Association of c-Src with p52Shc in mitotic NIH3T3 cells as revealed by Src-Shc binding site-specific antibodies

In a previous study, we presented evidence that the adaptor protein Shc interacts with and activates the tyrosine kinase c-Src without affecting the phosphorylation state of Tyr-527 in c-Src. Here we show that Shc-mediated c-Src activation occurs in mitotic NIH 3T3 cells. Co-immunoprecipitation studies demonstrate that the c-Src-p52Shc complex involves the activation segment/inter-DFG-APE (IDA) region of c-Src and the amino-terminal region of p52Shc. The complex formation contributes to the c-Src activation, because (i) specific activity of c-Src associated with p52Shc is higher than that of the total c-Src, and (ii) a recombinant protein containing the c-Src IDA sequence disrupts the complex and decreases the c-Src activity. Anti-Src IDA antibody can activate c-Src in vitro, and synthetic peptides that cover the carboxyl-terminal half of the Src IDA region interfere with the kinase-activating effect of anti-Src IDA antibody. These results support the idea that dephosphorylation-independent activation of c-Src by Shc is mediated by a molecular interaction involving the c-Src IDA region.