WO1996020211A1 - Procede de detection de phosphotyrosine et peptides utilises dans le procede - Google Patents

Procede de detection de phosphotyrosine et peptides utilises dans le procede Download PDF

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Publication number
WO1996020211A1
WO1996020211A1 PCT/US1995/016988 US9516988W WO9620211A1 WO 1996020211 A1 WO1996020211 A1 WO 1996020211A1 US 9516988 W US9516988 W US 9516988W WO 9620211 A1 WO9620211 A1 WO 9620211A1
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Prior art keywords
receptor
peptide
interferon
ifn
protein
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PCT/US1995/016988
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English (en)
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WO1996020211A9 (fr
Inventor
Andrew Frederick Wilks
Ailsa Gwendoline Harpur
Andrew Charles Oates
Martin Lackmann
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Ludwig Institute For Cancer Research
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Application filed by Ludwig Institute For Cancer Research filed Critical Ludwig Institute For Cancer Research
Priority to KR1019970704314A priority Critical patent/KR100239330B1/ko
Priority to JP8520598A priority patent/JPH10511399A/ja
Priority to NZ301466A priority patent/NZ301466A/en
Priority to AU46900/96A priority patent/AU703367B2/en
Priority to EP95944551A priority patent/EP0797582A4/fr
Publication of WO1996020211A1 publication Critical patent/WO1996020211A1/fr
Publication of WO1996020211A9 publication Critical patent/WO1996020211A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones

Definitions

  • This invention relates to an assay for molecules which are able to interact with cytokine receptor intracellular peptides, in particular receptors of the non- protein tyrosine kinase domain type, and to peptides suitable for use in this assay.
  • the assay relates to detection of molecules which are able to bind to cytokine receptors.
  • RTK receptor protein tyrosine kinase family
  • PCR Polymerase Chain Reaction
  • SH2 domains are regions of protein sequences (modules) of approximately 100 amino acids which bind independently to phosphotyrosine residues.
  • the binding demonstrates high affinity and amino acid sequence specificity.
  • the direct, sequence-specific interaction of SH2 domains with extracellular signal-induced phosphotyrosine residues has been recognised as a unifying theme in intracellular signal transduction.
  • the provision of high affinity and specificity in this interaction appears to be the most important factor defining the intracellular outcome of extracellular interactions.
  • RTKs which are members of the protein tyrosine kinase (PTK) family
  • PTK protein tyrosine kinase
  • JAK family of PTKs (Just Another Kinase or JAnus Kinase) were discovered by polymerase chain reaction- based screening using degenerate oligonucleotide primers based on highly conserved PTK catalytic domain motifs. See for example Australian Patent Application No. 88229/91.
  • members of the JAK family of PTKs are employed as receptor " ⁇ chains", and these molecules then act in much the same way as do the intrinsic PTK domains of the RTK family.
  • JAK PTKs do not possess obvious SH2 and SH3 domains, they do have a PTK domain (JH1) and a kinase-related domain (JH2) , as well as 5 other JH domains.
  • PTK family can be grouped together with the receptors for growth hormone, prolactin, leukaemia inhibitory factor, erythropoietin and ciliary neurotrophic factor, and other hormones and growth factors such as oncostatin M, to form an extended family of cytokine receptors.
  • Members of this family are characterised by the presence of four highly conserved cysteine residues in the extracellular domain, and, except for the growth hormone receptor, the characteristic sequence motif -WSKWS-, usually close to a single transmembrane domain.
  • the cytokine receptors have little significant amino acid sequence homology within their intracellular domain, they are clearly an evolutionarily-related set of cell surface receptors.
  • Type I cytokine receptors either require ligand- induced ho odimerization for signal transduction, for example erythropoietin receptor and growth hormone receptor, or require recruitment of at least two distinct sub-units for effective high-affinity ligand binding and signal transduction. In the latter group, in many cases several different ligand-specific a sub-units are capable of combining with the same ⁇ sub-unit. For example, the a sub-units of the receptors for IL-3, IL-5 and GM-CSF all combine with the same ⁇ sub-unit.
  • the Type II cytokine receptors include the receptor for interferon- ⁇ and interferon-j8 (the IFN- ⁇ //8 receptor) and the interferon- ⁇ receptor. While interferon-ot and interferon-jS appear to share a common receptor, interferon- ⁇ binds to a separate receptor, and there is little or no sequence homology between the two groups of interferons. However, the cellular response elicited by the two receptor types shares common features, suggesting that they may recruit very similar intracellular signal transduction pathways.
