Regulation of the cellular src protein tyrosine kinase: interactions of the carboxyl terminal sequences residing between the kinase domain and tyrosine-527.

Negative regulation of the cellular Src tyrosine kinase (pp60c-src) is mediated through the phosphorylation of a C-terminal tyrosine residue, Tyr-527. Current models predict that inhibition of c-Src kinase activity results from an interaction of phosphorylated Tyr-527 with the amino terminal SH2 domain. Tyr-527 is located 11 residues C-terminal from the end of the kinase domain. Insertion or deletion of residues within these 11 residues of pp60c-src activates kinase activity and induces morphological transformation. The resultant variant Src proteins also exhibit a reduced level of phosphorylation of Tyr-527. We have used antibodies to phosphotyrosine, susceptibility to tyrosine phosphatases and binding of mutant Src proteins to peptides mimicking the tyrosine phosphorylated C-terminus of pp60c-src to investigate the tyrosine phosphorylated and unphosphorylated forms of such insertion/deletion variants. The reactivity of variant proteins with phosphotyrosine antibodies and the susceptibility of phosphorylated Tyr-527 to tyrosine phosphatases were similar to that of wild type pp60c-src. In addition, the results of binding experiments performed with a C-terminal peptide containing phosphorylated Tyr-527 indicated that only dephosphorylated forms of variant Src proteins bound phospho-peptide. These data suggest that insertion or deletion mutations within the C-terminal region of pp60c-src do not substantially alter the interaction of phosphorylated Tyr-527 with the SH2 domain. Rather, the data are consistent with the hypothesis that the reduction of phosphorylation of Tyr-527 and the accompanying activation of these variants may be due to the action of a tyrosine phosphatase and the inefficient phosphorylation of Tyr-527 by a regulatory kinase.