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 GAPF457 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 GAPF457 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 GAPF457 was reduced approximately 3-fold compared with wild-type GAP. Subcellular fractionation experiments further demonstrated that Tyr-457 phosphorylation of GAP stabilized its association with p62 at cell membranes. Based on these findings, we propose that one role of tyrosine phosphorylation in GAP is to enhance its association with p62 at membranes, which in turn may contribute to regulation of Ras signal transduction pathways.