MAP KINASE PHOSPHORYLATION OF MSOS1 PROMOTES DISSOCIATION OF MSOS1-SHC AND MSOS1-EGF RECEPTOR COMPLEXES

The mouse protein mSos1 has a central Ras guanine nucleotide exchange domain, and a long proline-rich C-terminal tail which contains several potential binding sites for the SH3 domains of the adaptor protein, G rb2. In fibroblasts, growth factor stimulation results in the recruitm ent of Grb2-mSos1 into complexes with activated receptors and cytoplas mic phosphoproteins such as Shc, which are apparently involved in Ras activation, and subsequently to an increase in mSos1 phosphorylation o n serine and threonine, The catalytic and C-terminal domains of mSos1 contain several potential sites for phosphorylation by mitogen-activat ed protein kinases. In vitro, purified p42/p44 MAP-kinase selectively phosphorylated the C-terminal tail of mSos1. Comparative tryptic phosp hopeptide mapping of mSos1 phosphorylated in vitro by MAP kinase and o f mSos1 immunoprecipitated from EGF-stimulated cells, revealed several phosphopeptides in common. These common phosphorylation sites have be en mapped to a region encompassing the first three proline (pro)-rich motifs in the tail of mSos1. Furthermore, a region of mSos1 containing the first two pro-rich motifs could associate with MBP kinase activit y in vitro. Phosphorylation of mSos1 did not affect binding of Grb2 to mSos1, but appeared to decrease binding of the mSos1-Grb2 complex to She and the EGF-receptor. These findings suggest a potential inhibitor y role for MAP-kinase in attenuating nucleotide exchange on Ras, by un coupling mSos1 from membrane-bound receptor complexes that lead to Ras activation.