title: Selective binding of activated pp60c-src by an immobilized synthetic phosphopeptide modeled on the carboxyl terminus of pp60c-src.
abstract: Phosphorylation of the carboxyl terminus of pp60c-src, the product of the c-src protooncogene, at Tyr-527 suppresses its tyrosine kinase activity and transforming potential. It has been proposed that the phosphorylated carboxyl terminus of pp60c-src inhibits kinase activity by binding to the SH2 (src homology 2) domain. We have synthesized peptides corresponding to the carboxyl-terminal 13 residues of pp60c-src phosphorylated and nonphosphorylated at Tyr-527. A highly transforming mutant, pp60c-src(F527), in which Tyr-527 is mutated to Phe, bound to the phosphorylated peptide immobilized to Affi-Gel 10. Binding of the phosphorylated peptide was abolished by deletion of residues 144-175 in the SH2 domain but not by deletion of residues 93-143, which removes most of the SH3 domain. The phosphorylated peptide also bound to pp60v-src, the transforming protein of Rous sarcoma virus. Only traces of pp60v-src and pp60c-src(F527) bound to the corresponding nonphosphorylated c-src peptide. Normal pp60c-src bound much less efficiently to the phosphorylated peptide than did pp60c-src(F527). A phosphorylated peptide corresponding to the carboxyl terminus of the c-fgr protein also bound to pp60c-src(F527), but with weaker affinity. Furthermore, the phosphorylated synthetic carboxyl-terminal pp60c-src peptide markedly inhibited phosphorylation of pp60c-src(F527) during cytoskeletal kinase assays. These results provide direct evidence for models in which the phosphorylated carboxyl terminus of pp60c-src binds intramolecularly or intermolecularly to the SH2 domain of the c-src protein.
authors.affiliation: Department of Biochemistry, University of New Hampshire, Durham 03824.
authors.forename: R R
authors.initials: RR
authors.lastname: Roussel
authors.forename: S R
authors.initials: SR
authors.lastname: Brodeur
authors.forename: D
authors.initials: D
authors.lastname: Shalloway
authors.forename: A P
authors.initials: AP
authors.lastname: Laudano
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002846:Chromatography, Affinity; D002872:Chromosome Deletion; D004789:Enzyme Activation; D016391:Genes, src; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D011485:Protein Binding; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives
chemical_list: D010748:Phosphopeptides; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src)