Tyrosine phosphorylation of Ras GTPase-activating protein stabilizes its association with p62 at membranes of v-Src transformed cells

Abstract

Ras GTPase-activating protein (GAP) regulates the activity of Ras proteins, which have key roles in signal transduction pathways downstream of oncogenic and receptor tyrosine kinases. Previous studies indicated that Tyr-457 of bovine GAP (Tyr-460 of human GAP) is the major site of phosphorylation by viral Src (v-Src) kinase and epidermal growth factor receptor. The finding that Tyr-457 in GAP is located immediately adjacent to Src homology 2 (SH2) and 3 (SH3) domains led us to investigate the possibility that this specific phosphorylation regulates protein-protein interactions involving GAP. For this purpose, we constructed a full-length GAP mutant containing a substitution of Phe-457 in place of Tyr-457. Both wild-type GAP and mutant GAP(F457) were tagged with the KT3 epitope at the carboxyl terminus and were expressed in v-Src transformed rat fibroblasts. In vivo phosphorylation analyses established that GAP(F457) was weakly phosphorylated on tyrosine and, as expected, lacked the phosphopeptide containing Tyr-457. Analysis of GAP-associated proteins in anti-KT3 immunoprecipitates showed that GAP stably associated with two major phosphoproteins, p62 and p190, which have been previously described. Significantly, association of p62 with GAP(F457) was reduced approximately 3-fold compared with wild-type GAP. Subce