  • interferon-stimulated gene factor 3 ⁇ (ISGF-3 ⁇ ) , which in turn binds to an interferon-stimulated response element
  • ISRE ISRE
  • the ISGF-3 complex consists of three polypeptides, formerly designated pll3, p91 and p84, but now known respectively as STAT2, STATl ⁇ and STATljS, where STAT stands for Signal Transducers and Activators of
  • STAT1 has two alternatively spliced forms, previously designated p91 or STAT91 and p84 or STAT84 respectively. They are now called STATlor and STAT1S respectively.
  • STAT2 is encoded by a different gene, and the protein was previously known as pll3 or STAT113.
  • STAT3, STAT4, STAT5 and STAT6 have been identified, and encode proteins that are also involved in an analogous manner to STATl ⁇ , STAT1S and STAT2 in signal transcription from various cytokine receptors.
  • Pathways activated by EGF, PDGF, IFN- ⁇ , IFN- ⁇ , CSF-1 and IL-6 utilise homologous DNA promoter sequences and common transcription factor components. While the IFN- ⁇ receptor utilises STAT2, STATl ⁇ and STAT1/8, the receptors for IFN- ⁇ , EGF, PDGF and CSF-1 use STATl ⁇ and other as yet unidentified phosphoproteins. There is also redundancy or overlap of the DNA response elements involved. For example, a number of cytokines cause activation of DNA binding proteins which recognise a single region, designated the IFN- ⁇ response region (GRR) , located within the promoter of the high-affinity Fc- ⁇ receptor gene. Thus IL-3, I -5, IL-10 and GM-CSF all ultimately activate transcription factors which bind to sequences within a single element, further demonstrating the overlap and degeneracy involved in intracellular signal transduction.
  • IFN- ⁇ response region a number of cytokines cause activation of DNA binding proteins which recognise
  • cytokine receptors which attract members of the STAT family via their SH 2 domains are at present poorly defined.
  • STAT binding site of the IFN- ⁇ receptor The tyrosine located at amino acid position 440 in the human sequence has been shown to be the most important residue in defining the binding of STATl ⁇ , and therefore in the commitment of the IFN- ⁇ treated cell to respond to this cytokine in the appropriate way.
  • Y440 a 16-amino acid peptide was synthesized, designated Y440, whose sequence corresponds to part of the sequence of the IFN- ⁇ receptor (KAPTSFGYDKPHVLVD; SEQ. ID. NO.
  • a biosensor (BIAcore TM ) - based assay using the interaction of STAT1 from crude cellular extracts with Y440 on a BIAcore TM chip was developed.
  • the assay is simple (it can be used to assay crude cellular extracts, with Y440 on a BIAcore” chip) , reproducible (the signal is strong and easily read) , and dependent upon the presence of phosphotyrosine in the target peptide. It has been further demonstrated that the binding is exclusively due to STAT1, and that the binding is both specific and of high affinity.
  • a robust assay for agonists, antagonists, modulators and mimics of the STATl/Y440-type interaction has been devised.
  • This assay therefore provides a means of rational drug design and/or screening for agonists, antagonists, modulators or mimics of the effects of IFN- ⁇ on cell metabolism, including but not limited to screening of natural products.
  • the same basic mechanism is predicted to operate for all cytokines which act through the JAK kinase-STAT nexus.
  • the corresponding STAT2 binding site on the ⁇ chain of the IFN a/ ⁇ receptor has been identified.
  • This is IFN- ⁇ R [Y466p] , corresponding to residues 460-474 of the IFN- ⁇ receptor (FLRCINYVFFPSLKP; SEQ. ID. NO. 2) .
  • An IFN ⁇ //S, I -2, IL-4, IL-5, IL-9, IL-13, IL-15, GM-CSF, growth hormone or prolactin specific drug screen is envisaged along the same lines.
  • Antagonists of IL-4 and IL-5 are useful as anti- atopy drugs in the treatment of conditions such as asthma and psoriasis.
  • Antagonists of GM-CSF are useful in the treatment of inflammatory conditions such as rheumatoid arthritis.
  • Antagonists of interferon are useful in the treatment of autoimmune diseases such as insulin-dependent diabetes mellitus.
  • Agonists of interferon and other immune mediators are useful in the treatment of neoplastic disease, including leukaemias.
