GB2341182A - Protein comprising amino acid sequences having SH3 domain binding activity and nuclear localisation activity - Google Patents
Protein comprising amino acid sequences having SH3 domain binding activity and nuclear localisation activity Download PDFInfo
- Publication number
- GB2341182A GB2341182A GB9819038A GB9819038A GB2341182A GB 2341182 A GB2341182 A GB 2341182A GB 9819038 A GB9819038 A GB 9819038A GB 9819038 A GB9819038 A GB 9819038A GB 2341182 A GB2341182 A GB 2341182A
- Authority
- GB
- United Kingdom
- Prior art keywords
- seq
- fragment
- binding protein
- protein
- binding
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Peptides Or Proteins (AREA)
Abstract
A protein comprises: <SL> <LI>(i) an amino acid sequence having SH3 (src homology 3) domain binding activity selected from proline-rich SEQ.ID.No.1 to SEQ.ID.No.22 (not shown); <LI>(ii) an amino acid sequence having nuclear localisation activity, preferably selected from SEQ.ID.No.23 to SEQ.ID.No.26 (not shown). </SL> DNA sequences encoding the protein, and antibodies to said protein, are disclosed. The protein may be formulated into a pharmacutical for use in the therapy of proliferative disease states, especially cancer and chronic inflammatory diseases. The protein comprises sequences of a polypeptide of 70 kD, designated np70, concentrated in the nucleus in interphase cells and distributed throughout the cytoplasm in dividing cells.
Description
2341182 Protein The present invention relates to a novel SH3 binding
protein, hereinafter referred to as np70; polypeptide fragments thereof; DNA encoding the protein; antibodies specific for the protein; a pharmaceutical composition comprising the protein or fragments thereof; methods for its production; its use in a method for treatment of proliferative diseases such as cancer and chronic inflammatory diseases; and its use in the manufacture of a medicament for treatment of these diseases.
Amino acids and amino acid residues are represented herein by their standard codes as identified by the IUPAC-IUB Biochemical Nomenclature Commission and represent D and L amino acids, their analogues, mimetics and derivatives.
The word "comprises" is interpreted within the context of this specification to mean "includes or consists of- and it is not intended to mean "is limited to only".
Intermolecular associations are essential for the generation of intracellular signals resulting from activation stimuli-at the plasma membrane. A number of modular domains have now been described which mediate these interactions, and sequence homologies have indicated that these domains are widespread in signaling molecules. Examples of domains which are responsible for intracellular associations of signaling molecules include the src homology 2 (SH2) and src homology 3 (SH3) domains, originally identified as non-catalytic regions of the src oncogene whose presence were essential for regulation of its tyrosine kinase activity and the plekstrin homology (PH) domain which has now been identified in a number of signaling molecules (Cohen et al 1995; Pawson 1995).
2 PH domains are regions of about 100 amino acids which have low homology, but are structurally similar. Whilst the functions of these domains have not been totally established, there appears to be a preponderance for these domains to associate with small hydrophobic moieties. For example a number of PH domains associate with inositol phospholipids, and it has been proposed that PH domains are a mechanism for targeting proteins to membranes (Salim et al 1997; Lemmon et al 1996; Harlan et al 1994).
SH2 domains are regions of about 100 amino acids which bind to specific phosphotyrosine-containing peptide sequences, with the specificity being determined mainly by the residues immediately flanking the phosphorylated tyrosine residue (Songyang et al 1993, 1994; Moran et al 1995). Ligation of specific receptors at the cell surface results in the formation of complexes consisting of specific intracellular signaling molecules (Kypta et al 1990; Otsu et al 1991; Kaslauskas et al 1992), and so SH2 domains appear to target molecules to the plasma membrane by binding to proteins which contain phosphotyrosine. Thus both PH domains and SH2 domains appear to target proteins to membranes by distinct mechanisms. NMR studies and X-ray crystallography have revealed detailed structures of a number of SH2 domains and all consist of two antiparallel P sheets surrounded by two a-helices, with the phosphotyrosine and +3 residue tightly bound to two pockets on the domain surface (Waksman et al 1993).
SH3 domains are regions of about 50-75 amino acids which are also commonly found in signaling molecules, in cytoskeletal components and also in the phagocyte superoxide forming complex, the NADPH oxidase. Many proteins which contain PH and SH2 domains also contain an SH3 domain, however there are subsets of proteins which can have several permutations or multiples of these domains. SH3 domains bind to proline-rich sequences with a core motif of PXXP where P are conserved proline residues and X are commonly aliphatic residues (Ren et al 1993).
Whilst there is some promiscuity in the binding of some SH3 domains to different proline rich motifs (Gout et al 1993) other SH3 domains are highly selective and will bind to only a single specific sequence (Finan et al 1994, 1996a). All SH3 domain ligands adopt a type II polyproline helix conformation and structural studies have shown that proline-rich motifs fall into two classes which can bind to SH3 domains in either forward or reverse orientations (Booker et al 1993; Kohda et al 1993; Koyama et al 1993; Musacchio et al 1994; Noble et al 1993; Feng et al 1994; Yu et al 1994, Finan et al 1996a). The activation of the phagocyte NADPH oxidase is regulated by the SH3 domain mediated assembly of cytosolic components with a membrane protein containing a specific proline-rich SH3 domain binding site (McPhail 1994). Although it is well recognized that regulation of SH2 domain interactions occurs by phosphorylation, the regulation of SH3 domain interactions is less well understood and is probably at the level of conformational change leading to masking or unmasking of the domain or its ligand.
About 70-80 SH3 domain-containing proteins have now been described. The majority of these proteins fall into the categories of signaling proteins or protooncogenes, the archetype being pp6oc-src, and it has been shown that the presence of the SH3 domain of this protein is essential for DNA synthesis induced by platelet-derived growth factor and epidermal growth factor (Pawson and Scott 1997; Erpel et al 1996). Several cytoskeletal proteins also contain SH3 domains, for example myosin I and a-spectrin, and these domains may be involved in the intracellular localization of proteins (Bar-Sagi et al 1993). A third category of SH3 domain containing proteins are the components of the NADPH oxidase system of phagocytes. The SH3 domains of the cytoplasmic components p47 phOx and p67PhOx interact with the cytoplasmic tail of the membrane component p22a, and this subunit assembly results in an active holoenzyme (Finan et al 1994, 1996a; McPhail 1994, Wilson et al 1997). Less is known about the function of SH3 domain-binding proteins. A number of these are linked to intracellular signaling complexes found near the plasma membrane, for example proline-rich sequences of SOS bind to the SH3 domains of Grb2, and this leads to the activation of ras and thus is required for growth factor signaling (Rozakis-Adcock et al 1993). Several SH3 domain binding proteins have been identified in the activation pathway of small GTP-binding proteins, for example the first molecule identified as an SH3 domain binding protein, 3BP1, is a rasGAP (Cicchetti et al 1992) and dynamin, which binds to the SH3 domain of phosphatidylinositol kinase is a GTPase (Gout et al 1994) In most cases the localization of SH3 domain binding proteins has been shown to be either the plasma membrane or the cytoplasm, commonly near the plasma membrane, however more recently a family of other SH3 binding proteins with a different cellular localization has been described. Heteroribonucleoprotein K is an RNA-binding protein which also interacts with the SH3 domain of vav which may link receptor events with the cell nucleus (Hobert et al 1994). A protein recently identified as a target for c-src during mitosis, SAM68, is an RNA-binding protein which associates with a number of SH3 domains using several of its proline-rich sequences (Fumigalli et al 1994; Taylor and Shalloway 1994; Finan et al 1996b).
Moreover, SAM68 co-localizes with nck in the cell nucleus (Lawe et al 1997), thus there appears to be a subset of SH3 domain binding proteins which have some association with the'nucleus or nuclear material.
The present invention provides a novel protein (np70) or fragment thereof that binds to SH3 domains and is associated with the cell nucleus. It does not belong to the RNA-binding protein family and does not have extensive homology with any other protein present on available databases.
Whilst an extensive survey of cells has not been performed, np70 appears to be ubiquitous, and is present on all representatives of epithelial, fibroblastic and haemopoietic cells tested to date and in all tissues investigated.
