EP1841882A1 - Phosphorylation des proteines suivant des voies controlees par les egfr - Google Patents

Phosphorylation des proteines suivant des voies controlees par les egfr

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Publication number
EP1841882A1
EP1841882A1 EP04815061A EP04815061A EP1841882A1 EP 1841882 A1 EP1841882 A1 EP 1841882A1 EP 04815061 A EP04815061 A EP 04815061A EP 04815061 A EP04815061 A EP 04815061A EP 1841882 A1 EP1841882 A1 EP 1841882A1
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European Patent Office
Prior art keywords
rows
protein
corresponding column
tyrosine
column
Prior art date
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EP04815061A
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German (de)
English (en)
Inventor
Albrecht Moritz
Yu Li
Charles Farnsworth
Klarisa Rikova
Kimberly Lee
Ailan Guo
Roberto Polakiewicz
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Cell Signaling Technology Inc
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Cell Signaling Technology Inc
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Publication of EP1841882A1 publication Critical patent/EP1841882A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G01N2333/4706—Regulators; Modulating activity stimulating, promoting or activating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the invention relates generally to antibodies and peptide reagents for the detection of protein phosphorylation, and to protein phosphorylation in cancer.
  • Ligand binding induces homodimerization, leading to ATP-mediated autophosphorylation or transphosphorylation (by a partner kinase) of EGFR, which in turn activates its kinase function (Russo et al., 1985 J. Biol. Chem. 260: 5205-5208).
  • the sites reported to be most important in terms of receptor phosphorylation and activation are Tyr 1148, Tyr1173, Tyr1068, and Tyr1086 (Downward et al. (1984) Nature 311: 483-485; Margolis et al. (1989) J. Biol. Chem. 264: 10667-10671 ).
  • Herceptin® an inhibitor of HER2/neu
  • IressaTM ZD1839
  • TarcevaTM another small molecule inhibitor
  • NSCLC non-small cell lung carcinoma
  • the invention discloses 168 novel phosphorylation sites identified in signal transduction proteins and pathways downstream of, and including, EGFR, and provides new reagents, including phosphorylation- site specific antibodies and AQUA peptides, for the selective detection and quantification of these phosphorylated sites/proteins. Also provided are methods of using the reagents of the invention for the detection and quantification of the disclosed phosphorylation sites.
  • FIG. 3 - is an exemplary mass spectrograph depicting the detection of the tyrosine 998 phosphorylation site in EGFR (see Row 123 in Figure 2/Table 1 ), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); pY and Y* indicate the phosphorylated tyrosine (shown as lowercase "y" in Figure 2).
  • FIG. 5 - is an exemplary mass spectrograph depicting the detection of the tyrosine 1328 phosphorylation site in HER-3 (see Row 126 in Figure 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); pY and Y* indicate the phosphorylated tyrosine (shown as lowercase "y" in Figure 2).
  • the reagents provided by the invention is an isolated phosphorylation site-specific antibody that specifically binds the STAT3 transcription factor only when phosphorylated (or only when not phosphorylated) at tyrosine 539 (see Row 154 (and Columns D and E) of Table 1/ Figure 2).
  • the group of reagents provided by the invention is an AQUA peptide for the quantification of phosphorylated STAT3 protein, the AQUA peptide comprising phosphorylatable peptide sequence listed in Column E, Row 154, of Table 1/ Figure 2 (which encompasses the phosphorylatable tyrosine at position 539).
  • the invention provides a heavy-isotope labeled peptide (AQUA peptide) for the quantification of an EGFR-related signaling protein selected from Column A of Table 1 , said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1-168), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D of Table 1.
  • the phosphorylatable tyrosine within the labeled peptide is phosphorylated, while in other preferred embodiments, the phosphorylatable tyrosine within the labeled peptide is not phosphorylated.
  • antibodies and AQUA peptides for the detection/quantification of the following Adaptor/Scaffold protein phosphorylation sites are particularly preferred: CbI (Y445), IRS-2 (Y653, Y675, Y823), STAM1 (Y198), and STAM2 (Y113) (see SEQ ID NOs: 9, 13-15, and 25-26).
  • antibodies and AQUA peptides for the detection/quantification of the following Receptor Tyrosine Kinase phosphorylation sites are particularly preferred: EGFR (Y998), HER2 (Y923), and HER3 (Y1307, Y1328) (see SEQ ID NOs: 122-125).
  • EGFR Y998)
  • HER2 Y923
  • HER3 Y1307, Y1328
  • antibodies and AQUA peptides for the detection/quantification of the following Vesicle protein phosphorylation sites are particularly preferred: Syntaxin 4 (Y115) (see SEQ ID NO: 168).