  • Antagonists of haemopoietic growth factors are useful in the treatment of blood dy ⁇ plasias involving overproduction of blood cells, e.g. erythropoietin antagonists for treatment of overproduction of red cells, and antagonists of GM-CSF or CSF-1 for treatment of overproduction of white cells.
  • the invention provides a peptide corresponding to a tyrosine-containing region of the sequence of an intracellular domain of a cytokine or hormone receptor or of a JAK protein tyrosine kinase, wherein said peptide comprises a tyrosine residue which is able to be phosphorylated, and in its phosphorylated form is able to bind a cytoplasmic transcription factor of the STAT family.
  • the invention is applicable to any cytokine and its cognate STAT molecule.
  • some cytokines or hormones preferentially interact with STATl ⁇ , some with STAT13, and some with STAT2.
  • the skilled person will be able to identify the most appropriate STAT molecule to use with each desired cytokine receptor phosphopeptide, using methods known in the art or described herein.
  • the peptide is derived from a receptor for a protein selected from the group consisting of growth hormone, prolactin, leukaemia inhibitory factor, erythropoietin, ciliary neurotrophic factor, interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , IL-2, I -3, IL-4, IL-5, IL-6, IL-7, I -9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, GM-CSF, CSF-1, erythropoietin, EGF, PDGF and Oncostatin M.
  • a receptor for a protein selected from the group consisting of growth hormone, prolactin, leukaemia inhibitory factor, erythropoietin, ciliary neurotrophic factor, interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , IL-2, I -3, IL-4, IL-5, IL-6, IL-7, I
  • the peptide is derived from a receptor for a protein selected from the group consisting of growth hormone, prolactin, interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , IL-2, IL-4, IL-5, GM-CSF and erythropoietin.
  • the invention provides a peptide having the sequence KAPTSFGYDKPHVLVD (SEQ. ID. NO. 1), corresponding to residues Lys 433 to Asp 448 of the amino acid sequence of the interferon- ⁇ receptor protein, which in its phosphorylated form has the ability to bind STAT1.
  • the peptide and its phosphorylated form are referred to herein as IFN- ⁇ R [Y440] and IFN- ⁇ R [Y440 p ] receptor.
  • the peptide has the sequence F RCINYVFFPSLKP (SEQ. ID. NO. 2) , corresponding to residues Phe 460 to Pro 474 of the interferon- ⁇ receptor protein, which in its phosphorylated form has the ability to bind STAT2.
  • This peptide and its phosphorylated form are referred to herein as IFN- ⁇ R [Y466] and IFN- ⁇ R [Y466 p ] respectively.
  • homologues or variants of these peptide sequences are within the scope of the invention, provided that the relevant tyrosine is still present and able to be phosphorylated, and provided that the ability of the phosphorylated peptide to bind a STAT molecule is retained.
  • the peptides of the invention may be prepared by any convenient method; for example, they may be synthesised chemically, using conventional solid-phase techniques, they may be produced by recombinant DNA methods, or they may be prepared by cleavage of the appropriate receptor protein using appropriate enzyme techniques.
  • the receptor protein may be prepared by any convenient method.
  • the peptides of the invention are useful in the screening of molecules for their ability to interact with the receptor proteins.
  • the peptides of the invention may be used to screen for inhibitors of interferon- ⁇ , interferon- ⁇ , interferon-/8 or of other cytokines, such as interleukins, especially interleukin-4, interleukin-5 and interleukin-6, GM-CSF, G-CSF, other haemopoietins, including but not restricted to leukaemia inhibitory factor, and ciliary neurotrophic factor, and hormones and growth factors, such as growth hormone and EGF.
  • interferon- ⁇ , interferon- ⁇ , interferon-/8 or of other cytokines such as interleukins, especially interleukin-4, interleukin-5 and interleukin-6, GM-CSF, G-CSF, other haemopoietins, including but not restricted to leukaemia inhibitory factor, and ciliary neurotrophic factor,
  • cytokines which use the same basic signal transduction mechanisms as those of interferon- ⁇ are suitable for use with the peptides of this invention, especially IFN- ⁇ R [Y440 p ] .
  • any molecule whose activity is mediated by a STAT molecule or by the JAK-STAT pathway is within the scope of the invention.
  • the person skilled in the art will readily be able to test whether the receptor for a given protein belongs in this class, to identify a suitable phosphorylation site for use in the assay of the invention, and to prepare an appropriate peptide, using methods already known in the art.