The primary sequence of np70 shows that it preferably comprises a number of putative functional domains. Np70 contains several potential nuclear localization signal sequences, and this is consistent with immunofluorescence experiments.
Preferably the carboxy terminal half is highly prolinerich and contains a number of potential SH3 domain-binding sequences, and preferably there is also a sequence conforming to profilin-binding sites.
The carboxy terminal proline-rich sequences preferably fit with both classic type I and type II SH3 binding sequences, and so np70 preferably has the potential to bind to SH3 domains either in forward or reverse orientations (Feng et al 1994).
Immunofluorescence experiments have demonstrated that np70 is preferentially localized to the cell nucleus, and in particular is excluded from the nucleolus. The localization changes as cells undergo mitosis, and in fully mitotic cells np70 appears as a protein distributed throughout the cytoplasm. Biochemical extraction and subfractionation experiments have shown that np70 is associated with isolated nuclei, is resistant to DNase treatment or extraction in non- ionic detergents, but is solubilised by high salt or ammonium sulfate, treatments commonly used to isolate intermediate filaments and nuclear matrix material (He et al 1990) - These data are suggestive that np70 does not associate directly with DNA but associates with a matrix-like component of the cells.
Double immunofluorescence has revealed that np70 co localizes with pre-mRNA splicing factors in distinct nuclear speckles which appear to correspond to interchromatin granule clusters (IGC: Spector et al 1991) Like np7O, splicing factors in the speckles are resistant to extraction with non-ionic detergent or DNase I treatment, indicating an association with structural components of the nucleus (Spector et al 1991). Taken together with the findings that in the cytoplasm np70 associates with the intermediate filament protein vimentin, it is speculated that nuclear np70 is either a structural component or associates with structural components of the spliceosome. Higher power examination by immuno-electron microscopy has revealed that np70 associates with intermediate filaments, particularly those surrounding the nucleus.
The observat-ion that the relocation of np70 from a perinuclear to a cytoplasmic distribution coincides with the onset of mitosis, and therefore coincides with the breakdown of nuclear-associated intermediate filaments and the nuclear envelope, is also consistent with an association with a matrix component.
The subcellular distribution and sequence motifs suggest that np70 is a link between signaling from growth factors or other stimuli, via SH3 domain containing proteins to the nucleus and transcriptional activity. This indicates that np7O is involved in cell proliferation or mRNA splicing events, making this protein a potentially useful therapeutic target for proliferative diseases such as cancer and chronic inflammatory diseases, including but not limited to rheumatoid arthritis, nephritis, asthma and inflammatory bowel disease.
Accordingly the present invention provides an SH3 binding protein or fragment thereof which has (i) a sequence of amino acid residues having SH3 domain binding activity selected from the group which comprises:
434 PPPGAP -SEQ ID NO:1 202 PPPGPPPP -SEQ ID NO:12 PLPMP -SEQ ID NO:2 191 PRLPMRKPP -SEQ ID NO:13 474 PPPGLP -SEQ ID NO:3 484 PRGPPPRLPP -SEQ ID NO:14 406 PMMPP -SEQ ID NO:4 479 PMPPPRGPP -SEQ ID NO:15 482 PPPRGP -SEQ ID NO:5 417 PAPPLR -SEQ ID NO:16 487 PPPRLP -SEQ ID NO:6 480 PGPPPR -SEQ ID NO:17 410 PPPLGP -SEQ ID NO:7 508 RPPLVP -SEQ ID NO:18 462 PPPGRP -SEQ ID NO:6 197 MPPGP -SEQ ID NO:19 415 PPPAPP -SEQ ID NO:9 443 RPMMP -SEQ ID NO:20 492 PPPAPP -SEQ ID NO:10 422 RPMPP -SEQ ID NO:21 470 PPPWP -SEQ ID NO:11 466RPMPP SEQ ID NO:22 (the numbering refers to the position of the first amino acid residue in the full length np70 protein) (hereinafter refered to as group 1) and (ii) a sequence of amino acid residues having a nuclear localisation activity. In a second aspect the invention provides an SH3 binding protein or fragment thereof which has (i) a sequence of 25 amino acid residues having SH3 domain binding activity and (ii) a sequence of amino acid residues having a nuclear localisation activity selected from the group which comprises: 22 RKEARKRELKKNKK -SEQ ID NO:23 30 79 KDKRKK -SEQ ID NO:24 226 PPRRRDED -SEQ ID NO:25 314 PGMRKKK -SEQ ID NO:26 (the numbering refers to the position of the first amino acid residue in the full length np70 protein) (hereinafter refered to as group 2).
In a third aspect the invention provides a DNA sequence which encodes the SH3 binding protein or fragment thereof.
In a forth aspect the invention provides an antibody or fragment thereof which is specific for the SH3 binding protein or a fragment thereof.
In a fifth aspect the invention provides a pharmaceutical composition which comprises the SH3 binding protein or fragment thereof together with a pharmaceutically acceptable carrier, diluent or excipient.
In a sixth aspect the invention provides a method for production of the SH3 binding protein.
In a seventh aspect the invention provides an SH3 binding protein or fragment thereof for use in a method of prevention or treatment of proliferative diseases including cancer and chronic inflammatory diseases.
In an eigthth aspect the invention provides an SH3 binding protein or fragment thereof for use in the manufacture of a medicament for the treatment or prevention of proliferative diseases including cancer and chronic inflammatory diseases.
In a ninth aspect the invention provides a method of prevention or treatment of proliferative diseases including cancer and chronic inflammatory diseases which comprises administering a pharmaceutically effective amount of the SH3 binding protein or fragment thereof.
Preferably an embodiment of the SH3 binding protein or fragment thereof comprises at least one group 1 sequence and at least one group 2 sequence.
More preferably an embodiment of the SH3 binding protein or fragment thereof comprises at least two group 1 sequences and at least one group 2 sequence.
Preferably an embodiment of the SH3 binding protein or fragment thereof comprises a sequence selected from the group which comprises the full length np70 sequence:
MGRRSTSSTKSGKFMNPTDQARKEARKRELKKNKKQRMMVRAAVLKMKDPKQIIRDME KLDEMEFNPVQQPQLNEKVLKDKRKKLRETFERILRLYEKENPDIYKELRKLEVEYEQ KRAQLSQYFDAVKNAQHVEVESIPLPDMPHAPSNILIQDIPLPGAQPPSILKKTSAYG PPTRAVSILPLLGHGVPRLPPGRKPPGPPPGPPPPQVVQMYGRKVGFALDLPPRRRDE DMLYSPELAQRGHDDDVSSTSEDDGYPEDMDQDKHDDSTDDSDTDKS DGES DGDEFVH 15 RDNGERDNNEEKKSGLSVRFADMPGKSRKKKKNMKELTPLQAMMLRMAGQEIPEEGRE VEEFSEDDDEDDSDDSEAEKQSQKQHKEESHSDGTSTASSQQQAPPQSVPPSQIQAPP MPGPPPLGPPPAPPLRPPGPPTGLPPGPPPGAPPFLRPPGMPGLRGPLPRLLPPGPPP GRPPGPPPGPPPGLPPGPPPRGPPPRLPPPAPPGIPPPRPGMMRPPLVPPLGPAPPGL FPPAPLPNPGVLSAPPNLIQRPKADDTSAATIEKKATATISAKPQITNPKAEITRFVP 20 TALRVRRENKGATAAPQRKSEDDSAVPLAKAAPKSGPSVPVSVQTKDDVYEAFMKEME GLL -SEQ ID NO:27 and the carboxy terminal region thereof:
APPQSVPPSQIQAPPMPGPPPLGPPPAPPLRPPGPPTGLPPGPPPGAPPFLRPPGMPG LRGPLPRLLPPGPPPGRPPGPPPGPPPGLPPGPPPRGPPPRLPPPAPPGIPPPRPGMM RPPLVPPLGPAPPGLFPPAPLPNPGVLSAPPNLIQRPKADDTSAATIEKKATATISAK PQI TNPKAE I TRFVPTALRVRRENKGATAAPQRKSE DDSAVPLAKAAPKS GPSVPVSV QTKDWYEAFMKEMEGLL -SEQ ID NO:28 Preferably an embodiment of the SH3 binding protein or fragment thereof comprises a plurality of nuclear localisation signal sequences.