  • Antibody refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE, including F a b or antigen-recognition fragments thereof, including chimeric, polyclonal, and monoclonal antibodies.
  • Isolated phosphorylation site-specific antibodies that specifically bind an EGFR-related signaling protein disclosed in Column A of Table 1 only when phosphorylated (or only when not phosphorylated) at the corresponding amino acid and phosphorylation site listed in Columns D and E of Table 1 may now be produced by standard antibody production methods, such as anti-peptide antibody methods, using the phosphorylation site sequence information provided in Column E of Table 1. For example, two previously unknown HER3 kinase phosphorylation sites (tyrosines 1307 and 1328) (see Rows 125-126 of Table 1/Fig. 2) are presently disclosed.
  • the invention also provides immortalized cell lines that produce an antibody of the invention.
  • hybridoma clones constructed as described above, that produce monoclonal antibodies to the EGFR- related signaling protein phosphorylation sitess disclosed herein are also provided.
  • the invention includes recombinant cells producing an antibody of the invention, which cells may be constructed by well known techniques; for example the antigen combining site of the monoclonal antibody can be cloned by PCR and single-chain antibodies produced as phage-displayed recombinant antibodies or soluble antibodies in E. coli (see, e.g., ANTIBODY ENGINEERING PROTOCOLS, 1995, Humana Press, Sudhir Paul editor.)
  • Antibodies may be further characterized via immunohistochemical (IHC) staining using normal and diseased tissues to examine EGFR- related phosphorylation and activation status in diseased tissue.
  • IHC immunohistochemical staining may be carried out according to well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988). Briefly, paraffin-embedded tissue (e.g.
  • Phosphorylation-site specific antibodies of the invention specifically bind to a human EGFR-related signal transduction protein or polypeptide only when phosphorylated at a disclosed site, but are not limited only to binding the human species, perse.
  • the invention includes antibodies that also bind conserved and highly homologous or identical phosphorylation sites in respective EGFR-related proteins from other species (e.g. mouse, rat, monkey, yeast), in addition to binding the human phosphorylation site. Highly homologous or identical sites conserved in other species can readily be identified by standard sequence comparisons, such as using BLAST, with the human EGFR-related signal transduction protein phosphorylation sites disclosed herein.
  • novel EGFR-related signaling protein phosphorylation sites disclosed herein now enable the production of corresponding heavy- isotope labeled peptides for the absolute quantification of such signaling proteins (both phosphorylated and not phosphorylated at a disclosed site) in biological samples.
  • the production and use of AQUA peptides for the absolute quantification of proteins (AQUA) in complex mixtures has been described. See WO/03016861 , "Absolute Quantification of Proteins and Modified Forms Thereof by Multistage Mass Spectrometry," Gygi et al. and also Gerber et a/. Proc. Natl. Acad. Sci. U.S.A.
  • the second stage of the AQUA strategy is its implementation to measure the amount of a protein or modified protein from complex mixtures.
  • Whole cell lysates are typically fractionated by SDS-PAGE gel electrophoresis, and regions of the gel consistent with protein migration are excised. This process is followed by in-gel proteolysis in the presence of the AQUA peptides and LC-SRM analysis.
  • AQUA peptides are spiked in to the complex peptide mixture obtained by digestion of the whole cell lysate with a proteolytic enzyme and subjected to immunoaffinity purification as described above.
  • the retention time and fragmentation pattern of the native peptide formed by digestion e.g.
  • trypsinization is identical to that of the AQUA internal standard peptide determined previously; thus, LC-MS/MS analysis using an SRM experiment results in the highly specific and sensitive measurement of both internal standard and analyte directly from extremely complex peptide mixtures. Because an absolute amount of the AQUA peptide is added (e.g. 250 fmol), the ratio of the areas under the curve can be used to determine the precise expression levels of a protein or phosphorylated form of a protein in the original cell lysate.
  • the size of the peptide is also optimized to maximize ionization frequency. Thus, peptides longer than about 20 amino acids are not preferred. The preferred ranged is about 7 to 15 amino acids.
  • a peptide sequence is also selected that is not likely to be chemically reactive during mass spectrometry, thus sequences comprising cysteine, tryptophan, or methionine are avoided.
  • a peptide sequence that does not include a modified region of the target region may be selected so that the peptide internal standard can be used to determine the quantity of all forms of the protein.
  • a peptide internal standard encompassing a modified amino acid may be desirable to detect and quantify only the modified form of the target protein.