  • tyrosine residues susceptible to phosphorylation can readily be identified.
  • Tyrosine(s) which are important to interaction with the cognate STAT molecule can be rapidly identified using routine site-directed mutagenesis. Once the relevant tyrosine residue is identified, conveniently- sized peptides can rapidly be synthesized and tested.
  • the invention provides a method of screening of molecules for their ability to interact with a receptor peptide, comprising the step of exposing a receptor peptide to a molecule to be screened, and measuring the degree of interaction between the peptide and the molecule.
  • the invention also provides a method of measuring the ability of a molecule to inhibit or promote interaction between a receptor peptide and a molecule known to be able to bind to said receptor, comprising the step of exposing the receptor peptide to a known molecule having the ability to bind to said receptor in the presence of a putative inhibitor or promoter, and measuring the ability of the putative inhibitor to inhibit or promote said binding.
  • the term "interact" is to be understood to encompass promoting or inhibiting the activity of the receptor peptide.
  • a molecule which interacts with a receptor peptide may act as an agonist or antagonist of the receptor, may mimic the activity of the receptor, or may modulate its activity directly or indirectly.
  • the interaction may take place either during the screening process, or may take place prior to screening.
  • this aspect of the invention includes within its scope detection of interactions which take place in vitro or in vivo, before preparation of the sample which is actually subjected to the assay, as well as interactions taking place within the sample. It is particularly envisaged that cell or tissue cultures whose medium is to be assayed, or an animal from which a tissue sample or biological fluid is to be assayed, may be pretreated with the molecule to be screened for its ability to modulate the interaction between a receptor peptide with its cognate STAT.
  • the molecules to be screened may be of either synthetic, recombinant or natural origin, and may be of a wide variety of structures.
  • the binding may be measured by any convenient means.
  • either the receptor peptide or the molecule to be tested for binding may be labelled with a detectable marker, such as a radioactive label, a fluorescent label, or a marker detectable by way of an enzyme reaction.
  • a detectable marker such as a radioactive label, a fluorescent label, or a marker detectable by way of an enzyme reaction.
  • detectable marker such as a radioactive label, a fluorescent label, or a marker detectable by way of an enzyme reaction.
  • detectable marker such as a radioactive label, a fluorescent label, or a marker detectable by way of an enzyme reaction.
  • detectable marker such as a radioactive label, a fluorescent label, or a marker detectable by way of an enzyme reaction.
  • detection systems are known in the art.
  • assay systems which are suitable for use in the methods of the second and third aspects of the invention include, but are not limited to, immunoassays such as enzyme linked immunosorbent assay (ELISA) ; affinity-type as
  • One particularly rapid and convenient test system uses an optical biosensor, such as the BIAcore" (Pharmacia Biosensor AB, Uppsala, Sweden) , which enables the use of proteins or peptides immobilized to a sensor chip.
  • the biosensor assay is very simple, reproducible and rapid, while having high specificity.
  • the biosensor assay enables a very high throughput of samples, and is amenable to automation, and to use with relatively crude samples, such as biological fluids, or cell or tissue extracts. It is therefore suitable for screening of natural products for their ability to inhibit or promote binding, or for activity as agonists or antagonists of cytokines. It is also suitable for use with biological fluids, for example in clinical assays, or culture media, including medium from recombinant bacterial or cell cultures.
  • the BIAcoreTM assay of the invention permits qualitative or quantitative analysis of the effects of putative modulators, including agonists and antagonists, on the kinetics of the STAT/STAT and STAT/receptor peptide interaction. Potentiation of STAT binding or decrease in affinity for STAT are characteristics which may be desirable in a given potential therapeutic agent.
  • FCS foetal calf serum
  • RU response units STAT signal transducers and activators of transcription.
  • Figure 1 shows a sensorgram profile of immunological detection of the phosphorylated tyrosine- containing peptide IFN- ⁇ R [Y440 p ] immobilized to a BIAcore sensor chip, using a phosphotyrosine-specific MAb ⁇ 1 ⁇ g/ml antiphosphotyrosine monoclonal antibody,
  • IFN- ⁇ R [Y440] -derivatised sensor chip O 1 ⁇ g/ml anti-STATl monoclonal antibody, peptide IFN- ⁇ R [Y440 p ] -derivatised sensor chip.
