Quantitative analysis of SH2 domain binding. Evidence for specificity and competition.
We report the development of a quantitative assay for measuring SH2 domain binding in vitro. Using this assay we have analyzed the binding of purified recombinant SH2 domains from ras GTPase activating protein (GAP) and the 85-kDa subunit of phosphatidylinositol 3-kinase (p85) to proteins from epidermal growth factor-stimulated and v-src-transformed cells. The purified recombinant SH2 domains from GAP and p85 bind to the tyrosine phosphorylated epidermal growth factor receptor with nanomolar affinities. Moreover, competition studies suggest that these two proteins bind to equivalent or overlapping sites on this receptor. In v-src-transformed cells the purified recombinant SH2 domains from GAP and p85 bind to distinct but overlapping sets of proteins.
Wood
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Division of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
McDonald
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Sahyoun
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D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D066246:ErbB Receptors / Q000378:metabolism; D020690:GTPase-Activating Proteins; D016391:Genes, src; D006801:Humans; D007700:Kinetics; D051379:Mice; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D011506:Proteins; D011994:Recombinant Proteins; D020703:ras GTPase-Activating Proteins; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src)