Amersham Biosciences, Inc.) and 2,2'-azino-jb/s(3-ethylbenzothiazoline-6- sulfonic acid) (ABTS) substrate (Calbiochem). Plates are read at 405nm.
Alternatively, a competitive reverse phage ELISA is used, essentially as described by Barrett et al, Anal. Biochem. 204, 357-364, 1992 or Carcamo et al, Proc. Natl. Acad. Sci. )U. S. A.) 95, 11146-11151, 1998. In a competitive reverse phage ELISA, purified GST fusion protein is added to a plate precoated with anti-GST polyclonal antibody and blocked with casein in Tris-buffered saline. The peptide phage are concentrated by polyethylene glycol/NaCI precipitation, The synthetic peptides are mixed at different concentrations (1, 10, and 100 μM) with peptide phage (1 x 106 transforming units/well) before adding to the GST fusion protein bound to the 96-well plate. Bound phage are detected as described above. Percent inhibition of binding achieved by the synthetic free peptides is calculated.
7. Assaying for modulatory activity of peptides
The relative ability of the free synthetic SH3-binding peptides or PRR-binding peptides to inhibit the formation of a protein complex described herein, particularly a complex comprising cortactin and CD2AP is determined by measuring the level of immunoprecipitated complex in the presence and absence of a synthetic free peptide comprising the SH3-binding or PRR-binding sequence.
Briefly, whole cell extracts are made from MDA-MB-231 cells. Alternatively, HeLa cells, optionally transfected with cortactin-encoding nucleic acid, are grown until 70- 80% confluence in T-75 or T-150 flasks. After overnight starvation in serum-free media, the cells are stimulated with EGF for 2-5 min at 37 °C. After stimulation, the cells are washed twice with phosphate-buffered saline, lysed with ice-cold lysis buffer (1% Triton X-100, 50 mM Hepes, 50 mM NaCl, 10% glycerol, 1.5mM MgCI2, 1 mM EGTA, 10 mM sodium pyrophosphate, 20 mM NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate) for 10 min on ice, and collected using a cell scraper. The insoluble debris is separated by microfuging lysates for 5 min at 4 °C and 13,000 rpm ,and the supernatant is collected and frozen at -80 °C. Protein concentration of the lysate is determined by a Bradford- based protein assay (Bio-Rad). The EGF-stimulated cell lysate (0.5-1 mg) is incubated with 20 μl of protein A-Sepharose to preclear the lysates. The precleared lysate is incubated with increasing amounts of free synthetic peptide or no peptide (control) for 1 h at 4 °C and then immunoprecipitated with an amount (eg., 2 μg) of a polyclonal or monoclonal antibody against CD2AP or cortactin (UBI; Santa Cruz Biotechnology) overnight at 4°C. The immune precipitated protein complexes are collected by incubation with 50 μl of protein A-Sepharose 4B beads (Zymed Laboratories Inc.) for 1 h at 4 °C. The immune complexes are collected by centrifugation, washed five times in cold lysis buffer, and eluted with sample buffer at 100 °C for 5 min. The immune complexes are then subjected to Western blot analysis on a 7.5% SDS-polyacrylamide gel using one or more of the following primary antibodies: horseradish peroxidase-conjugated anti-phosphotyrosine recombinant antibody (BD Transduction Laboratories); anti-cortactin polyclonal antibody (Santa Cruz Biotechnology) or anti-cortactin monoclonal antibody (UBI); anti-CD2AP polyclonal antibody (Santa Cruz Biotechnology); anti-Cbl polyclonal antibody (Santa Cruz Biotechnology); anti-endophilin polyclonal antibody (Santa Cruz Biotechnology); and anti-EGFR polyclonal antibody (Santa Cruz Biotechnology);. All protein bands are detected using an ECL chemiluminescence kit (Amersham Biosciences, Inc.). Densitometric analyses of the autoradiographs are performed using the Fluor-S Multilmager with Quantity One 4.2.1 software (Bio-Rad).