Preferably the carboxy terminal half of the protein or fragment according to an embodiment of the invention is proline rich. "Carboxy terminal half" is interpreted to mean residues 393 to 641 of the protein, that is the sequence identified by SEQ ID NO:28. "Proline rich is interpreted to mean that the amino acid sequence comprises at least three proline residues and more preferably includes a sequence which falls into the consensus motif for SH3 domain binding, commonly PXPXXP, PXXPXXRXXP, PXXPXR and RXPXXP.
Preferably the protein or fragment thereof according to an embodiment of the invention comprises a plurality of SH3 domain-binding sequences.
Preferably the protein or fragment thereof according to an embodiment of the invention comprises a prolifin-binding site.
Preferably the DNA sequence which encodes the SH3 binding protein according to an embodiment of the invention is cDNA.
Preferably the DNA sequence according to an embodiment of the invention comprises the sequence as shown in figure 2.
Preferably a pharmaceutical composition according to an embodiment of the invention is formulated in accordance with routine procedures for administration by any route, particularly parenteral, especially by intravenous infusion but also including the oral, sublingual, subcutaneous, intraperioneal and transdermal routes or by inhalation. The compositions may be in the form of tablets, capsules, powders, granules, lozenges, creams or liquid preparations, such as oral or sterile parenteral solutions or suspensions or in the form of a spray, aerosol or other conventional method for inhalation.
Preferably a topical formulation according to an embodiment of the present invention may be presented as, for instance, ointment, -cream or lotion and eye or ear drop, impregnated dressings and aerosols and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointment and cream.
Preferably the formulation according to an embodiment of the invention may also contain compatible conventional carriers, such as cream or ointment bases and ethanol or oleyl alcohol for lotions. Such carriers may be present as from about 1% up to about 98% of the formulation. More usually they will form up to about 80% of the formulation.
Preferably tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventi-onal excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrollidone; fillers, for example lactose, sugar, maizestarch, calcium phosphate, sorbitol or glycine; tabletting lubricants,_for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. Tablets may also contain agents for the stabilisation of polypeptide drugs against proteolysis and absorbtion-enhancing agents for macromolecules. The tablets may be coated according to methods well known in normal pharmaceutical practice.
Preferably suppositories according to an embodiment of the invention contain conventional suppository bases, e.g. cocoa-butter or other glyceride.
Preferably formulations for parenteral administration are in fluid unit dosage forms and are prepared using the compound and a sterile vehicle, water being preferred. The compound, depending on the vehicle and concentration used, is dissolved in the vehicle. In preparing solutions the compound can be dissolved in water for injection and filter sterilised before filling into a suitable vial or ampoule and sealing.
Preferably parenteral formulations include sustainedrelease systems such as encapsulation within microspheres of biodegradable polymers such as poly-lactic co-glycolic acid.
Preferably agents such as a local anaesthetics, preservatives and buffering agents can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. The dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use. More preferably, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.
Preferably compositions according to an embodiment of this invention may also suitably be presented for administration to the respiratory tract as a snuff or an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case the particles of active compound suitably have diameters of less than 50 microns, preferably less than 10 microns for example diameters in the range of 1-50 microns, 1-10 microns or 1-5 microns. Where appropriate, small amounts of anti-asthmatics and bronchodilators, for example sympathomimetic amines such as isoprenaline, isoetharine, salbutamol, phenylephrine and ephedrine; xanthine derivatives such as theophylline and aminophylline and corticosteroids such as prednisolone and adrenal stimulants such as ACTH may be included.
Preferably microfine powder formulations may suitably be administered in an aerosol as a metered dose or by means of a suitable breath-activated device or transdermally by ballistic techniques.
Preferably suitable metered dose aerosol formulations comprise conventional pro15611ants, cosolvents, such as ethanol, surfactants such as oleyl alcohol, lubricants such as oleyl alcohol, desiccants such as calcium sulphate and density modifiers such as sodium chloride.
Preferably suitable solutions for a nebulizer are isotonic sterilised solutions, optionally buffered, at for example between pH 4-7, containing up to-20mg ml-l of compound but more generally 0.1 to 10mg ml-l, for use with standard nebulisation equipment.
The quantity of material administered will depend upon the potency of the derivative and the nature of the complaint be decided according to the circumstances by the physician supervising treatment. However, in general, an effective amount of the polypeptide for the treatment of a disease or disorder is in the dose range of 1-100 mg/kg per day, preferably 1-10 mg/kg per day, administered in up to five doses or by infusion.
No adverse toxicological effects are indicated with the compounds of the invention within the above described dosage range.
Preferably an embodiment of the invention also provides a derivative of the invention for use as a medicament.
Preferably an embodiment of the method of producing the SH3 binding protein or fragment thereof comprises the steps of purifying it from cells by carrying out SH3 affinity chromatography. Preferaboly the protein of fragment thereof is separated by SDS-PAGE, identified by Coomassie Blue staining, excised and digested with Lys-C in the gel slice. Preferably peptides are extracted from the gel, separated by tandem ion exchange and reverse phase HPLC using a Hewlett-Packard 1090M and diode array detection system, and the purified peptides are preferably sequenced using a fast cycle, automated sequencer.
Preferably the cDNA sequence of the protein or fragment thereof is found, placed in an appropriate vector and expressed using an appropriate host.
Embodiments of the present invention will now be described with reference to the accompanying drawings in which:
Figure 1 shows a profile of SH3 domain-binding proteins.
GST-SH3 fusion proteins were immobilized to glutathione beads as described below in Materials and Methods. Lysates from Namalwa cells were adsorbed to the beads and the bound proteins were separated by SDS-PAGE under reducing conditions followed by Coomassie blue staining. The 55kD protein which was analyzed further is denoted with an asterisk. M: markers; BEADS: glutathione beads alone; GST:
glutathione beads with immobilized GST; p47NC: glutathione beads with immobilized with the tandem N and C-terminal hox SH3 domains of p47P Figure 2 shows the complete nucleic acid sequence of np70 Figure 3 shows complete predicted amino acid sequence of np70. The peptide sequences derived from microsequencing of the Sh3 domain-binding protein are shown underneath.
Figure 4 shows northern blotting of np70 on human tissues. RNAs from a panel of human tissue (Clontech) were run on an acrylamide gel and probed with a full length np70 probe. The lower panel shows the blot stripped and reprobed from P-actin. 10 Figure 5 shows characterization of antibodies against np70 and its cellular distribution. A: Total SDSPAGE extracts from Namalwa, COS or 293 cell lines were immunoblotted using either anti-np70 peptide antiserum, or animal-matched preimmune serum. I represents immune anti-peptide sera and P represents animal-matched pre- immune serum. B: Cells transfected with myc-tagged np70 were lysed and immunoprecipitated with anti-myc, followed by immunoblotting with either anti-myc (tracks 1 and 2) or anti np70 antibodies (tracks 3 and 4). 20 Figure 6 shows selectivity of np70 binding to SH3 domains. The binding of np70 from Namalwa cell lysates to a panel of immobilized SH3 domains was performed as described for Figure 1 and the bound np70 detected by western blotting as described for Figure 3. Glutathione beads were 25 immobilized with GST fusion proteins containing the following SH3 domains. CSRC: c-src; N-SRC: Nsrc; P85a: the SH3 domain of the 85kD subunit of phosphatidylinositol 3'-kinase; PLCyl: the SH3 domain of phospholipase Cyl; FGR: c-fgr; p47N: the N-terminal SH3 domain of p47 Phox 30 p47C: the C-terminal SH3 domain of p47P"'; p47N+C: the tandem N-terminal and C- terminal SH3 domains of p47 Phox p67N: the N-terminal SH3 domain of p67P"'; p67C: the C- terminal SH3 domain of p67 Phox.