  • Peptide standards for both modified and unmodified regions can be used together, to determine the extent of a modification in a particular sample (i.e. to determine what fraction of the total amount of protein is represented by the modified form).
  • peptide standards for both the phosphorylated and unphosphorylated form of a protein known to be phosphorylated at a particular site can be used to quantify the amount of phosphorylated form in a sample.
  • the peptide is labeled using one or more labeled amino acids (Ae. the label is an actual part of the peptide) or less preferably, labels may be attached after synthesis according to standard methods.
  • the label is a mass-altering label selected based on the following considerations: The mass should be unique to shift fragments masses produced by MS analysis to regions of the spectrum with low background; the ion mass signature component is the portion of the labeling moiety that preferably exhibits a unique ion mass signature in MS analysis; the sum of the masses of the constituent atoms of the label is preferably uniquely different than the fragments of all the possible amino acids.
  • Peptide internal standards are characterized according to their mass-to-charge (m/z) ratio, and preferably, also according to their retention time on a chromatographic column (e.g. an HPLC column). Internal standards that co-elute with unlabeled peptides of identical sequence are selected as optimal internal standards.
  • the internal standard is then analyzed by fragmenting the peptide by any suitable means, for example by collision-induced dissociation (CID) using, e.g., argon or helium as a collision gas.
  • CID collision-induced dissociation
  • the fragments are then analyzed, for example by multi-stage mass spectrometry (MS ⁇ ) to obtain a fragment ion spectrum, to obtain a peptide fragmentation signature.
  • MS ⁇ multi-stage mass spectrometry
  • peptide fragments have significant differences in m/z ratios to enable peaks corresponding to each fragment to be well separated, and a signature is that is unique for the target peptide is obtained. If a suitable fragment signature is not obtained at the first stage, additional stages of MS are performed until a unique signature is obtained.
  • Each isolated peptide is then examined by monitoring of a selected reaction in the MS. This involves using the prior knowledge gained by the characterization of the peptide internal standard and then requiring the MS to continuously monitor a specific ion in the MS/MS or MS n spectrum for both the peptide of interest and the internal standard. After elution, the area under the curve (AUC) for both peptide standard and target peptide peaks are calculated. The ratio of the two areas provides the absolute quantification that can be normalized for the number of cells used in the analysis and the protein's molecular weight, to provide the precise number of copies of the protein per cell. Further details of the AQUA methodology are described in Gygi et al., and Gerber et al. supra.
  • AQUA internal peptide standards may now be produced, as described above, for any of the 168 novel EGFR-related signaling protein phosphorylation sites disclosed herein (see Table 1/ Figure 2).
  • Peptide standards for a given phosphorylation site e.g. the tyrosine 653 site in IRS-2 - see Row 114 of Table 1
  • may be produced for both the phosphorylated and non-phosphorylated forms of the site e.g. see IRS-2 site sequence in Column E, Row 114 of Table 1
  • such standards employed in the AQUA methodology employed in the AQUA methodology to detect and quantify both forms of such phosphorylation site in a biological sample.
  • Heavy-isotope labeled equivalents of an of the peptides enumerated in Table 1/ Figure 2 can be readily synthesized and their unique MS and LC-SRM signature determined, so that the peptides are validated as AQUA peptides and ready for use in quantification experiments.
  • AQUA peptides provided by the invention are described above (corresponding to particular protein types/groups in Table 1 , for example, Receptor Tyrosine Kinases or Transcription Factor proteins).
  • Example 4 is provided to further illustrate the construction and use, by standard methods described above, of exemplary AQUA peptides provided by the invention.
  • the above-described AQUA peptides corresponding to the both the phosphorylated and non-phosphorylated forms of the disclosed STAT3 tyrosine 593 phosphorylation site may be used to quantify the amount of phosphorylated STAT3(Tyr593) in a biological sample, e.g. a tumor cell sample (or a sample before or after treatment with a test drug).
  • Immunoassay formats and variations thereof that may be useful for carrying out the methods disclosed herein are well known in the art. See generally E. Maggio, Enzyme-lmmunoassay, (1980) (CRC Press, Inc., Boca Raton, FIa.); see also, e.g., U.S. Pat. No. 4,727,022 (Skold et al., "Methods for Modulating Ligand-Receptor Interactions and their
  • the cancer cell lines expressing elevated levels of EGFR and stimulated with EGF were chosen to mimic signaling pathway activity in cancers involving activated EGFR. Tryptic phosphotyrosine peptides were purified and analyzed from extracts of the each of the eleven cell lines mentioned above as follows. Cells were cultured in DMEM medium or RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells at about 80% confluency were starved in medium without serum for 16 hours and stimulated with 100 ng/ml EGF for 5 minutes.