Those peptides that modify the number of proteins present in a cortactin immune precipitate or CD2AP immune precipitate, such as, for example, by preventing the association/recruitment of endophilin or Cbl to cortactin and/or CD2AP, or that prevent the recruitment of a complex comprising cortactin and Cbl and CD2AP to the EGFR are retained. Similarly, those peptides that modify the amount of a protein that is recruited to a cortactin immune precipitate or CD2AP immune precipitate, such as, for example, by enhancing the level of recruitment of Cbl to cortactin and/or CD2AP, or that enhances the recruitment of a complex comprising cortactin and Cbl and CD2AP to the EGFR are retained.

Claims

WE CLAIM:
1. An isolated or recombinant protein complex comprising:
(iii) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1 , FISH and Sam68; and
(b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1 , FISH and Sam68 or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and (iv) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
(b) a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide.
2. The isolated or recombinant protein complex of claim 1 comprising:
(i) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide; and (ii) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a cortactin polypeptide or a portion of a cortactin polypeptide.
3. The isolated or recombinant protein complex of claim 1 comprising:
(i) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a Cbl polypeptide; and
(ii) a Cbl polypeptide or a portion of a Cbl polypeptide sufficient to bind to a CD2AP polypeptide or a portion of a CD2AP polypeptide.
4. The isolated or recombinant protein complex of claim 2 further comprising a Cbl polypeptide.
5. The isolated or recombinant protein complex of claim 2 or 4 further comprising an endophilin polypeptide.
6. The isolated or recombinant protein complex of claim 4 comprising:
(i) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide; (ii) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide; and (b) a Cbl polypeptide or a portion of a Cbl polypeptide; and (iii) a Cbl polypeptide or a portion of a Cbl polypeptide sufficient to bind to a
CD2AP polypeptide or a portion of a CD2AP polypeptide.
7. The isolated or recombinant protein complex of claim 5 comprising:
(i) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide; (ii) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide; and (b) an endophilin polypeptide or a portion of an endophilin polypeptide; and (iii) an endophilin polypeptide or a portion of an endophilin polypeptide sufficient to bind to a CD2AP polypeptide or a portion of a CD2AP polypeptide.
8. The isolated or recombinant protein complex according to any one of claims 4 to 7 comprising:
(i) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide;
(ii) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide; and (b) an endophilin polypeptide or a portion of an endophilin polypeptide; (iii) an endophilin polypeptide or a portion of an endophilin polypeptide sufficient to bind to (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide; and (b) a Cbl polypeptide or a portion of a Cbl polypeptide; and (iv) a Cbl polypeptide or a portion of a Cbl polypeptide sufficient to bind to an endophilin polypeptide or a portion of an endophilin polypeptide.
9. The isolated or recombinant protein complex according to any one of claims 4 to 7 comprising:
(i) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide; (ii) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide, (b) an endophilin polypeptide or a portion of an endophilin polypeptide, and (c) a Cbl polypeptide or a portion of a Cbl polypeptide; (iii) an endophilin polypeptide or a portion of an endophilin polypeptide sufficient to bind to (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide and to (b) a Cbl polypeptide or a portion of a Cbl polypeptide; and
(iv) a Cbl polypeptide or a portion of a Cbl polypeptide sufficient to bind to
(a) a CD2AP polypeptide or a portion of a CD2AP polypeptide, and (b) an endophilin polypeptide or a portion of an endophilin polypeptide.
10. The isolated or recombinant protein complex according to any one of claims 4 to 7 comprising:
(i) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide;
(ii) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide,
(b) an endophilin polypeptide or a portion of an endophilin polypeptide, and (c) a Cbl polypeptide or a portion of a Cbl polypeptide;
(iii) an endophilin polypeptide or a portion of an endophilin polypeptide sufficient to bind to a CD2AP polypeptide or a portion of a CD2AP polypeptide; and
(iv) a Cbl polypeptide or a portion of a Cbl polypeptide sufficient to bind to a CD2AP polypeptide or a portion of a CD2AP polypeptide.
11. The isolated or recombinant protein complex according to any one of claims 4 to 7 comprising:
(i) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a CD2AP polypeptide; (ii) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to (a) a cortactin polypeptide or a portion of a cortactin polypeptide, and (b) a Cbl polypeptide or a portion of a Cbl polypeptide; (iii) a Cbl polypeptide or a portion of a Cbl polypeptide sufficient to bind to
(a) a CD2AP polypeptide or a portion of a CD2AP polypeptide; and (b) an endophilin polypeptide or a portion of an endophilin polypeptide; and
(iv) an endophilin polypeptide or a portion of an endophilin polypeptide sufficient to bind to a Cbl polypeptide or a portion of a Cbl polypeptide.