- 16 Figure 7 shows double fluorescence of np70 and DNA in MDCK cells. Cells were stained with a nti-np70 (a,d,g,j) or propidium. iodide (b,e,h). a-c: interphase cells showing nuclear distribution of np70 but exclusion from nucleoli.
d-f: Early metaphase cell showing fragmentation of nuclear np70 (arrows) and condensation of DNA. g-i; Late anaphase cell showing cytoplasmic distribution of np70 (arrows) and its-exclusion from condensing chromosomes.
J,k: nuclear localization of overexpressed recombinant np7O. 1: A digital confocal image Of a 1im optical slice of np70 in an interphase cell showing the granularity of nuclear staining. Bar = 20pm Figure 8 shows extraction of np70 from MDCK nuclei. MDCK cells on coverslips were treated with 1% NP40 (a,b,c), DNase I (d,e,f), 2M NaCl (g,h,i) or 0.25M ammonium sulfate (j,k,l). np70 in the treated cells was detected by staining with anti-np7O (a,d,g,j) and DNA detected by staining with propidium. iodide (b,e,h,k) as described in Materials and Methods. Bar = 20pm.
Figure 9 shows subcellular fractionation and quantitation of np70 extraction from MDCK cells. A: Cells were treated as described for Figure 4, then the insoluble material was processed for np70 immunoblotting as described in Materials and Methods. Equal cell equivalents from 101 cells were run-on each track. The blot show np70 remaining with the cell after extraction with: 1% NP40 (91%); DNase 1 (94%); 2M NaCl (track S; 23%) or 0.25M NH3SO4 (11%) compared with total cellular np70 (track T; 100%). The numbers in parentheses represent the percentage of np70 remaining after extraction, as determined by scanning densitometry. B: Whole cell lysates (TOTAL) or lysates from cells subjected to fractionation into nuclear, post nuclear membranous (MEM) and cytosolic fractions followed by SDSPAGE were immunoblotting for np70.
Figure 10 shows double immunofluorescence of np70 and vimentin. Cells were fixed in methanol and labeled with anti- vimentin (a,c) or anti- np70 (b,d). The figures in the upper panel show normal epifluorescence, and the figures in the lower panel show a 1 pm optical section using digital confocal microscopy software. Bar = 20pm.
Figure 11 shows double immunofluorescence of np7O (a,c,e,g) and the pre-mRNA splicing factors sc35 (b, f) and M"(d,h). In panels a-d the cells were fixed in 3.7% formaldehyde and permeabili,sed with 0.5% NP40. In panels e-h the cells were permeabilised prior to fixation. The cells were subjected to digital confocal immunofluorescence microscopy and representative deconvolved images are shown. Bar = 20pm.
is MATERIA.LS AND METHODS Cells and Reagents The bacterial expression vector pGEX-4T-1 was purchased from Pharmacia. Glutathione-agarose beads were obtained from Sigma. HEL, Namalwa, Jurkat and U937 cells were originally obtained from the American Type Culture Collection and cultured in RPMI 1640 medium (Flow Laboratories), and COS and 293 cells were grownin Dulbecco's modified Eagles medium containing 10% heat inactivated fetal calf serum (Imperial Laboratories) at 37'C in 5% C02. All chemicals were Analar grade or better (BDH), and all other reagents were purchased from Sigma unless stated otherwise.
Glutathione S-Transferase Fusion Proteins cDNA sequence encoding the SH3 domains of PLCyl, p85a, c src, n-src, c-fgr, p47P"', and p67P111 as glutathione-S transferase (GST) fusion proteins were generated and used as described previously (Finan et al 1994, 1996a).
18 - Constructs were transformed into Escherichia coli XLl-Blue and expression of GST fusion proteins performed as described previously (Smith and Johnson 1988).
GST-SH3 binding assays Affinity matrices were prepared by immobilizing 25 mg of fusion protein on 50ml of glutathione-agarose beads (Sigma). Cells were pelleted and washed twice in phosphate buffered saline. Cell pellets were solubilised in lysis buffer (50mM Tris PH 7.5, 5 mM EGTA, 2% (v/v) Triton X- 100, 75 mM NaCl, 0.5 mM PMSF) and clarified by centrifugation at 14,000g for 15 min at 40C. Cell lysate was mixed with the GST-SH3 affinity matrices for 3 hours at 40C. The beads were then washed extensively in wash buffer (50 mM Tris PH 7.5, 0.1 % (v/v) Triton X-100, 10% (v/v) glycerol). Binding proteins were eluted by boiling in SDS-PAGE sample buffer, resolved by SDS-PAGE and prepared for microsequencing as described (Finan et al 1994; 1996a).
Sequencing of 90kD SH3 domain-binding protein A 90kD polypeptide that bound to the SH3 domain of p47P"Ox was purified from 109 Namalwa cells by SH3 affinity chromatography as described above. The protein sample was separated by SDS-PAGE, identified by CoGmassie Blue staining, excised and digested with Lys-C in the gel 25 slice. Peptides were extracted from the gel and separated by tandem ion exchange and reverse phase HPLC using a Hewlett-Packard 1090M and diode array detection system. Purified peptides were sequenced using fast cycle, automated Edman chemistry on an Applied Biosystems 477A 30 sequencer, modified as described (Totty et al 1992).
Western blotting Anti-peptide antibodies raised against the final fifteen carboxy terminal residues from np70 were generated in rabbits and used for western blotting from cell lysates.
SDS-PAGE separated proteins from 101 cells were transferred to nitrocellulose filters and after blocking with 5% non fat milk powder for 1 hour the filters were overlaid with a 1:1000 dilution of anti-np70 in PBS containing 0.05% Tween 20 for 1 hour. The filters were washed three times in PBS/Tween 20 and overlaid with a peroxidase-conjugated goat anti-rabbit Ig (Biorad). The bands were detected using the ECL system (Amersham). Images were scanned using a Microtek Scanmaker II, densitometry was performed and peaks integrated using ScanAnalysis linked to an Apple Macintosh Quadra.
Immunofluorescence Cells were grown for 24 hours on glass coverslips and fixed in 4% parafomaldehyde (Fluka) in PBS for 10 minutes.
After 3 washes in PBS the cells were permeabilised by immersion in 0.2% NP-40 in PBS for 5 minutes. Staining was performed by a 1:200 dilution of rabbit anti-np70 in PBS plus 2% BSA for 45 minutes, followed by a 1:100 dilution of FITC-labeled goat anti-rabbit Ig with three washes in PBS between antibodies. After an additional three washes in PBS the cells were mounted in Citifluor and viewed using a Zeiss axioplan microscope equipped with epifluorescence. In some experiments cells were viewed using a Zeiss Axiovert microscope equipped with Biovision Digital Confocal Microscopy software (Improvision Ltd Warwick, UK). Double immunofluorescence was performed using monoclonal antibodies to the pre-mRNA splicing factors sc35 and U2B" in combination with anti-np70 antibodies followed by a mix of FITC-labeled goat anti rabbit Ig and TRITC-labeled goat anti- mouse Ig. The FITC labeled goat anti-rabbit Ig and TRITC-labeled goat anti- - mouse Ig secondary antibodies were cross absorbed against mouse Ig and rabbit Ig respectively Northern blotting The np70 full length construct was cut out of the pCDNA3.1 Myc His tagged vector (Invitrogen) using Eco Rl and Not 1 (Gibco), the 2kb insert was gel purified using a Qiaquick kit (Qiagen) and radio labeled to a specific activity of -2xlO'dpm/mg using a rediprime random prime labeling kit (Amersham). A Multiple tissue northern blot containing Poly A+ mRNA isolated from various human tissues (Clontech) was pre- hybridized with RapidHyb (Amersham) for 30min at 600C. The unincorporated nucleotides (32 P dCTP) were removed using a nick spin column (Bio-Rad) and the denatured probe added to the RapidHyb buffer. After overnight hybridization at 600C, the blot was washed at high stringency (twice in 2 x SSC/0.1% (w/v) SDS for 20 min each and once in 0.1 x SSC/0. 1% (w/v) SDS for 20 min). The blot was exposed for 2hrs at -701C using Hyperfilm-MP (Amersham). The blot was subsequently stripped by adding boiling 1% (w/v) SDS and allowing to cool to room temperature. The stripping was confirmed by overnight autoradiography at - 701C and re-probed using a RediPrime labeled (as before) actin probe supplied with the Multiple tissue blot. Films were processed using a Compact X4 system (Xograph) automated film processor and the resulting autoradiographs scanned using a Microtech scanner using PaintShop Pro.
