Evidence for autoinhibitory regulation of the c-src gene product. A possible interaction between the src homology 2 domain and autophosphorylation site.

In the previous study (Sato, K., Miki, S., Tachibana, H., Hayashi, F., Akiyama, T., and Fukami, Y. (1990) Biochem. Biophys. Res. Commun. 171, 1152-1159), we found a synthetic peptide, termed peptide A, that inhibited the kinase activity of p60v-src. The peptide A sequence corresponds to residues 137 to 157 of p60v-src which are included in the amino-terminal portion of the src homology 2 domain. In this study, we attempted to specify the inhibitory sequence in this domain and to identify its target site. The most potent peptide A derivative was one that corresponds to residues 140 through 157. The target site of peptide A was assumed to reside in the autophosphorylation site of p60v-src, since synthetic peptides containing the sequence Phe424-Pro-Ile-Lys-Trp428 which is present downstream of the autophosphorylated Tyr416 partially counteracted the inhibitory effect of peptide A. An antibody was prepared against one of such target peptides, termed pepY. Cross-linking experiments showed that 125I-labeled peptide A could bind to p60v-src blotted on a membrane, and the binding was blocked by the anti-pepY antibody but not by other anti-p60v-src antibodies. Conversely, immunoblotting of p60v-src with anti-pepY antibody was blocked by the cross-linking of peptide A to p60v-src. To our surprise, anti-pepY antibody did not affect the p60v-src activity. Furthermore, p60c-src was activated 2- to 6-fold by this antibody. These results suggest that the pepY region in the catalytic domain of p60v-src or of p60c-src is not essential for the catalytic activity but rather is involved in the negative regulation of the kinase activity of p60c-src.