12. An isolated or recombinant protein complex selected from the group consisting of: (i) a complex comprising CD2AP and cortactin; (ii) a complex comprising
CD2AP and Cbl; (iii) a complex comprising CD2AP and endophilin; (iv) a complex comprising CD2AP and endophilin and cortactin; (v) a complex comprising CD2AP and Cbl and cortactin; (vi) a complex comprising CD2AP and endophilin and Cbl; (vii) a complex comprising CD2AP and endophilin and Cbl and cortactin; (viii) a complex comprising cortactin and ASAP1; (ix) a complex comprising cortactin and N-WASP; (x) a complex comprising cortactin and FISH; and (xi) a complex comprising cortactin and Sam68.
13. The isolated or recombinant protein complex according to any one of claims 1 to 12 wherein the CD2AP polypeptide comprises an amino acid sequence having at least about 80% identity to SEQ ID NO: 1 or SEQ ID NO: 2.
14. The isolated or recombinant protein complex according to any one of claims 1 to 12 wherein the cortactin polypeptide comprises an amino acid sequence having at least about 80% identity to SEQ ID NO: 3 or SEQ ID NO: 4.
15. The isolated or recombinant protein complex according to any one of claims 1 to 12 wherein the Cbl polypeptide comprises an amino acid sequence having at least about 80% identity to SEQ ID NO: 5 or SEQ ID NO: 6.
16. The isolated or recombinant protein complex according to any one of claims 1 to 12 wherein the endophilin polypeptide comprises an amino acid sequence having at least about 80% identity "to SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9.
17. The isolated or recombinant protein complex according to any one of claims 1 to 12 wherein the ASAP1 polypeptide comprises an amino acid sequence having at least about 80% identity to SEQ ID NO: 10 or SEQ ID NO: 11.
18. The isolated or recombinant protein complex according to any one of claims 1 to 12 wherein the N-WASP polypeptide comprises an amino acid sequence having at least about 80% identity to SEQ ID NO: 12 or SEQ ID NO: 13.
19. The isolated or recombinant protein complex according to any one of claims 1 to 12 wherein the FISH polypeptide comprises an amino acid sequence having at least about 80% identity to SEQ ID NO: 14 or SEQ ID NO: 15.
20. The isolated or recombinant protein complex according to any one of claims 1 to 12 wherein the sam68 polypeptide comprises an amino acid sequence having at least about 80% identity to SEQ ID NO: 16 or SEQ ID NO: 17.
21. The isolated or recombinant protein complex according to any one of claims 1 to 12 wherein one or more polypeptides of the protein complex are mammalian polypeptides.
22. The isolated or recombinant protein complex according to any one of claims 1 to 12 wherein one or more polypeptides of the protein complex are human polypeptides.
23. The isolated or recombinant protein complex according to any one of claims 1 to 12 wherein one or more polypeptides of the protein complex are murine polypeptides.
24. The isolated or recombinant protein complex according to any one of claims 1 to 12 wherein one or more polypeptides of the protein complex are rat polypeptides.
25. The isolated or recombinant protein complex according to any one of claims 1 to 24 wherein the portion of a cortactin polypeptide comprises an SH3 domain of a cortactin polypeptide.
26. The isolated or recombinant protein complex according to claim 25 wherein the SH3 domain comprises an amino acid sequence having about 80% identity to SEQ ID NO: 18 or SEQ ID NO: 19.
27. The isolated or recombinant protein complex according to any one of claims 1 to 24 wherein the portion of an endophilin polypeptide comprises an SH3 domain of an endophilin polypeptide.
28. The isolated or recombinant protein complex according to claim 27 wherein the SH3 domain comprises an amino acid sequence having about 80% identity to SEQ ID NO: 20 or SEQ ID NO: 21.