Extraction of np7O from cell nuclei Cells were plated on to glass coverslips overnight and subjected to one of the following extraction procedures: CSK buffer (10 mM PIPES, pH 6.8, 300mM sucrose, 100mm NaCl, 3mM MgC12, 1 mM EGTA, 1% NP40) for 5 minutes on ice; CSK buffer plus 500 units/ml DNase I for 40 minutes at 37'C; CSK buffer plus 0.25M ammonium sulfate for 5 minutes on ice; CSK buffer plus 2M NaCl for 5 minutes on ice. imm AEBSF was included in all buffers to limit proteolytic digestion. After extraction the cells were washed in ice cold CSK buffer, fixed and stained as described above.
Alternatively, the cells were grown in 60mm petri dishes and after extraction the cells were lysed directly into SDS sample buffer, boiled for 5 minutes, and subjected to SDS PAGE for western blotting.
RESULTS SH3 domain binding proteins from a B cell line Namalwa cells were lysed into Triton X-100-containing buffer, and after centrifugation of insoluble material the lysates were incubated with immobilized fusion protein constructs of GST-SH3 domains. Figure I shows that the Namalwa cell proteins which bound to the tandem N-terminal and C-terminal SH3 domains of p47PIII (p47ph"N/C) and the SH3 domain of PLCyl gave distinct profiles. Both domains bound a polypeptide of approximately 60kD and 85kD. Polypeptides of approximately 7OkD and 75kD associated only with 47 PhOxN/C, and there was an additional prominent band was detected at about 90kD which also bound to the immobilized P47PIlOxN/C-SH3 domain-containing construct (arrowhead). This 90kD prote-in did not bind to beads alone, to GST immobilized beads or to the SH3 domain of PLCyl. Larger quantities of the 90kD band were prepared from 101 cells and purified by binding to p47PIII N/C SH3 domain followed by SDS-PAGE. The band was excised and subjected to microsequencing as described in the Materials and Methods section.
Cloning and sequencing of np70 Ten peptides were derived from microsequencing of the 90kD polypeptide bound to p47P"O" SH3 domain (Fig. 3). Database searching revealed several expressed sequencing tagged sequences with identity to the predicted consensus nucleotide sequence of the peptides. These sequences allowed construction of oligonucleotide PCR primers which gave about 70% of the full sequence, and the rest of the sequence was derived by 5' RACE PCR. The complete nucleic acid sequence of cDNA of the 90kD polypeptide is shown in Figure 2. The complete amino acid sequence of the 90kD polypeptide is shown in Fig. 3. The polypeptide has 641 residues and the predicted amino- acid sequence encodes a protein with a molecular weight of 69,997kD. The polypeptide has a theoretical pI of 8.28, and contains an unusually high proportion of proline residues (18.6%). The amino acid sequence reveals several possible functional regions in np70. Four potential putative nuclear localization sites are found in the amino terminal half of the protein (residues 2-39; 78-83; 226-233; 314-322; The carboxy terminal region contains proline-rich sequences which are likely to be the SH3 domain binding sequences (residues 410-531). In addition there is a - potential profilin binding sequence in the carboxy terminal half (residues 461-489). No signal sequences or other binding motifs were detected. Database searching revealed no homology with any other known protein. The peptide sequences obtained from microsequencing were distributed along the length of the predicted sequence for the cloned polypeptide, confirming that the cloned sequence was correct and in frame.
TiSsue distribution of np70 Northern blotting experiments on a number of human tissues were performed to examine the tissue expression of np70 mRNA (Fig 4). Np70 message (a single band of approximately 3kb) was detectable on all tissues examined, however, longer exposures showed the presence of further transcripts at approximately 4 and 5kb. The highest level of mRNA can be seen in the heart, whilst liver and lung can be seen to have the lowest levels of mRNA. Western blot data using human tissue extracts also showed high level of np70 staining in the heart samples whilst the liver and lung samples had lower levels of protein In order to gain more information about the function of np70 rabbit antipeptide antibodies were raised against the fifteen carboxy terminal residues. Western blotting using anti-peptide antisera revealed the detection of a single band which runs about 90kD on SDSPAGE (Fig 5A), whereas matched preimmune sera gave no such band. Confirmation that the anti-peptide antibodies recognized the cloned 15 protein was obtained by transfection of myc-tagged np70 cDNA into 3T3 cells. Immune precipitation using anti-myc antibodies or antinp70 antibodies followed by western blotting with antinp70 antibodies gave identical results showing the presence of a polypeptide of about 90kD (Fig. 20 5B).Thus np70 tuns with a higher apparent molecular weight than would be predicted from the primary sequence. The reason for this is unknown. Examination of a number of cell lines showed that np70 was expressed in all cells tested which included representatives of B cells, T 25 cells, monoblastoid cells, reticuloendothelial cells and fibroblasts (Fig. 5A; data not showrj). Western blotting of a number of human tissues revealed a ubiquitous distribution, consistent with the results of Northern blotting 30 Select:lvity of np70 binding to SH3 domains The binding of np70 to a panel of 12 immobilized SH3 domains was performed and the association detected by immunoblotting (Figure 6). np70 bound selectively to the N-terminal SH3 domain derived from the NADPH oxidase component p47phox, and to a construct containing both the N terminal and C-terminal SH3 domains of p4711111. No binding was detected to the SH3 domains derived from c-src, n-src, p85a, PLCyl, fgr, the C-terminal SH3 domain of p47phox, or either the N-terminal or C-terminal domains of p67phox.
Intracellular localization of np70 Preliminary immunofluorescence experiments in a number of cell types revealed nuclear staining of T lymphoid cells, B lymphoblastic cells, monocytic cells, fibroblasts and epithelial cells. MDCK cells were chosen for further investigation because they are relatively large cells with prominent nuclei and thin cytoplasm, which allows good intracellular localization at the light microscope level.
Antibody staining of formaldehyde fixed, permeabilised cells revealed that np70 was localized prominently in the nucleus, and had a granular appearance when viewed at high power or using digital confocal microscopy, with little or no staining in the cytoplasm of interphase cells (Fig.
7a,l). Double staining with propidium iodide showed that np7O did not co-localize specifically with DNA, since it was excluded from nucleoli where DNA staining was strongest (Fig. 7a,b,c). The localization of np70 in dividing cells was different from that of interphase cells. In prophase cells where DNA condensation had just been initiated the nuclear np70 staining became more diffuse throughout the cell (Fig. 7d,e,f), and in metaphase or anaphase cells np70 was distributed throughout the cytoplasm, but excluded from the condensing chromatids (Fig. 7g, h, i). In order to examine whether the cloned gene product had a similar function to endogenous np70, cDNA for np70 was microinjected into MDCK cells and 24 hours later the cells were processed for immunofluorescence. immunofluorescence microscopy revealed that the recombinant protein was localized predominantly in the nucleus, however there was some excess protein in the cytoplasm, suggesting that the nuclear transport mechanism had been saturated by the overproduction of np70 (Fig 7j, k). Digital conf ocal microscopy revealed that np70 had a granular, non-random distribution within the nucleus (Fig. 71) Extraction of np70 from cell nuclei.
