DOI: 10.1073/pnas.97.1.179. Analysis of receptor signaling pathways by mass spectrometry: Identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

Analysis of receptor signaling pathways by mass spectrometry: Identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

10.1073/pnas.97.1.179

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry. Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling. Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR. We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR in vivo . Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.

Bibliography

Pandey, A., Podtelejnikov, A. V., Blagoev, B., Bustelo, X. R., Mann, M., & Lodish, H. F. (2000). Analysis of receptor signaling pathways by mass spectrometry: Identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors. Proceedings of the National Academy of Sciences, 97(1), 179â184.

Authors 6

Akhilesh Pandey (first)
Alexandre V. Podtelejnikov (additional)
Blagoy Blagoev (additional)
Xosé R. Bustelo (additional)
Matthias Mann (additional)
Harvey F. Lodish (additional)

References 49 Referenced 362

10.1016/0092-8674(91)90638-F
10.1016/0092-8674(90)90801-K
10.1038/373573a0
10.1126/science.1700866
10.1016/0092-8674(91)90461-7
10.1073/pnas.93.25.14440
10.1038/nbt1196-1579
10.1073/pnas.94.2.385
10.1016/S0962-8924(97)01031-3
10.1038/1700
10.1126/science.279.5354.1216
10.1093/emboj/17.4.967
10.1021/bi982093r
10.1002/elps.1150150161
10.1111/j.1469-1809.1995.tb01603.x
K E Schuebel, X R Bustelo, D A Nielsen, B J Song, M Barbacid, D Goldman, I J Lee Oncogene 13, 363–371 (1996). / Oncogene by Schuebel K E (1996)
10.1021/ac950914h
10.1002/(SICI)1097-0231(199608)10:11<1371::AID-RCM682>3.0.CO;2-5
10.1021/ac00091a044
10.1021/ac970896z
10.1038/379466a0
10.1021/ac9509519
10.1002/(SICI)1097-0231(19970615)11:9<1015::AID-RCM958>3.0.CO;2-H
10.1021/ac00096a002
10.1016/S0968-0004(99)01400-0
10.1016/S0021-9258(17)45429-9
10.1016/S0021-9258(18)31593-X
10.1016/0092-8674(92)90536-L
10.1016/0092-8674(91)90410-Z
10.1016/0092-8674(92)90455-L
10.1128/MCB.15.1.186
10.1038/343377a0
W T Wong, M H Kraus, F Carlomagno, A Zelano, T Druck, C M Croce, K Huebner, P P Di Fiore Oncogene 9, 1591–1597 (1994). / Oncogene by Wong W T (1994)
10.1128/MCB.15.11.6213
T J Blake, M Shapiro, H C Morse, W Y Langdon Oncogene 6, 653–657 (1991). / Oncogene by Blake T J (1991)
10.1074/jbc.271.24.14554
10.1615/CritRevOncog.v7.i1-2.50
10.1126/science.256.5060.1196
10.1038/356068a0
10.1038/356071a0
10.1016/S0021-9258(19)37146-7
10.1182/blood.V84.12.4135.bloodjournal84124135
10.1002/j.1460-2075.1995.tb06999.x
10.1038/385169a0
10.1038/374467a0
10.1038/374474a0
10.1038/374470a0
10.1002/j.1460-2075.1995.tb06969.x
10.1093/emboj/17.22.6608

Dates

Type	When
Created	23 years ago (July 26, 2002, 10:41 a.m.)
Deposited	3 years, 4 months ago (April 13, 2022, 5:24 p.m.)
Indexed	4 weeks, 2 days ago (July 23, 2025, 8:22 a.m.)
Issued	25 years, 7 months ago (Jan. 4, 2000)
Published	25 years, 7 months ago (Jan. 4, 2000)
Published Online	25 years, 7 months ago (Jan. 4, 2000)
Published Print	25 years, 7 months ago (Jan. 4, 2000)