29. The isolated or recombinant protein complex according to claim 27 wherein the
SH3 domain comprises an amino acid sequence having about 80% identity to a sequence selected from the group consisting of:
(i) a sequence from about position 327 to about position 340 of SEQ ID NO: 7;
(ii) a sequence from about position 328 to about position 341 of SEQ ID NO: 8; and
(iii) a sequence from about position 322 to about position 335 of SEQ ID NO: 9.
30. The isolated or recombinant protein complex according to any one of claims 1 to 24 wherein the portion of a CD2AP polypeptide comprises an SH3 domain of a CD2AP polypeptide.
31. The isolated or recombinant protein complex according to claim 30 wherein the SH3 domain comprises an amino acid sequence having about 80% identity to a sequence selected from the group consisting of:
(i) a sequence from about position 97 to about position 111 of SEQ ID NO: 1 ; (ii) a sequence from about position 144 to about position 157 of SEQ ID NO: 2; (iii) a sequence from about position 261 to about position 274 of SEQ ID NO: 1 ; and (ii) a sequence from about position 306 to about position 319 of SEQ ID NO: 2.
32. The isolated or recombinant protein complex according to any one of claims 1 to 24 wherein the portion of an ASAP1 polypeptide comprises an SH3 domain of an ASAPI polypeptide.
33. The isolated or recombinant protein complex according to claim 32 wherein the SH3 domain comprises an amino acid sequence having about 80% identity to a sequence from about position 1121 to about position 1138 of SEQ ID NO: 10.
34. The isolated or recombinant protein complex according to any one of claims 1 to 24 wherein the portion of a CD2AP protein comprises a proline rich region (PRR) of a CD2AP polypeptide.
35. The isolated or recombinant protein complex of claim 34 wherein the PRR comprises an amino acid sequence having at least about 80% identity to a sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 25.
36. The isolated or recombinant protein complex according to any one of claims 1 to 24 wherein the portion of a Cbl protein comprises a proline rich region (PRR) of a Cbl polypeptide.
37. The isolated or recombinant protein complex of claim 37 wherein the PRR of a Cbl polypeptide comprises an amino acid sequence having at least about 80% identity to the sequence of a protein region selected from the group consisting of:
(i) amino acid residues 494-500 of SEQ ID NO: 5; (ii) amino acid residues 531-537 of SEQ ID NO: 5; (iii) amino acid residues 540-551 of SEQ ID NO: 5; (iv) amino acid residues 531-551 of SEQ ID NO: 5; (v) amino acid residues 512-518 of SEQ ID NO: 6; and
(vi) amino acid residues 512-532 of SEQ ID NO: 6.
38. The isolated or recombinant protein complex according to any one of claims 1 to 37 further comprising an epidermal growth factor receptor (EGFR) polypeptide.
39. Use of an isolated or synthetic peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ
ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO:24 in the preparation of a reagent for producing the protein complex according to any one of claims 1 to 38.
40. Use of an isolated or synthetic peptide in the preparation of a reagent for producing the protein complex according to any one of claims 1 to 38, said peptide comprising an amino acid sequence selected from the group consisting of: (i) a sequence from about position 327 to about position 340 of SEQ ID NO: 7;
(ii) a sequence from about position 328 to about position 341 of SEQ ID NO: 8; (iii) a sequence from about position 322 to about position 335 of SEQ ID NO: 9; (iv) a sequence from about position 97 to about position 111 of SEQ ID NO: 1 ; (ii) a sequence from about position 144 to about position 157 of SEQ ID NO: 2; (v) a sequence from about position 261 to about position 274 of SEQ ID NO: 1 ; (vi) a sequence from about position 306 to about position 319 of SEQ ID NO: 2; (vii)a sequence from about position 494 to about position 500 of SEQ ID NO: 5; (viii) a sequence from about position 531 to about position 537 of SEQ ID NO:
5;
(ix) a sequence from about position 540 to about position 551 of SEQ ID NO: 5; (x) a sequence from about position 531 to about position 551 of SEQ ID NO: 5; (xi) a sequence from about position 512 to about position 518 of SEQ ID NO: 6; and
(xii) a sequence from about position 512 to about position 532 of SEQ ID NO: 6.