The intracellular localization of np70 suggested that it was not a direct DNA-binding protein since its immunofluorescence staining profile did not exactly coincide with propidium iodide staining. To investigate this further the biochemical characteristics of np70 association with the nucleus of MDCK cells were studied by the use of a variety of extraction procedures. np70 nuclear localization was resistant to extraction with 1% NP40 in cytoskeleton buffer for 5 minutes, a procedure which extracts several nuclear proteins such as proliferating cell nuclear antigen (data not shown; Fig 8a,b,c). np70 nuclear localization was also resistant to digestion with DNase I under conditions which removed virtually all the DNA from the nucleus (Fig. 8d, e, f). In contrast, substantial amounts of np70 were extracted from MDCK nuclei by either 2M sodium chloride (Fig. 8g,h,i) or 0.25M ammonium sulfate(Fig. 8j,k,l), conditions which had no effect on nuclear DNA staining. Additional experiments also revealed that the cytosolic np7O in mitotic cells was readily removed by a brief NP40 extraction. The extraction of np70 from these cells was quantified by western blotting. Fig. 9A shows that NP40-treated or DNase I-treated MDCK cells retained greater than 90% of np70 whereas 77% of np70 was solubilised by 2M NaCl and 89% was solubilised by 0.25M ammonium sulfate.
Association of np70 with cell nuclei 26 - The previous experiments were performed in cells which had been extracted with detergent prior to further biochemical fractionation. To further assess the association of np70 with the nucleus, MDCK cells were subjected to hypotonic shock followed by dounce homogenization. A low speed spin produced isolated nuclei whilst a high speed spin of the post-nuclear supernatant produced a particulate fraction consisting mainly the membranous components of the cells and a cytosolic fraction (Fig 9B). Western blotting revealed that np70 was predominantly associated with the nuclear fraction, with a minority associated with the membranous fraction. No detectable np70 was found in the cytosol. Immunostaining of nuclei for np70 gave results consistent with the blotting data, with np7Q nuclear localization essentially similar to that found in whole cells. Anti-np70 antibodies stained the nuclei of cells which had not been permeabilised, although the staining was stronger when the cells were extracted with triton X100. We interpret this as showing that np70 is localized both in the cytoplasmic face of the nucleus, but also within the nucleus.
Association of cytoplasmic np70 with vimentin In methanol fixed cells therewas some cytoplasmic filamentous staining reminiscent of intermediate filaments. The association of cytoplasmic np70 with vimentin-containing intermediate filaments was verified by double immunofluorescence in methanol fixed cells. Some np70 could be detected co-localized with vimentin filaments (Fig 10a,b) using normal epifluorescence, however this could be seen more clearly using digital confocal microscopy (Fig. l0c,d). Co-localization could not be detected in formaldehyde fixed, Triton X-100 extracted cells, either because formaldehyde destroyed the association, or because Triton X-100 solubilised much of the cytoplasmic nplOO.
Nuclear nplOO co-localizes with pre-mRNA splicing factors.
The nuclear staining of nplOO was granular, and on close examination using digital confocal microscopy, was localized to 20-30 distinct speckles within the nucleus, reminiscent of the distribution of pre-mRNA splicing factors. To determine whether np70 associated with these spliceosome factors double immunofluorescence was performed using antibodies to np70 and the pre- mRNA splicing factors sc35 and M". The staining patterns of sc35 and U2B" showed the expected speckles distributed throughout the nucleus (Fig llb,d), and digital confocal fluorescence experiments showed an almost 100% colocalization of np70 with both the pre-mRNA splicing factors sc35 (Fig lla,b) and U2B" (FiglOc,d). This co- localization was even more distinct when the cells were pre-extracted with Triton X-100 prior to fixation (Fig 10e-h). Not only was there evident co-localization in the nucleus, the size and intensity of the pre- mRNA splicing factor speckles correlated closely with that of np70.
Specificity control experiments using the.anti-mouse secondary antibodies with rabbit primary antibodies and vice versa showed only faint background fluorescence.
REFERENCES Bar-Sagi, D., Rotin, D., Batzer A., Mandiyan, V. and Schlessinger, J. 1993. SH3 domains direct cellular localization of signaling molecules. Cell 74:83-91.
Booker, G.W., Gout, I., Downing, A.K., Driscoll, P.C., Boyd, J., Waterfield, M.D. and-Campbell, I.D. 1993. Solution structure and LigandBinding Site of the SH3 Domain of the p85a Subunit of Phosphatidylinositol 3 Kinase. Cell, 73:1-20.
Cicchetti, P., Mayer b. J., Theil, G. and Baltimore D. 1992. Identification of a protein that binds to the SH3 region of abl and is similar to bcr and Gap-rho. Science 257:803-806. Cohen, G.B., Ren, R. and Baltimore, D. 1995. Modular 15 binding domains in signal transduction proteins. Cell, 80:237-248. Erpel, T., Alonso, G., Roche, S. and Courtneidge, S. A. 1996. The src SR3 domain is required for DNA synthesis induced by platelet-derived growth factor and epithelial 20 growth factor. J. Biol. Chem. 271:16807-16812.
Feng, S., Chen, J.K., Yu, H., -Simon, J.A. and Schreiber, S.L. 1994. Two Binding Orientations for Peptides to the Src SH3 Domain: Development of a General Model for SH3Ligand Interactions. Science, 266:1241-1247.
Finan, P.M., Shimizu, Y., Gout, I., Hsuan, J., Truong, 0., Butcher, C., Bennett, P., Waterfield, M.D. and Kellie, S. 1994. An SH3 domain and proline- rich sequence mediate an interaction between two components of the phagocyte NADPH oxidase complex J. Biol. Chem. 269:13752-13755.
Finan, P., Koga, H., Zvelebil, M. J., Waterfield, M. D. and Kellie S. 1996a. The C-terminal SH3 domain of p67phox binds its natural ligand in reverse orientation. J. mol Biol. 261:173-180.
Finan, P., Hall, A. and Kellie, S. 1996b. Sam68 from an imortalised B cell line associates with a subset of SH3 domains. FEBS Letters 389:141- 144. Fumigalli, S., Totty, N. F., Hsuan, J. and Courtniedge, S. A. 1994. A target for src in mitosis. Nature 368:871-874 10 Gout, I., Dhand, R., Hiles, I.D., Fry, M.J., Panayatou, G., Das, P., Truong, 0., Totty, N.F., Hsuan, J., Booker, G.W., Campbell, I.D., and Waterfield, M.D. 1993. The GTPase dynamin binds to and is activated by a subset of SH3 domains. Cell 75:25-36. 15 Harlan, J. E., Hajduk, P. J., Yoon, H. S., and Fesik, S. W. 1994. Pleckstrin homology domains bind to phosphatidylinositol-4,5bisphosphate. Nature 371:168-170. He, D., Nickerson, J. A. and Penman, S. 1990. Core filaments of the nuclear matrix. J Cell Biol. 110:569-581.
Hobert, 0., Jallal, B., Schlessinger, J. and Ullrich, A. 1994. Novel signaling pathway suggested by SH3 domainmediated P95"'/heteroribonucleoprotein K interaction. J. Biol. Chem. 269:2022520228.
Kaslauskas, A., Kashishian, A., Cooper, J. A. and Valius M. 1992. GTPase-activating protein and phosphatidylinositol 3-kinase bind to a distinct region of the platelet-derived growth factor receptor P subunit.
Mol. Cell Biol. 12:2534-2544.
Kohda, D., Hatanak, H., Odaka, M., Mandiyan, V., Ullrich, A., Schlessinger J., Inagaki, F. 1993. Solution structure of the SH3 domain of phospholipase Cy. Cell 72:953-960.
Koyama, S., Yu, H., Dalgarno, D.C., Shin, T.B., Zydowsky, L.D. and Schreiber, S.L. 1993. Structure of the P13K SH3 Domain and Analysis of the SH3 FamilY. Cell, 72:945-952 Kypta, R. M., Goldberg, Y., Ulug, E.,r. and Courtneidge, S.
A. 1990. Association between the PDGF receptor and members of the src family of tyrosine kinases. Cell:62 481-492.
Lawe, D. C., Hahn, C. and Wong, A. J. 1997. The nck SH2/SH3 adaptor protein is prerent in the nucleus and associates with the nuclear protein SAM68. Oncogene 14:223-2312.
Lawson D. 1984. Distribution of epinamin in colloidal gold-labelled, quick-frozen deep-etched fibroblast cytoskeletons. J. Cell Biol. 99:1451-1460.
Lemmon, M. A., Ferguson, K. M., and Schlessinger, J. 1996.
PH domains: Diverse sequences with a common fold recruit signaling molecules to the cell surface. Cell 85:621-624.
