Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 97, Issue 1, Pages 179-184Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.97.1.179
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Funding
- NCI NIH HHS [K01 CA75447] Funding Source: Medline
- NHLBI NIH HHS [P01 HL32262, P01 HL032262] Funding Source: Medline
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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.
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