  • Sonicated cell lysates were cleared by centrifugation at 20,000 x g, and proteins were reduced with DTT at a final concentration of 4.1 mM and alkylated with iodoacetamide at 8.3 mM.
  • protein extracts were diluted in 20 mM HEPES pH 8.0 to a final concentration of 2 M urea and soluble TLCK-trypsin (Worth ington) was added at 10-20 ⁇ g/mL Digestion was performed for 1-2 days at room temperature.
  • Polyclonal antibodies that specifically bind an EGFR-related signal transduction protein only when phosphorylated at the respective phosphorylation site disclosed herein are produced according to standard methods by first constructing a synthetic peptide antigen comprising the phosphorylation site sequence and then immunizing an animal to raise antibodies against the antigen, as further described below. Production of exemplary polyclonal antibodies is provided below.
  • IRS-2 (tyrosine 823).
  • Isolated phospho-specific polyclonal antibody does not recognize the target protein when not phosphorylated at the appropriate phosphorylation site in the non-stimulated cells (e.g. IRS-2 is not bound when not phosphorylated at tyrosine 823).
  • Monoclonal antibodies that specifically bind a EGFR-related signal transduction protein only when phosphorylated at the respective phosphorylation site disclosed herein are produced according to standard methods by first constructing a synthetic peptide antigen comprising the phosphorylation site sequence and then immunizing an animal to raise antibodies against the antigen, and harvesting spleen cells from such animals to produce fusion hybridomas, as further described below. Production of exemplary monoclonal antibodies is provided below.
  • This peptide is then coupled to KLH and used to immunize animals and harvest spleen cells for generation (and subsequent screening) of phospho-specific monoclonal EphB4(tyr574) antibodies as described in Immunization/ Fusion/Screening below.
  • B. MINK tyrosine 906
  • This peptide is then coupled to KLH and used to immunize animals and harvest spleen cells for generation (and subsequent screening) of phospho-specific monoclonal PTP-kappa (tyr858) antibodies as described in Immunization/Fusion/ Screening below.
  • Ron(tyr1238) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated Ron(tyr1238) in the sample, as further described below in Analysis & Quantification.
  • the PI3K P85-beta(tyr467) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated PI3K P85-beta(tyr467) in the sample, as further described below in Analysis & Quantification.
  • the Annexin A4(tyr164) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated Annexin A4(tyr164) in the sample, as further described below in Analysis & Quantification.
  • the Taiin 1 (tyr26) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated Talin 1 (tyr26) in the sample, as further described below in Analysis & Quantification.
  • a desired AQUA peptide described in A-D above are purified by reversed- phase C18 HPLC using standard TFA/acetonitrile gradients and characterized by matrix-assisted laser desorption ionization-time of flight (Biflex III, Bruker Daltonics, Billerica, MA) and ion-trap (ThermoFinnigan, LCQ DecaXP) MS.
  • Samples are then directly loaded onto the microcapillary column by using a FAMOS inert capillary autosampler (LC Packings, San Francisco) after the flow split. Peptides are reconstituted in 6% acetic acid/0.01% TFA before injection.
  • Target protein e.g. a phosphorylated protein of A-D above
  • AQUA peptide as described above.
  • the IAP method is then applied to the complex mixture of peptides derived from proteolytic cleavage of crude cell extracts to which the AQUA peptides have been spiked in.
  • LC-SRM of the entire sample is then carried out.
  • MS/MS may be performed by using a ThermoFinnigan (San Jose, CA) mass spectrometer (LCQ DecaXP ion trap or TSQ Quantum triple quadrupole).
  • LCQ DecaXP ion trap or TSQ Quantum triple quadrupole On the DecaXP, parent ions are isolated at 1.6 m/z width, the ion injection time being limited to 150 ms per microscan, with two microscans per peptide averaged, and with an AGC setting of 1 x 10 8 ; on the Quantum, Q1 is kept at 0.4 and Q3 at 0.8 m/z with a scan time of 200 ms per peptide.