McPhail, L.C. 1994. SH3-dependent Assembly of the Phagocyte NADPH Oxidase. J. Exp.Med., 180:2011-2015. - Moran, M.F., Koch, C. A., Anderson D., Ellis C., England L., Martin G.S. and Pawson, T. 1995. SRC homology region 2 domains direct protein-protein interactions in signal transduction. Proc. Natl. Acad. Sci. USA 87:8622-8626.
Musacchio, A., Saraste, M. and Wilmanns, M. 1994. Highresolution crystal structures of tyrosine kinase SH3 domains complexed with proline-rich peptides. Nature Structural Biology. 1:546-551.
Noble, M.E.M., Musacchio, A., Saraste, M., Courtneidge, S.A. and Wierenga, R.K. 1993. Crystal structure of the SH3 domain in human Fyn; comparison of the threedimensional structures of SH3 domains in tyrosine kinases and spectrin. EMBO J, 12:2617-2624 Otsu, M., Hiles I., Gout I., Fry, M. J., Ruiz-Larrea, Panatotou, G., Thompson, A., Dhand, R., Hsuan, J., Totty, N., Smith, A. D., Morgan, S., Courtneidge, S., Parker, P. J. and Waterfield M. D. 1991. Characterization of two 85kD proteins that associate with receptor tyrosine kinase, middle T/pp60'-"' complexes and P13 kinase. Cell 65:91104.
Pawson, T.. 1995. Protein modules and signaling networks. Nature 373:573580.
Pawson, T and Scott, J. D. 1997. Signaling through scaffold, anchoring and adaptor proteins. Science 278:2075-2080.
Ren, R., Mayer, B. J., Cichetti, P. and Baltimore D. 1993. Identification of a ten amino acid proline-rich SH3 binding site. Science 259:1157-1161.
Rosakis-Adcock, M., Fernley, R., Wade, J., Pawson, T and Bowtell, D. 1993. Ther SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the ras activator mSosl. Nature 363:83-85.
Salim, K., Bottomley, M. J., Querfurth E., Zvelebil, M. J., Gout, I., Scaife, R., Gigg, R., Driscoll, P. C., Waterfield, M. D., and Panayotou, G. 1997. Distinct specificity in the recognition region of phosphoinositides by the pleckstrin homology (PH) domains of dynamin and Bruton's tyrosine kinase (Btk). EMBO J. 15:6241-6250.
Smith, D. B., and Johnson, K.S. 1988. Single step purification of polypeptides expressed in Escherichia coli as fusions with glutathione Stransferase. Gene 67:31-40.
Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel B. J. , Birge, R. B., Fajardo, F. E., Chou, M. M., Hanafusa, H., Schaffhausen, B. and Cantley, L. 1993. SH2 domains recognize specific phosphopeptide sequences. Cell 72:767-778. Songyang, Z., Shoelson, S. E., MeGlade, J., Olivier, P., Pawson, T., Bustelo, X. R., Barbacid, M., Sabe, H., Hanafusa, H., Yi, T., Ren, R., Baltimore, D., Ratnofsky, 15 S., Feldman R. A. and Cantley L. C. 1994. Specific motifs recognized by the SH2 domains of csk, 3BP2, fps/fes, grb2, HCP, shc, syk and vav. M61 Cell. Biol. 14:2777-2785. Spe. ctor, D. L., Fu, X. D. and Maniatis T. 1991. Associations between distinct pre-mRNA splicing components 20 and the cell nucleus. EMBO J. 10:3467-3481 Taylor, S. J. and Shalloway, D. 1994. An RNA-binding protein associated with src through its SH2 and SH3 domain in mitosis. Nature 368:867-871.
Totty, N. F., Waterfield, M. D. and Hsuan, J. J. 1992.
Accelerated high-sensitivity microsequencing of proteins and peptides using a miniature reaction cartridge. Protein Scl. 1:1215-1224.
Waksman G., Shoelson, S. E., Pant, N., Cowburn, D. and Kuriyan J. 1993. Binding of a high affinity phosphotyrosyl peptide to the src SH2 domain: Crystal structures of the complexed and peptide-free forms. Cell 72:779-790.
Wilson, L., Butcher, C., Finan, P. and Kellie, S. 1997. SH3 domainmediated interactions involving the phox components of the NADPH oxidase. Inflammation Res. 46:265271.
Yu, H., Chen, J.K., Feng, S., Dalgarno, D.C., Brauer, A.W. and Schreiber, S.L. 1994. Structural Basis for the Binding of Proline-Rich Peptides to SH3 Domains. Cell 76:933-945.
Claims (19)
1. An SH3 binding protein or fragment thereof which has (i) a sequence of amino acid residues having SH3 domain binding activity selected from the group which comprises:
PPPGAP -SEQ ID NO:l PPPGPPPP -SEQ ID NO:12 PLPDMP -SEQ ID NO:2 PRLPPGRKPP -SEQ ID NO:13 PPPGLP -SEQ ID NO:3 PRGPPPRLPP -SEQ ID NO:14 PMPGPP -SEQ ID NO:4 PPGPPPRGPP -SEQ ID NO:15 PPPRGP -SEQ ID NO:5 PAPPLR -SEQ ID NO:16 PPPRLP -SEQ ID NO:6 PGPPPR -SEQ ID NO:17 PPPLGP -SEQ ID NO:7 RPPLVP -SEQ ID NO:18 PPPGRP -SEQ ID NO:8 RKPPGP -SEQ ID NO:19 PPPAPP -SEQ ID NO:9 RPPGMP -SEQ ID NO:20 PPPAPP -SEQ ID NO:10 RPPGPP -SEQ ID NO:21 PPPGPP -SEQ ID NO:11 RPPGPP -SEQ ID NO:22 and (ii) a sequence of amino acid residues having a nuclear localisation activity.
2. An SH3 binding protein or fragment thereof which has (i) a sequence of amino acid residues having SH3 domain binding activity and (ii) a sequence of amino acid residues having a nuclear localisation activity selected from the group which comprises:
RKEARKRELKKNKK -SEQ ID NO:23 KDKRKK -SEQ ID NO:24 PPRRRDED -SEQ ID NO:25 PGKSRKKK -SEQ ID NO:26.
3. An SH3 binding protein or fragment thereof comprises at least one amino acid sequence of claim 1 having SH3 binding activity and at least one amino acid sequence of claim 2 having nuclear localisation activity.
4. An SH3 binding protein or fragment thereof comprises at least two amino acid sequences of claim 1 having SH3 binding activity and at least one amino acid sequence of claim 2 having nuclear localisation activity.
5. An SH3 binding protein or fragment thereof comprises a sequence selected from the group which comprises the full length np70 sequence:
MGRRSTSSTKSGKFMNPTDQARKEARKRELKKNKKQRMMVRAAVLKMKDPKQ IIRDMEKLDEMEFNPVQQPQLNEKVLKDKRKKLRETFERILRLYEKENPDIY KELRKLEVEYEQKRAQLSQYFDAVKNAQHVEVESIPLPDMPHAPSNILIQDI PLPGAQPPSILKKTSAYGPPTRAVSILPLLGHGVPRLPPGRKPPGPPPGPPP PQVVQMYGRKVGFALDLPPRRRti-EDMLYSPELAQRGHDDDVSSTSEDDGYPE DMDQDKHDDSTDDSDTDKSDGESDGDEFVHRDNGERDNNEEKKSGLSVRFAD MPGKSRKKKKNMKELTPLQAMMLRMAGQEIPEEGREVEEFSEDDDEDDSDDS EAEKQSQKQHKEESHSDGTSTASSQQQAPPQSVPPSQIQAPPMPGPPPLGPP PAPPLRPPGPPTGLPPGPPPGAPPFLRPPGMPGLRGPLPRLLPPGPPPGRPP GPPPGPPPGLPPGPPPRGPPPRLPPPAPPGIPPPRPGMMRPPLVPPLGPAPP GLFPPAPLPNPGVLSAPPNLIQRPKADDTSAATIEKKATATISAKPQITNPK AEITRFVPTALRVRRENKGATAAPQRKSE-DDSAVPLAKAAPKSGPSVPVSVQ TKDDMAFMKEMEGLL -SEQ ID NO:27 and the carboxy terminal region thereof:
APPQSVPPSQIQAPPMPGPPPLGPPPAPPLRPPGPPTGLPPGPPPGAPPFLR PPGMPGLRGPLPRLLPPGPPPGRPPGPPPGPPPGLPPGPPPRGPPPRLPPPA PPGIPPPRPGMMRPPLVPPLGPAPPGLFPPAPLPNPGVLSAPPNLIQRPKAD DTSAATIEKKATATISAKPQITNPKAEITRFVPTALRVRRENKGATAAPQRK SEDDSAVPLAKAAPKSGPSVPVSVQTKDDVYEAFMKEMEGLL -SEQ ID NO:28.
