Tyrosine phosphorylation of Ras GAP by the Src kinases.

Cellular Ras has an essential function in oncogenic signal transduction pathways downstream of the viral Src kinase. GTPase-activating protein (GAP), discovered as a key regulator of Ras, also interacts with the Src kinase. To study the functional role of GAP in Src oncogenesis, interactions between the Src kinase and GAP were investigated using a baculovirus expression system. In this system, it was demonstrated that the viral and cellular Src kinases specifically associate with and phosphorylate GAP. Further studies using GAP deletion mutants suggest that the Src homology 2 (SH2) region of GAP interacts with phosphorylated residues in Src. To determine the precise residues in GAP phosphorylated by the Src kinases, phosphopeptide mapping experiments using either in vitro or in vivo labeled proteins were performed. This analysis showed that Tyr-457 in bovine GAP is the major site phosphorylated by the Src kinases both in vitro and in vivo. Tyr-457 in GAP is located immediately adjacent to SH2 domains implicated in GAP association with cellular phosphoproteins, including p62. To investigate whether this specific phosphorylation in GAP is involved in its association with cellular proteins, an epitope-tagged GAP mutant containing a substitution of Phe-457 in place of Tyr-457 was constructed and expressed in Src transformed rodent fibroblasts. GAP-associated proteins were analyzed using epitope specific antibodies reacting only with exogenously expressed GAP. These experiments showed that Tyr-457 phosphorylation of GAP stabilizes its association with p62, particularly at cell membranes. Therefore, one role of tyrosine phosphorylation in GAP may be to recruit p62 to cell membranes where Ras is localized. These results are consistent with a model in which phosphorylation of GAP by activated tyrosine kinases stimulates Ras signalling pathways through formation of GAP complexes with p62 at cell membranes.PhDMicrobiology and ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103823/1/9409778.pdfDescription of 9409778.pdf : Restricted to UM users only