41. A kit for producing or detecting a protein complex said kit comprising a first polypeptide consisting of cortactin or a portion thereof and a second peptide or polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH, Sam68, a portion of CD2AP, a portion of N-WASP, a portion of ASAP1 , a portion of FISH and a portion of Sam68, wherein said portion of said second polypeptide is sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide.
42. The kit of claim 41 further comprising an antibody or ligand that binds to the first or second polypeptide or to the complex formed between said first and said second polypeptide.
43. A kit for producing or detecting a protein complex said kit comprising a first compartment comprising cortactin or a portion thereof and a second compartment comprising an antibody or ligand that binds to a peptide or polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH, Sam68, a portion of CD2AP, a portion of N-WASP, a portion of ASAP1, a portion of FISH and a portion of Samδδ, wherein said portion of said second polypeptide is sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide, or an antibody that binds to a complex formed between the content of the first compartment and the content of the second compartment.
44. A kit for producing or detecting a protein complex said kit comprising a first compartment comprising a polypeptide selected from the group consisting of
CD2AP, N-WASP, ASAP1, FISH, Sam68, a portion of CD2AP, a portion of N- WASP, a portion of ASAP1 , a portion of FISH and a portion of Samδδ, wherein said portion of said second polypeptide is sufficient to bind to said cortactin polypeptide or said portion .of a cortactin polypeptide and a second compartment comprising an antibody or ligand that binds to a polypeptide selected from the group consisting of cortactin, CD2AP, N-WASP, ASAP1, FISH and Samδδ, or an antibody that binds to a complex formed between cortactin and a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1, FISH and Sam68.
45. A kit for producing or detecting a protein complex said kit comprising a first polypeptide consisting of CD2AP or a portion thereof sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin, and a second polypeptide consisting of a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion thereof sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide.
46. The kit of claim 45 further comprising an antibody or ligand that binds to the first polypeptide or the second polypeptide, or to a protein complex comprising said first and second polypeptide.
47. A kit for producing or detecting a protein complex said kit comprising:
(c) a first compartment comprising a CD2AP polypeptide or a portion thereof sufficient to form a protein complex selected from the group consisting of: (i) CD2AP and cortactin; (ii) CD2AP and endophilin; (iii)
CD2AP and Cbl; (iv) CD2AP and endophilin and cortactin; (v) CD2AP and cortactin and Cbl; and (vi) CD2AP and cortactin and Cbl and endophilin; and (d) a second compartment comprising an antibody or ligand that binds to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin, or an antibody or ligand that binds to a protein complex selected from the group consisting of: (i) CD2AP and cortactin; (ii) CD2AP and endophilin; (iii) CD2AP and Cbl; (iv) CD2AP and endophilin and cortactin; (v) CD2AP and cortactin and Cbl; and (vi) CD2AP and cortactin and Cbl and endophilin, wherein said antibody or ligand that binds to a protein complex does not bind to Cbl in the absence of CD2AP.
48. A kit for producing or detecting a protein complex said kit comprising a first compartment comprising an endophilin polypeptide or a portion thereof sufficient to bind CD2AP and a second compartment comprising an antibody or ligand that binds to CD2AP.
49. A kit for producing or detecting a protein complex said kit comprising a first compartment comprising a cortactin polypeptide or a portion thereof sufficient to bind CD2AP and a second compartment comprising an antibody or ligand that binds to CD2AP.
50. A kit for producing or detecting a protein complex said kit comprising a first compartment comprising a Cbl polypeptide or a portion thereof sufficient to bind CD2AP and a second compartment comprising an antibody or ligand that binds to CD2AP.
51. A kit for producing or detecting a protein complex said kit comprising:
(c) a first compartment comprising an isolated or recombinant protein complex selected from the group consisting of: (i) CD2AP and cortactin; (ii) CD2AP and endophilin; (iii) CD2AP and Cbl; (iv) CD2AP and endophilin and cortactin; (v) CD2AP and cortactin and Cbl; and (vi)
CD2AP and cortactin and Cbl and endophilin; and
(d) a second compartment comprising an (i) antibody or ligand that binds to a polypeptide selected from the group consisting of CD2AP, cortactin, Cbl and endophilin; or (ii) an antibody or ligand that binds to one or more protein complexes (a).