  • Figure 2 shows the relative response of IFN- ⁇ R [Y440 p ] peptides to crude lysates of HeLa 3 cells.
  • Figure 3 shows the correlation between the relative response of an IFN- ⁇ R [Y440 p ] -coupled biosensor chip with the presence of immunoreactive STAT1 in the cytosolic fraction of a HeLa S3 cell extract.
  • Figure 4 shows the effect of depletion of STAT1 from a nuclear extract of HeLa S3 cells on the response of a biosensor coupled to IFN- ⁇ R [Y440 p ] .
  • Figure 5 shows a sensorgram demonstrating immunodetection of STAT1 binding to an IFN- ⁇ R [RY440 p ] - coupled sensor chip.
  • A Immunoblot detection of STAT1 in cytosolic extracts of cell lines expressing STAT1, but not in a mutant cell line which does not express this protein;
  • B Sensorgrams showing immunodetection of
  • BIAcore TM biosensor for the analysis of biomolecular interactions (Fagerstam, 1991; J ⁇ nsson et al, 1991; J ⁇ nsson and Malmg ist, 1992), in particular for the analysis of phosphopeptide/protein interactions, has been described elsewhere (Felder et al, 1993; End et al, 1992; Panayotou et al, 1993) and in application notes issued by Pharmacia. Unless otherwise stated, the running buffer used in the BIAcore TM experiments was 20 mM HEPES, pH 7.4, 150 mM NaCl, 2.8 mM EDTA, 0.005% Tween 20.
  • the phosphotyrosine-specific MAb and the anti-STATl MAb were diluted into BIAcore TM running buffer at 1 ⁇ g/ml prior to injection.
  • the anti-STATl MAb was provided at 0.25 mg/ml in a solution containing 1.0 mg/ml BSA and 50% glycerol as stabilizers. The presence of these additives does not have an effect on the immunoreactivity of the MAb (see Example 5) , but is evident in the sensorgram as a substantial refractive index change.
  • a 35 ⁇ l sample containing 1 ⁇ g/ml of a phosphotyrosine-specific MAb (Upstate Biotechnology Incorporated, Lake Placid, NY, USA. Cat. No. 05-321) was passed at 5 ⁇ l/min over a BIAcore TM sensor chip which had been derivatised with approximately 0.5 ng of IFN- ⁇ receptor-derived peptide [Y440] . Following the sample pulse the chip surface was washed for 750 s with running buffer followed by injection of 30 ⁇ l of 20 mM phenylphosphate to desorb phosphopeptide-bound MAb. The sensorgram profile is shown in Figure 1. Injection of sample, end of injection and injection of desorption solution are indicated with A l, ⁇ 2, ⁇ 3, respectively.
  • the freeze-dried phosphopeptide IFN- ⁇ R [Y440 p ] was dissolved in a volume of 700 ⁇ l at 2 mg/ml into DMSO and incubated with 500 ⁇ l of Affi-10 NHS-activated agarose (BioRad) for 8 hours at room temperature. Non-reacted NHS groups of the affinity resin were blocked by incubation with 1 M ethanolamine-HCl, pH 8.5.
  • the immobilization of peptide was monitored by microbore RP-HPLC (with on-line UV-spectroscopy) of samples taken prior to and following the coupling and blocking steps, and coupling efficiency was estimated from the peak area (absorbance at 214 nm) obtained for the analysed peptide solutions. The coupling efficiency under these conditions was 20%, yielding 280 ⁇ g peptide/ml of agarose.
  • IFN- ⁇ R peptide [Y440] was seen in any of the fractions.
  • IFN- ⁇ causes both a net increase in IFN- ⁇ R [Y440 p ] binding (RU) and a shift of the binding factor to an apparently higher M_, as determined by size exclusion-HPLC. This change correlates with an increased quantity and apparent M_ of immunoreactive STATl under non-denaturing conditions during size exclusion HPLC, suggesting its identity with the binding factor.
  • Example 2 but activated with IFN- ⁇ only 15 minutes prior to extraction.
  • Nuclear extracts were prepared as described by Dignam et al (1983), with the following modifications. The insoluble material removed after homogenization was resuspended in High-salt Buffer [20 mM Hepes pH 7.9, 25% glycerol, 1.5 mM MgCl 2 , 1.2 M KC1, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT] , and allowed to incubate for 30 minutes on ice. Nuclear matrices were removed by centrifugation for 20 minutes at 25,000 g. Nuclear extract and the anti-STATl affinity eluate were exchanged into assay buffer, as described in Example 2.