Funders 0

None

BibTeX

@article{Pandey_2000, title={Analysis of receptor signaling pathways by mass spectrometry: Identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors}, volume={97}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.97.1.179}, DOI={10.1073/pnas.97.1.179}, number={1}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Pandey, Akhilesh and Podtelejnikov, Alexandre V. and Blagoev, Blagoy and Bustelo, Xosé R. and Mann, Matthias and Lodish, Harvey F.}, year={2000}, month=jan, pages={179–184} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        7,
        23
      ]
    ],
    "date-time": "2025-07-23T12:22:52Z",
    "timestamp": 1753273372490
  },
  "reference-count": 49,
  "publisher": "Proceedings of the National Academy of Sciences",
  "issue": "1",
  "content-domain": {
    "domain": [
      "www.pnas.org"
    ],
    "crossmark-restriction": true
  },
  "published-print": {
    "date-parts": [
      [
        2000,
        1,
        4
      ]
    ]
  },
  "abstract": "<jats:p>\n            Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry. Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling. Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR. We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR\n            <jats:italic>in vivo</jats:italic>\n            . Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.\n          </jats:p>",
  "DOI": "10.1073/pnas.97.1.179",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2002,
        7,
        26
      ]
    ],
    "date-time": "2002-07-26T14:41:48Z",
    "timestamp": 1027694508000
  },
  "page": "179-184",
  "update-policy": "http://dx.doi.org/10.1073/pnas.cm10313",
  "source": "Crossref",
  "is-referenced-by-count": 362,
  "title": "Analysis of receptor signaling pathways by mass spectrometry: Identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors",
  "prefix": "10.1073",
  "volume": "97",
  "author": [
    {
      "given": "Akhilesh",
      "family": "Pandey",
      "sequence": "first",
      "affiliation": [
        {
          "name": "Whitehead Institute for Biomedical Research, Cambridge, MA 02142; Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115; Protein Interaction Laboratory, Center for Experimental Bioinformatics, University of Southern Denmark, Odense M, DK-5230, Denmark; and Department of Pathology, State University of New York, Stony Brook, NY 11794"
        }
      ]
    },
    {
      "given": "Alexandre V.",
      "family": "Podtelejnikov",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Whitehead Institute for Biomedical Research, Cambridge, MA 02142; Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115; Protein Interaction Laboratory, Center for Experimental Bioinformatics, University of Southern Denmark, Odense M, DK-5230, Denmark; and Department of Pathology, State University of New York, Stony Brook, NY 11794"
        }
      ]
    },
    {
      "given": "Blagoy",
      "family": "Blagoev",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Whitehead Institute for Biomedical Research, Cambridge, MA 02142; Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115; Protein Interaction Laboratory, Center for Experimental Bioinformatics, University of Southern Denmark, Odense M, DK-5230, Denmark; and Department of Pathology, State University of New York, Stony Brook, NY 11794"
        }
      ]
    },
    {
      "given": "Xos\u00e9 R.",
      "family": "Bustelo",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Whitehead Institute for Biomedical Research, Cambridge, MA 02142; Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115; Protein Interaction Laboratory, Center for Experimental Bioinformatics, University of Southern Denmark, Odense M, DK-5230, Denmark; and Department of Pathology, State University of New York, Stony Brook, NY 11794"
        }
      ]
    },
    {
      "given": "Matthias",
      "family": "Mann",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Whitehead Institute for Biomedical Research, Cambridge, MA 02142; Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115; Protein Interaction Laboratory, Center for Experimental Bioinformatics, University of Southern Denmark, Odense M, DK-5230, Denmark; and Department of Pathology, State University of New York, Stony Brook, NY 11794"
        }
      ]
    },
    {
      "given": "Harvey F.",
      "family": "Lodish",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Whitehead Institute for Biomedical Research, Cambridge, MA 02142; Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115; Protein Interaction Laboratory, Center for Experimental Bioinformatics, University of Southern Denmark, Odense M, DK-5230, Denmark; and Department of Pathology, State University of New York, Stony Brook, NY 11794"
        }
      ]
    }
  ],
  "member": "341",
  "published-online": {
    "date-parts": [
      [
        2000,
        1,
        4
      ]
    ]
  },
  "reference": [
    {
      "key": "e_1_3_3_1_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(91)90638-F"
    },
    {
      "key": "e_1_3_3_2_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(90)90801-K"
    },
    {
      "key": "e_1_3_3_3_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/373573a0"
    },
    {
      "key": "e_1_3_3_4_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.1700866"
    },
    {
      "key": "e_1_3_3_5_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(91)90461-7"
    },
    {
      "key": "e_1_3_3_6_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.93.25.14440"
    },
    {
      "key": "e_1_3_3_7_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/nbt1196-1579"
    },
    {
      "key": "e_1_3_3_8_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.94.2.385"
    },
    {
      "key": "e_1_3_3_9_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0962-8924(97)01031-3"
    },
    {
      "key": "e_1_3_3_10_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/1700"
    },
    {
      "key": "e_1_3_3_11_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.279.5354.1216"
    },
    {
      "key": "e_1_3_3_12_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/emboj/17.4.967"