6. An SH3 binding protein or fragment thereof according to any preceding claim which comprises a plurality of nuclear localisation signal sequences.
7. An SH3 binding protein or fragment thereof according to any preceding claim wherein the carboxy terminal half of the protein or fragment is proline rich or comprises a consensus motif for SH3 domain binding selected from the group which comprises PXPXXP, MPMRMP, MPXR and RXPXXP.
8. An SH3 binding protein or fragment thereof according to any preceding claim which comprises a plurality of SH3 domain-binding sequences.
9. An SH3 binding protein or fragment thereof according to any preceding claim which comprises a prolifin binding site.
10. A DNA sequence which encodes the SH3 binding protein or fragment thereof according to any one of claims 1 to 9.
11. A DNA sequence according to claim 10 which comprises cDNA.
-
12. A DNA sequence according to claim 10 or 11 which comprises the sequence shown in figure 2.
13. An antibody or fragment thereof which is specific for the SH3 binding protein or a fragment thereof according to any one of claims 1 to 9.
14. A pharmaceutical composition which comprises the SH3 binding protein or fragment thereof according to any one of claims 1 to 9 together with a - 37 pharmaceutically acceptable carrier, diluent or excipient.
15. A pharmaceutical composition according to claim 14 formulated in accordance with routine procedures for administration in the form of tablets, capsules, powders, granules, lozenges, creams or liquid preparations, such as oral or sterile parenteral solutions or suspensions or in the form of a spray, aerosol or other conventional method for inhalation.
16. A method for production of the SH3 binding protein or fragment thereof according to any one of claims 1 to 9 which comprises the steps of lysing cells, carring out a binding assay with the lysate, eluting, sequencing and cloning the bound protein.
17. An SH3 binding protein or fragment thereof according to any one of claims 1 to 9 for use in a method of prevention or treatment of proliferative diseases including cancer and chronic inflammatory diseases.
18. An SH3 binding protein or fragment thereof according to any one of claims 1 to 9 for use in the manufacture of a medicament for the treatment or prevention of proliferative diseases including cancer and chronic inflammatory diseases.
19. A method of prevention or treatment of proliferative diseases including cancer and chronic inflammatory diseases which comprises administering a pharmaceutically effective amount of an SH3 binding protein or fragment thereof according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 14 or 15.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9819038A GB2341182A (en) | 1998-09-01 | 1998-09-01 | Protein comprising amino acid sequences having SH3 domain binding activity and nuclear localisation activity |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9819038A GB2341182A (en) | 1998-09-01 | 1998-09-01 | Protein comprising amino acid sequences having SH3 domain binding activity and nuclear localisation activity |
Publications (2)
Publication Number | Publication Date |
---|---|
GB9819038D0 GB9819038D0 (en) | 1998-10-28 |
GB2341182A true GB2341182A (en) | 2000-03-08 |
Family
ID=10838169
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB9819038A Withdrawn GB2341182A (en) | 1998-09-01 | 1998-09-01 | Protein comprising amino acid sequences having SH3 domain binding activity and nuclear localisation activity |
Country Status (1)
Country | Link |
---|---|
GB (1) | GB2341182A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1082415A1 (en) * | 1998-05-29 | 2001-03-14 | The Scripps Research Institute | Methods and compositions useful for modulation of angiogenesis using tyrosine kinase src |
US10376557B2 (en) * | 2006-06-13 | 2019-08-13 | Helix Biomedix Inc. | Peptide fragments for inducing synthesis of extracellular matrix proteins |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997030074A1 (en) * | 1996-02-16 | 1997-08-21 | Cytogen Corporation | Isolation and use of sh3 binding peptides |
-
1998
- 1998-09-01 GB GB9819038A patent/GB2341182A/en not_active Withdrawn
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997030074A1 (en) * | 1996-02-16 | 1997-08-21 | Cytogen Corporation | Isolation and use of sh3 binding peptides |
Non-Patent Citations (5)
Title |
---|
Biochemical and Biophysical Research Communications 1997,235(3),794-798 * |
Biochemical and Biophysical Research Communications 1997,239(2),488-492 * |
J.Biological Chemistry 1994,269(21),15092-15100 * |
Oncogene 1995,10,2367-2377 * |
Proc.Natl.Acad.Sci.USA 1997,94,7493-7498 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1082415A1 (en) * | 1998-05-29 | 2001-03-14 | The Scripps Research Institute | Methods and compositions useful for modulation of angiogenesis using tyrosine kinase src |
EP1082415A4 (en) * | 1998-05-29 | 2003-07-09 | Scripps Research Inst | Methods and compositions useful for modulation of angiogenesis using tyrosine kinase src |
US10376557B2 (en) * | 2006-06-13 | 2019-08-13 | Helix Biomedix Inc. | Peptide fragments for inducing synthesis of extracellular matrix proteins |
Also Published As
Publication number | Publication date |
---|---|
GB9819038D0 (en) | 1998-10-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7141417B1 (en) | Compositions, kits, and methods relating to the human FEZ1 gene, a novel tumor suppressor gene | |
JP2002531384A (en) | Peptide that modulates the interaction between B class ephrin and PDZ region | |
JP2003274984A (en) | IKK-beta-PROTEIN, NUCLEIC ACID AND METHOD | |
JPH08509119A (en) | TATA binding protein-related factor, nucleic acid encoding TAF and method of use | |
US5463023A (en) | Composition for inhibition of intracellular transcription | |
KR100565819B1 (en) | Compounds that inhibit interaction between signal-transducing proteins and the glgfpdz/dhr domain and uses thereof | |
US5744313A (en) | Assay employing novel protein domain which binds tyrosine phosphorylated proteins | |
AU745504B2 (en) | Disruption of the mammalian Rad51 protein and disruption of proteins that associate with mammalian Rad51 for hindering cell proliferation | |
US20180273585A1 (en) | Medical Use | |
US20030077262A1 (en) | Methods and compositions for modulating apoptosis | |
US5849580A (en) | Nucleic acid encoding a NF-κB activation regulatory protein, IκBβ | |
AU773957B2 (en) | Protein-protein interactions and methods for identifying interacting proteins and the amino acid sequence at the site of interaction | |
US20060228365A1 (en) | Protein and gene involved in myocyte differentiation | |
MXPA01008626A (en) | A novel inhibitor of programmed cell death. | |
CA2167303A1 (en) | Regulation of transcription factor, nf-il6/lap | |
US6172187B1 (en) | CD40 associated proteins | |
GB2341182A (en) | Protein comprising amino acid sequences having SH3 domain binding activity and nuclear localisation activity | |
US20020156258A1 (en) | Transcription factor of MHC class II genes, substances capable of inhibiting this new transcription factor and medical uses of these substances | |
PT1361439E (en) | Screening for peptides inhibiting pp1c binding to bcl-2, bcl-xl and bcl-w proteins | |
AU8072194A (en) | Receptor recognition factors, protein sequences and methods of use thereof | |
AU2018302855B2 (en) | Peptide derivative and pharmaceutical composition containing same | |
WO1999037768A1 (en) | Capon: a protein associated with neuronal nitric oxide synthase | |
US7060682B2 (en) | Receptor recognition factors, protein sequences and methods of use thereof | |
US20110117576A1 (en) | Mct-1, a human oncogene | |
AU2003231196C1 (en) | Androgen-regulated PMEPA1 and cancer |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WAP | Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1) |