  • the dilution of the extract prior to affinity absorption was chosen to yield a BIAcore response within the same range as the other samples.
  • IFN- ⁇ R [Y440 p ] derivatised sensor chip The nuclear extract of IFN- ⁇ activated HeLa cells was purified on an IFN- ⁇ R [Y440 p ] affinity resin and the phenylphosphate eluate of the affinity matrix was exchanged into the appropriate assay buffer, as described in Example 2. The eluate was injected either directly (A, B) or after incubation with anti-STATl MAb (C) over a sensor chip derivatised with IFN- ⁇ R [Y440 ] . In a second injection immediately following the first sample plug and prior to the start of the dissociation phase, either buffer (A, C) or anti-STATl MAb (B) was injected on to the same channel of the sensor chip.
  • Figure 5 illustrates an overlay of BIAcore responses normalised to the same baseline.
  • the individual injections are marked with an arrow labelled "1" (first injection) and "2" (second injection) .
  • the response levels at the start of the second injection are indicated.
  • Activated HeLa cells were fractionated as described in Example 4, and a 4 ml sample of the nuclear fraction was extracted on 0.5 ml IFN- ⁇ R [Y440 p ] -agarose for 30 minutes at 0°C as described in Example 2.
  • the affinity resin was washed quickly with 8 column volumes of assay buffer and eluted with one column volume of phenylphosphate (20 mM) , collected in a single 0.5 ml fraction. The breakthrough from this column was loaded again under identical conditions. This second binding step increased recovery from the lysate to 90%. Both eluates were pooled, and a 90 ⁇ l aliquot incubated for 30 minutes at room temperature with 10 ⁇ l of anti-STATl MAb
  • the cells were grown to 80% confluency, and harvested, following 2 washes in ice cold PBS supplemented with 0.1 mM Na 3 V0 4 , in 1 ml tryp ⁇ in-versene. Following trypsinisation, cells were washed twice in DMEM plus 10% foetal calf serum, and twice in ice-cold PBS supplemented with 0.1 mM Na 3 V0 4 . HeLa S3 cells were grown as described in Example 2. Cytoplasmic extracts were then prepared as described in Example 2.
  • BIAcoreTM detection of IFN- ⁇ R [Y440 p ] binding activity in the above samples was carried out as follows. Samples of 10-fold concentrated cytosolic extracts, prepared as described above, were incubated either with IFN- ⁇ R [Y440 p ] affi agarose or IFN- ⁇ R [Y440] affi agarose, aspirated after 30 minutes at room temperature, and then injected at 5 ⁇ l/minutes onto BIAcoreTM sensor chips, derivatised on parallel channels with IFN- ⁇ R [Y440] or IFN- ⁇ R [Y440 p ] as described in Example 1.
  • Example 7 Other Potential STAT Binding Sites Tyrosine-containing amino acid sequences in which the tyrosine can be phosphorylated are present in a number of proteins which have been reported in the literature. Some of these phosphotyrosine residues are located upon cytokine receptors, whereas others are found towards the C- terminal end of the STATs themselves. The putative STAT binding sites are summarized in Table 1, with the location of the phosphotyrosine identified in brackets. This location uses sequence numbering as indicated by mutation analysis.
  • STATl ⁇ Y701 Only the STATl ⁇ Y701 is demonstrably a STAT binding site, while the other sites have been shown to be strong candidates for STAT binding by virtue of the fact that either phenylalanine substitution of the tyrosine results in loss of STAT binding (for example in the case of the EPO-R Y343 or the PRL-R Y580) , or the sites lie in a region which appears to be required for signal transduction down-stream of a particular cytokine receptor (for example in the case of the LIF-R and gpl30) .
  • a further class of predicted sites has been derived on the basis of what is known about STAT binding sites, coupled with what is known in the field of cytokine receptor-mediated signal transduction (usually denoted as Harpur, 1995) .
  • IFN- ⁇ Treatment of the cells with IFN- ⁇ causes both a net increase in IFN- ⁇ R [Y440 p ] binding (RU) and a shift of the binding factor to an apparently higher M_ as determined by size exclusion-HPLC. This change correlates with an increased quantity and apparent M r of immunoreactive STATl, suggesting its identity with the binding factor (Example 3) .
