REGULATION OF C-SRC TYROSINE KINASE-ACTIVITY BY THE SRC SH2 DOMAIN

The protein-tyrosine kinase activity of pp60c-src (c-Src) is inhibited by phosphorylation of tyr527, within the c-Src c-terminal tail. Genet ic and biochemical data have suggested that this negative regulation r equires an intact Src homology 2 (SH2) domain. Since SH2 domains recog nize phosphotyrosine, it is possible that these two noncatalytic domai ns associate, and thereby repress c-Src kinase activity. Consistent wi th this model, an isolated Src SH2 domain expressed in bacteria as a G ST fusion protein bound in vitro to a synthetic phosphotyrosine-contai ning peptide modeled on the C-terminal 13 residues of the c-Src tail. Binding was absolutely dependent on phosphorylation of tyr527 in the t ail peptide, and was modified by both the length and sequence of the p eptide. Competition experiments indicated only a moderate binding affi nity between the Src SH2 domain and the phosphorylated tail. A distinc t phosphotyrosine-containing peptide previously identified as binding the Src SH2 domain with high affinity stimulated c-Src tyrosine kinase activity in vitro, possibly by competing with the endogenous tail pho sphorylation site for binding to the SH2 domain. Indeed, this activati on was competitively inhibited by purified bacterial Src SH2 domain. T hese data provide direct evidence that the c-Src tail has an intrinsic affinity for the Src SH2 domain, and suggest that such an interaction in the intact molecule contributes to maintaining c-Src in an inactiv e form.