52. An isolated antibody that binds to a protein complex selected from the group consisting of:
(i) a complex comprising CD2AP and cortactin; (ii) a complex comprising CD2AP and Cbl; (iii) a complex comprising CD2AP and endophilin; (iv) a complex comprising CD2AP and endophilin and cortactin; (v) a complex comprising CD2AP and Cbl and cortactin; (vi) a complex comprising CD2AP and endophilin and Cbl; (vii) a complex comprising CD2AP and endophilin and
Cbl and cortactin; (viii) a complex comprising cortactin and ASAP1; (ix) a complex comprising cortactin and N-WASP; (x) a complex comprising cortactin and FISH; and (xi) a complex comprising cortactin and Samδδ, wherein said antibody binds to said complex in the presence or absence of EGFR.
53. The antibody of claim 52 wherein said antibody recognizes a conformational epitope of the protein complex.
54. An isolated antibody that binds to the antibody of claim 52 or 53.
55. A method for isolating or identifying a protein selected from the group consisting of CD2AP, ASAP1, N-WASP, FISH and Samδδ from a cell comprising contacting an extract of said cell with a cortactin polypeptide or a portion thereof that binds to said protein for a time and under conditions sufficient for a protein complex to form and then isolating the protein complex formed.
56. The method of claim 55 wherein the portion comprises an SH3 domain of cortactin.
57. The method of claim 56 wherein the SH3 domain of cortactin comprises an amino acid sequence having about 80% identity to SEQ ID NO: 18 or SEQ ID NO: 19.
58. A method for isolating or identifying a protein selected from the group consisting of cortactin, Cbl and endophilin from a cell comprising contacting an extract of said cell with a CD2AP polypeptide or a portion thereof that binds to said protein for a time and under conditions sufficient for a protein complex to form and then isolating the protein complex formed.
59. The method of claim 58 wherein the portion comprises an SH3 domain of CD2AP.
60. The method of claim 59 wherein the SH3 domain of CD2AP comprises an amino acid sequence having about 30% identity to a sequence selected from the group consisting of:
(i) a sequence from about position 97 to about position 111 of SEQ ID NO: 1 ; (ii) a sequence from about position 144 to about position 157 of SEQ ID NO: 2;
(iii) a sequence from about position 261 to about position 274 of SEQ ID NO: 1 ; and (ii) a sequence from about position 306 to about position 319 of SEQ ID NO: 2.
61. The method of claim 5δ wherein the portion of CD2AP comprises a proline rich region (PRR) of a CD2AP polypeptide.
62. The method of claim 61 wherein the PRR comprises an amino acid sequence having at least about 60% identity to a sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 25.
63. The method according to any one of claims 55 to 62 wherein the cell is a cancer cell.
64. The method of claim 63 wherein the cancer cells is a carcinoma cell, breast cancer cell, head and neck cancer cell, adenocarcinoma cell, squamous lung cancer cell, gastrointestinal cancer cell, renal cell cancer cell, bladder cancer cell, ovarian cancer cell, prostate cancer cell, squamous cell carcinoma cell, non-squamous carcinoma cell, glioblastoma cell, or medulloblastoma cell.
65. A method of isolating or identifying the protein complex according to any one of claims 1 to 22 from a cell comprising incubating an extract of said cell with a first antibody that binds to a cortactin protein and a second antibody that binds to a protein selected from the group consisting of CD2AP, Cbl, and endophilin for a time and under conditions sufficient for antigen-antibody complexes to form and then detecting the antigen-antibody complexes formed.
66. A method of isolating or identifying the protein complex according to any one of claims 1 to 22 from a cell comprising incubating an extract of said cell with a first antibody that binds to a CD2AP protein and a second antibody that binds to a protein selected from the group consisting of cortactin, Cbl, and endophilin for a time and under conditions sufficient for antigen-antibody complexes to form and then detecting the antigen-antibody complexes formed.
67. The method according to claim 65 or 66 further comprising incubating the cellular extract with an antibody against EGFR.
6δ. The method according to any one of claims 65 to 67 wherein the cell is a cancer cell.
