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Phosphorylation of the carboxyl terminus of pp60C'T, 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 pp60111 inhibits kinase activity by binding to the SH2 (src homology 2) domain. We have synthesized-peptides corresponding to the carboxyl-terminal 13 residues of pp60'w phosphorylated and nonphosphorylated at Tyr-527. A highly transforming mutant, pp6OC`src(F527), in which Tyr-527 is mutated to Phe, bound to the phosphorylated peptide innobilized 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 pp6OvSrc, the transforming protein of Rous sarcoma virus. Only traces of pp60v7 and pp6Oc 527) bound to the corresponding nonphosphorylated c-src peptide. Normal pp60'1 bound much less efficiently to the phosphorylated peptide than did pp60`8)c(FS27). A phosphorylated peptide corresponding to the carboxyl terminus of the c-fgr protein also bound to pp60cSrc(F527), but with weaker affinity. Furthermore, the phosphorylated synthetic carboxyl-terminal pp6OC4re peptide markedly inhibited phosphorylation of pp604rC(F527) during cytoskeletal kinase assays. These results provide direct evidence for models in which the phosphorylated carboxyl terminus of pp60C`rc binds intramolecularly or intermolecularly to the SH2 domain of the c-src protein.

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