  • SH2 domains exhibit high-affinity binding to tyrosine- phosphorylated peptides yet also exhibit rapid dissociation and exchange
  • Ligand-induced IFNg receptor tyrosine phosphorylation couples the receptor to its signal transduction system
  • MOLECULE TYPE peptide
  • HYPOTHETICAL no
  • FRAGMENT TYPE internal

Abstract

L'invention concerne un procédé de détection de moléculles capables d'exercer une interaction avec des peptides intracellulaires du récepteur de cytokine, ainsi que les peptides pouvant être utilisés dans ledit procédé. Ces peptides correspondent à des régions contenant tyrosine de la séquence d'un domaine intracellulaire d'une cytokine, d'un récepteur d'hormone ou d'une tyrosine kinase de protéine de la famille JAK et comprennent une tyrosine capable d'être phosphorylée. Sous la forme phosphorylée, ces peptides peuvent se fixer à des facteurs de transcription cytoplasmique de la famille STAT.
PCT/US1995/016988 1994-12-23 1995-12-22 Procede de detection de phosphotyrosine et peptides utilises dans le procede WO1996020211A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
KR1019970704314A KR100239330B1 (ko) 1994-12-23 1995-12-22 포스포티로신 분석 및 여기에서 사용하기 위한 펩티드
JP8520598A JPH10511399A (ja) 1994-12-23 1995-12-22 ホスホチロシン・アッセイおよびそれに使用するペプチド
NZ301466A NZ301466A (en) 1994-12-23 1995-12-22 Assay for interacting with cytokine receptor intracellular peptides ifn-gamma
AU46900/96A AU703367B2 (en) 1994-12-23 1995-12-22 Phosphotyrosine assay and peptides for use therein
EP95944551A EP0797582A4 (fr) 1994-12-23 1995-12-22 Procede de detection de phosphotyrosine et peptides utilises dans le procede

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AUPN0249A AUPN024994A0 (en) 1994-12-23 1994-12-23 Assay and peptides for use therein
AUPN0249 1994-12-23

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WO1996020211A1 true WO1996020211A1 (fr) 1996-07-04
WO1996020211A9 WO1996020211A9 (fr) 1996-08-29

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EP (1) EP0797582A4 (fr)
JP (1) JPH10511399A (fr)
KR (1) KR100239330B1 (fr)
AU (2) AUPN024994A0 (fr)
CA (1) CA2208318A1 (fr)
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000050631A2 (fr) * 1999-02-25 2000-08-31 Cyclacel Limited Compositions et methodes de controle de la modification de partenaires de liaison naturels
WO2000050902A2 (fr) * 1999-02-25 2000-08-31 Cyclacel Limited Dosage a haut rendement
WO2000050630A2 (fr) * 1999-02-25 2000-08-31 Cyclacel Limited Dosage permettant une mesure simultanee de l'activite de differentes enzymes
WO2000050631A3 (fr) * 1999-02-25 2000-12-07 Fluorescience Ltd Compositions et methodes de controle de la modification de partenaires de liaison naturels
WO2000050902A3 (fr) * 1999-02-25 2000-12-14 Fluorescience Ltd Dosage a haut rendement
WO2000050630A3 (fr) * 1999-02-25 2000-12-21 Fluorescience Ltd Dosage permettant une mesure simultanee de l'activite de differentes enzymes
US6656696B2 (en) 1999-02-26 2003-12-02 Cyclacel Compositions and methods for monitoring the phosphorylation of natural binding partners
US7166475B2 (en) 1999-02-26 2007-01-23 Cyclacel Ltd. Compositions and methods for monitoring the modification state of a pair of polypeptides
WO2000052057A1 (fr) * 1999-03-04 2000-09-08 Biomolecular Engineering Research Institute Cristaux et coordonnees structurales d'un complexe proteique et utilisation desdites coordonnees structurales
US7795383B2 (en) * 2004-06-16 2010-09-14 Affinergy, Inc. IFBM's to promote the specific attachment of target analytes to the surface of orthopedic implants

Also Published As

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CA2208318A1 (fr) 1996-07-04
JPH10511399A (ja) 1998-11-04
EP0797582A1 (fr) 1997-10-01
AU703367B2 (en) 1999-03-25
AU4690096A (en) 1996-07-19
EP0797582A4 (fr) 1999-06-16
AUPN024994A0 (en) 1995-01-27
NZ301466A (en) 1998-01-26
KR100239330B1 (ko) 2000-02-01

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