ABERRANT RAS REGULATION AND REDUCED P190 TYROSINE PHOSPHORYLATION IN CELLS LACKING P120-GAP

The Ras guanine nucleotide-binding protein functions as a molecular sw itch in signalling downstream of protein-tyrosine kinases. Ras is acti vated by exchange of GDP for GTP and is turned off by hydrolysis of bo und GTP to GDP. Ras itself has a low intrinsic GTPase activity that ca n be stimulated by GTPase-activating proteins (GAPs), including p120-G ap and neurofibromin. These GAPs possess a common catalytic domain but contain distinct regulatory elements that may couple different extern al signals to control of the Ras pathway. p120-Gap, for example, has t wo N-terminal SH2 domains that directly recognize phosphotyrosine moti fs on activated growth factor receptors and cytoplasmic phosphoprotein s. To analyze the role of p120-Gap in Ras regulation in vivo, we have used fibroblasts derived from mouse embryos with a null mutation in th e gene for p120-Gap (Gap). Platelet-derived growth factor stimulation of Gap(-/-) Cells led to an abnormally large increase in the level of Ras-GTP and in the duration of mitogen-activated protein (MAP) kinase activation compared with wild-type cells, suggesting that p120-Gap is specifically activated following growth factor stimulation. Induction of DNA synthesis in response to platelet-derived growth factor and mor phological transformation by the v-src and EJ-ras oncogenes were not s ignificantly affected by the absence of p120-Gap. However, we found th at normal tyrosine phosphorylation of p190-rhoGap, a cytoplasmic prote in that associates with the p120-Gap SH2 domains, was dependent on the presence of p120-Gap. Our results suggest that p120-Gap has specific functions in downregulating the Ras/MAP kinase pathway following growt h factor stimulation, and in modulating the phosphorylation of p190-rh oGap, but is not required for mitogenic signalling.