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Abstract

La présente invention décrit 168 nouveaux sites de phosphorylation identifiés parmi les protéines de transduction de signal ainsi que dans les voies en aval de la EGFR kinase, incluant la EGFR kinase elle-même. La présente invention concerne également des anticorps spécifiques des sites de phosphorylation et des peptides marqués à l'aide d'isotopes lourds (peptides AQUA) permettant la détection et la quantification sélectives de ces sites et/ou protéines phosphorylés, ainsi que des méthodes d'emploi de réactifs pour aboutir à ce résultat. Parmi les sites de phosphorylation identifiés se trouvent des sites contenus dans les types de protéines suivants : protéines Liant l'Actine, protéines Adaptatrices/Pièges, Protéines Liant le Calcium, protéines de Régulation du Cycle Cellulaire, protéines du Cytosquelette, protéines de Liaison et de Réplication de l'ADN, protéines d’Activation de la GTPase, protéines de type Facteur d'Échange Nucléotidique de la Guanine, Lipide Kinases, Récepteurs Tyrosine Kinases, ligands de Récepteurs Tyrosine Kinases, Protéine Kinases, Phosphatases de Protéines et de Récepteurs, protéines de type Facteur de Transcription, protéines de type Élimination de Tumeurs, et protéines de Vésicule.
EP04815061A 2004-12-21 2004-12-21 Phosphorylation des proteines suivant des voies controlees par les egfr Withdrawn EP1841882A1 (fr)

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CN102460152B (zh) * 2009-01-15 2014-11-26 美国控股实验室公司 通过测量Her-3确定患者反应的方法
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DATABASE UniProt [online] 1 May 1991 (1991-05-01), "Receptor tyrosine-protein kinase erbB-3 precursor (EC <A HREF="http://srs.ebi.ac.uk/srsbin/cgi-bin/wgetz?[enzyme-ECNumber:2.7.10.1]+-e">2.7.10.1</A>) (c- erbB3) (Tyrosine kinase-type cell surface receptor HER3).", XP002467292, retrieved from EBI accession no. UNIPROT:P21860 Database accession no. P21860 *
DATABASE UniProt [online] 15 December 1998 (1998-12-15), "Receptor tyrosine-protein kinase erbB-3 precursor (EC <A HREF="http://srs.ebi.ac.uk/srsbin/cgi-bin/wgetz?[enzyme-ECNumber:2.7.10.1]+-e">2.7.10.1</A>) (c- erbB3).", XP002467293, retrieved from EBI accession no. UNIPROT:Q62799 Database accession no. Q62799 *
HELLYER N J ET AL: "Cloning of the rat ErbB3 cDNA and characterization of the recombinant protein", GENE, vol. 165, no. 2, 20 November 1995 (1995-11-20), pages 279 - 284, XP004043156 *
KIM H H ET AL: "Epidermal growth factor-dependent association of phosphatidylinositol 3-kinase with the erbB3 gene product.", J BIOL CHEM, vol. 269, no. 40, 7 October 1994 (1994-10-07), pages 24747 - 24755, XP002467290 *
KRAUS M H ET AL: "Isolation and characterization of ERBB3, a third member of the ERBB/epidermal growth factor receptor family: evidence for overexpression in a subset of human mammary tumors.", PNAS USA, vol. 86, no. 23, December 1989 (1989-12-01), pages 9193 - 9197 *
OLAYIOYE M: "The Erbbeta Signaling Network: Receptor Heterodimerization in Development and Cancer", EMBO J, vol. 19, no. 13, January 2000 (2000-01-01), pages 3159 - 3167, XP002985171 *
PRIGENT S A ET AL: "Identification of c-erbB-3 binding sites for phosphatidylinositol 3'-kinase and SHC using an EGF receptor/c-erbB-3 chimera", EMBO J, vol. 13, no. 12, 1994, pages 2831 - 2841, XP002467288 *
See also references of WO2006068640A1 *
UPSTATE: "Antibodies for Phosphorylation & Beyond", INTERNET ARTICLE, June 2004 (2004-06-01), pages 1 - 16, XP002334236, Retrieved from the Internet <URL:http://www.upstate.com/img/pdf/antibodies_phos.pdf> [retrieved on 20050630] *
VADLAMUDI R K ET AL: "Heregulin and HER2 signaling selectively activates c-Src phosphorylation at tyrosine 215", FEBS LETT, vol. 543, no. 1-3, 22 May 2003 (2003-05-22), pages 76 - 80, XP004425037 *
VIJAPURKAR U ET AL: "Roles of mitogen-activated protein kinase and phosphoinositide 3'-kinase in ErbB2/ErbB3 coreceptor-mediated heregulin signaling.", EXP CELL RES, vol. 284, no. 2, 1 April 2003 (2003-04-01), pages 291 - 302, XP002467289 *
VIJAPURKAR U. ET AL: "Mutation of a Shc binding site tyrosine residue in ErbB3/HER3 blocks heregulin-dependent activation of mitogen-activated protein kinase", J BIOL CHEM, vol. 273, no. 33, 14 August 1998 (1998-08-14), pages 20996 - 21002, XP001074635 *

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