    },
    {
      "key": "e_1_3_3_13_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi982093r"
    },
    {
      "key": "e_1_3_3_14_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/elps.1150150161"
    },
    {
      "key": "e_1_3_3_15_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1469-1809.1995.tb01603.x"
    },
    {
      "key": "e_1_3_3_16_2",
      "first-page": "363",
      "volume": "13",
      "author": "Schuebel K E",
      "year": "1996",
      "unstructured": "K E Schuebel, X R Bustelo, D A Nielsen, B J Song, M Barbacid, D Goldman, I J Lee Oncogene 13, 363\u2013371 (1996).",
      "journal-title": "Oncogene"
    },
    {
      "key": "e_1_3_3_17_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/ac950914h"
    },
    {
      "key": "e_1_3_3_18_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/(SICI)1097-0231(199608)10:11<1371::AID-RCM682>3.0.CO;2-5"
    },
    {
      "key": "e_1_3_3_19_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/ac00091a044"
    },
    {
      "key": "e_1_3_3_20_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/ac970896z"
    },
    {
      "key": "e_1_3_3_21_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/379466a0"
    },
    {
      "key": "e_1_3_3_22_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/ac9509519"
    },
    {
      "key": "e_1_3_3_23_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/(SICI)1097-0231(19970615)11:9<1015::AID-RCM958>3.0.CO;2-H"
    },
    {
      "key": "e_1_3_3_24_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/ac00096a002"
    },
    {
      "key": "e_1_3_3_25_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0968-0004(99)01400-0"
    },
    {
      "key": "e_1_3_3_26_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0021-9258(17)45429-9"
    },
    {
      "key": "e_1_3_3_27_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0021-9258(18)31593-X"
    },
    {
      "key": "e_1_3_3_28_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(92)90536-L"
    },
    {
      "key": "e_1_3_3_29_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(91)90410-Z"
    },
    {
      "key": "e_1_3_3_30_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(92)90455-L"
    },
    {
      "key": "e_1_3_3_31_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/MCB.15.1.186"
    },
    {
      "key": "e_1_3_3_32_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/343377a0"
    },
    {
      "key": "e_1_3_3_33_2",
      "first-page": "1591",
      "volume": "9",
      "author": "Wong W T",
      "year": "1994",
      "unstructured": "W T Wong, M H Kraus, F Carlomagno, A Zelano, T Druck, C M Croce, K Huebner, P P Di Fiore Oncogene 9, 1591\u20131597 (1994).",
      "journal-title": "Oncogene"
    },
    {
      "key": "e_1_3_3_34_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/MCB.15.11.6213"
    },
    {
      "key": "e_1_3_3_35_2",
      "first-page": "653",
      "volume": "6",
      "author": "Blake T J",
      "year": "1991",
      "unstructured": "T J Blake, M Shapiro, H C Morse, W Y Langdon Oncogene 6, 653\u2013657 (1991).",
      "journal-title": "Oncogene"
    },
    {
      "key": "e_1_3_3_36_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1074/jbc.271.24.14554"
    },
    {
      "key": "e_1_3_3_37_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1615/CritRevOncog.v7.i1-2.50"
    },
    {
      "key": "e_1_3_3_38_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.256.5060.1196"
    },
    {
      "key": "e_1_3_3_39_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/356068a0"
    },
    {
      "key": "e_1_3_3_40_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/356071a0"
    },
    {
      "key": "e_1_3_3_41_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0021-9258(19)37146-7"
    },
    {
      "key": "e_1_3_3_42_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1182/blood.V84.12.4135.bloodjournal84124135"
    },
    {
      "key": "e_1_3_3_43_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/j.1460-2075.1995.tb06999.x"
    },
    {
      "key": "e_1_3_3_44_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/385169a0"
    },
    {
      "key": "e_1_3_3_45_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/374467a0"
    },
    {
      "key": "e_1_3_3_46_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/374474a0"
    },
    {
      "key": "e_1_3_3_47_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/374470a0"
    },
    {
      "key": "e_1_3_3_48_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/j.1460-2075.1995.tb06969.x"
    },
    {
      "key": "e_1_3_3_49_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/emboj/17.22.6608"
    }
  ],
  "container-title": "Proceedings of the National Academy of Sciences",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://pnas.org/doi/pdf/10.1073/pnas.97.1.179",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2022,
        4,
        13
      ]
    ],
    "date-time": "2022-04-13T21:24:45Z",
    "timestamp": 1649885085000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://pnas.org/doi/full/10.1073/pnas.97.1.179"
    }
  },
  "subtitle": [],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        2000,
        1,
        4
      ]
    ]
  },
  "references-count": 49,
  "journal-issue": {
    "issue": "1",
    "published-print": {
      "date-parts": [
        [
          2000,
          1,
          4
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1073/pnas.97.1.179"
  ],
  "URL": "http://dx.doi.org/10.1073/pnas.97.1.179",
  "relation": {},
  "ISSN": [
    "0027-8424",
    "1091-6490"
  ],
  "subject": [],
  "container-title-short": "Proc. Natl. Acad. Sci. U.S.A.",
  "published": {
    "date-parts": [
      [
        2000,
        1,
        4
      ]
    ]
  },
  "assertion": [
    {
      "value": "1999-10-29",
      "order": 1,
      "name": "accepted",
      "label": "Accepted",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    },
    {
      "value": "2000-01-04",
      "order": 2,
      "name": "published",
      "label": "Published",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    }
  ]
}