69. The method of claim 6δ wherein the cancer cells is a carcinoma cell, breast cancer cell, head and neck cancer cell, adenocarcinoma cell, squamous lung cancer cell, gastrointestinal cancer cell, renal cell cancer cell, bladder cancer cell, ovarian cancer cell, prostate cancer cell, squamous cell carcinoma cell, non-squamous carcinoma cell, glioblastoma cell, or medulloblastoma cell.
70. The method according to any one of claims 65 to 69 wherein the antibody binds to an SH3 domain of a polypeptide selected from the group consisting of cortactin, CD2AP, endophilin and ASAP1.
71. The method according to any one of claims 65 to 69 wherein the antibody binds to a proline rich region (PRR) of a polypeptide selected from the group consisting of CD2AP, Cbl, ASAP1, N-WASP, FISH and Sam63.
72. A method for producing the protein complex according to any one of claims 1 to 33 comprising incubating a cell that comprises introduced nucleic acid that comprises a nucleotide sequence encoding a polypeptide of said protein complex in operable connection with a suitable promoter sequence for a time and under conditions sufficient for expression and assembly of the protein complex to occur.
73. The method of claim 72 wherein the introduced nucleic acid encodes a fusion protein comprising two or more polypeptide partners of the protein complex.
74. A method for determining a predisposition for disease or a disease state comprising detecting the protein complex according to any one of claims 1 to 38 in a cell of a human subject.
75. A method for determining a predisposition for disease or a disease state comprising performing the method according to any one of claims 55 to 71.
76. A method for determining a modulator of the activity, formation or stability of a protein complex comprising:
(iii) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of
CD2AP, N-WASP, ASAP1, FISH and Sam68; and (b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1 , FISH and Samδδ or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and
(iv) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and (b) a polypeptide selected from the group consisting of cortactin,
Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide, wherein said protein complex further optionally comprises EGFR, said method comprising determining the level or association or dissociation of said protein complex in the absence of a candidate compound or candidate antibody and determining the level of said protein complex in the presence of a candidate compound or in the presence of said candidate antibody, wherein a difference in the level of said protein complex in the absence and presence of the candidate compound or candidate antibody indicates that the candidate compound or candidate antibody is a modulator of said interaction.
77. The method of claim 76 wherein the modulator modulates the activity, formation or stability of a protein complex selected from the group consisting of: (i) a complex comprising CD2AP and cortactin; (ii) a complex comprising
CD2AP and Cbl; (iii) a complex comprising CD2AP and endophilin; (iv) a complex comprising CD2AP and endophilin and cortactin; (v) a complex comprising CD2AP and Cbl and cortactin; (vi) a complex comprising CD2AP and endophilin and Cbl; (vii) a complex comprising CD2AP and endophilin and Cbl and cortactin; (viii) a complex comprising cortactin and ASAP1; (ix) a complex comprising cortactin and N-WASP; (x) a complex comprising cortactin and FISH; and (xi) a complex comprising cortactin and Samδδ.
78. The method of claim 77 comprising: (v) determining the level of a protein complex comprising cortactin or a portion of cortactin and a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH and Samδδ or a portion of said polypeptide in the absence of a candidate compound or candidate antibody; and (vi) determining the level of said protein complex in the presence of a candidate compound or in the presence of said candidate antibody wherein a difference in the level of said protein complex at (i) and (ii) indicates that the candidate compound or candidate antibody is a modulator of said interaction.
79. The method of claim 77 comprising:
(i) determining the level of a protein complex selected from the group consisting of: (i) a complex comprising CD2AP and cortactin; (ii) a complex comprising CD2AP and endophilin; (iii) a complex comprising CD2AP and Cbl; (iv) a complex comprising CD2AP and endophilin and cortactin; (v) a complex comprising CD2AP and Cbl and cortactin; and
(vi) a complex comprising CD2AP and endophilin and Cbl and cortactin in the absence of a candidate compound or candidate antibody; and (ii) determining the level of said protein complex in the presence of a candidate compound or in the presence of said candidate antibody wherein a difference in the level of said protein complex at (i) and (ii) indicates that the candidate compound or candidate antibody is a modulator of said interaction.
δO. The method according to any one of claims 76 to 79 wherein the modulator is an agonist of complex formation or stability.
