Aberrant Ras regulation and reduced p190 tyrosine phosphorylation in cells lacking p120-Gap.
The Ras guanine nucleotide-binding protein functions as a molecular switch in signalling downstream of protein-tyrosine kinases. Ras is activated by exchange of GDP for GTP and is turned off by hydrolysis of bound GTP to GDP. Ras itself has a low intrinsic GTPase activity that can be stimulated by GTPase-activating proteins (GAPs), including p120-Gap and neurofibromin. These GAPs possess a common catalytic domain but contain distinct regulatory elements that may couple different external signals to control of the Ras pathway. p120-Gap, for example, has two N-terminal SH2 domains that directly recognize phosphotyrosine motifs on activated growth factor receptors and cytoplasmic phosphoproteins. 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 the 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 morphological transformation by the v-src and EJ-ras oncogenes were not significantly affected by the absence of p120-Gap. However, we found that normal tyrosine phosphorylation of p190-rhoGap, a cytoplasmic protein 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 growth factor stimulation, and in modulating the phosphorylation of p190-rhoGap, but is not required for mitogenic signalling.
van der Geer
P
P
Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Henkemeyer
M
M
Jacks
T
T
Pawson
T
T
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009687:Nuclear Proteins / Q000378:metabolism*; D009857:Oncogenes; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins; D015398:Signal Transduction; D014170:Transformation, Genetic; D014443:Tyrosine / Q000378:metabolism; D020703:ras GTPase-Activating Proteins; D018631:ras Proteins / Q000378:metabolism*; D020666:ras-GRF1
C513041:Arhgap35 protein, mouse; C513042:Arhgap5 protein, mouse; D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011506:Proteins; C513037:Rasgrf1 protein, mouse; D012097:Repressor Proteins; D020703:ras GTPase-Activating Proteins; D020666:ras-GRF1; D014443:Tyrosine; D006160:Guanosine Triphosphate; D004247:DNA; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018631:ras Proteins