81. The method of claim 80 wherein the agonist enhances EGFR endocytosis in a cell, or prevents or reduces an impairment of EGFR endocytosis in a cell, or prevents or reduces a reduced or limited EGFR endocytosis in a cell.
δ2. The method of claim δO or δ1 wherein the agonist compound comprises a polypeptide of a protein complex comprising:
(i) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1, FISH and Samδδ; and
(b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1 , FISH and Samδδ or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and (ii) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
(b) a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide.
δ3. The method of claim 60 or δ1 wherein the agonist comprises nucleic acid encoding a polypeptide of a protein complex comprising: (i) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of CD2AP, N-WASP, ASAP1 , FISH and Samδδ; and (b) a polypeptide selected from the group consisting of CD2AP, N-
WASP, ASAP1 , FISH and Samδδ or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and (ii) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
(b) a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said CD2AP polypeptide or said portion of a CD2AP polypeptide.
δ4. The method of claim δO or δ1 wherein the agonist is a mimetic of Cbl capable of binding to a protein complex cortactin and CD2AP.
δ5. The method of claim 80 or 81 wherein the agonist is a mimetic of CD2AP capable of binding to cortactin and/or Cbl.
86. The method of claim δO or δ1 wherein the agonist is an antibody. δ7. The method according to any one of claims 76 to 79 wherein the modulator is an antagonist of complex formation or stability.
δδ. The method of claim δ7 wherein the antagonist comprises UCS15A.
69. A method of treatment of a subject comprising administering to a subject an amount of a modulator of the activity, formation or stability of a protein complex comprising:
(iii) (a) a cortactin polypeptide or a portion of a cortactin polypeptide sufficient to bind to a polypeptide selected from the group consisting of
CD2AP, N-WASP, ASAP1, FISH and Sam6δ; and (b) a polypeptide selected from the group consisting of CD2AP, N- WASP, ASAP1 , FISH and Samδδ or a portion of said polypeptide sufficient to bind to said cortactin polypeptide or said portion of a cortactin polypeptide; and
(iv) (a) a CD2AP polypeptide or a portion of a CD2AP polypeptide sufficient to bind to a polypeptide selected from the group consisting of cortactin, Cbl and endophilin; and
(b) a polypeptide selected from the group consisting of cortactin, Cbl and endophilin or a portion of said polypeptide sufficient to bind to said
CD2AP polypeptide or said portion of a CD2AP polypeptide.
90. The method of claim 89 wherein the modulator modulates EGFR endocytosis in a cell.
91. The method of claim 89 or 90 wherein the subject has a condition in which EGFR endocytosis is impaired, limited or reduced.
92. The method of claim δ9 or 90 wherein the subject has a disease or condition characterized by elevated EGFR levels, or elevated or sustained EGF- mediated signalling.
93. The method according to any one of claims 39 to 92 wherein the subject has cancer or a predisposition to cancer.
94. The method according to any one of claims δ9 to 93 wherein the modulator is an agonist of the formation or stability of a protein complex selected from the group consisting of:
(i) a complex comprising cortactin and CD2AP and Cbl; and
(ii) a complex comprising cortactin and CD2AP and Cbl and endophilin.
95. A method of enhancing EGFR endocytosis in a cancer cell of a subject said cancer characterized by elevated expression of EGFR and elevated expression of cortactin, said method comprising administering to a subject an amount of a Cbl polypeptide or portion thereof and/or a CD2AP polypeptide or portion thereof for a time and under conditions sufficient for EGFR endocytosis to be enhanced in said cell.
96. The method according to claim 95 further comprising administering to the subject an amount of UCS15A sufficient to reduce the formation of a protein complex that does not bind to the EGFR or that does not facilitate or enhance receptor endocytosis in a cell of said subject but not sufficient to disrupt recruitment of cortactin and CD2AP and Cbl to the EGFR.
97. A method of enhancing EGFR endocytosis in a cancer cell of a subject said cancer characterized by elevated expression of EGFR and elevated expression of cortactin, said method comprising administering to a subject an amount of a nucleic acid encoding a Cbl polypeptide or portion thereof and/or nucleic acid encoding a CD2AP polypeptide or portion thereof for a time and under conditions sufficient for said nucleic acid to be expressed to thereby enhance EGFR endocytosis in said cell.
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