Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc.
Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known about the tyrosine phosphorylations involved, although Src activation and Shc phosphorylation are thought to be critical. We find that agonist-induced Src activation in Rat-1 cells is not mediated by Gi and shows no correlation with Ras/MAP kinase activation. Furthermore, LPA-induced tyrosine phosphorylation of Shc is PTX-insensitive and Ca2+-dependent in COS cells, but undetectable in Rat-1 cells. Expression of dominant-negative Src or Shc does not affect MAP kinase activation by LPA. Thus, Gi-mediated Ras/MAP kinase activation in fibroblasts and COS cells involves neither Src nor Shc. Instead, we detect a 100 kDa tyrosine-phosphorylated protein (p100) that binds to the C-terminal SH3 domain of Grb2 in a strictly Gi- and agonist-dependent manner. Tyrosine kinase inhibitors and wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, prevent p100-Grb2 complex formation and MAP kinase activation by LPA. Our results suggest that the p100-Grb2 complex, together with an upstream non-Src tyrosine kinase and PI 3-kinase, couples Gi to Ras/MAP kinase activation, while Src and Shc act in a different pathway.
Kranenburg
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Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam.
Verlaan
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Hordijk
P L
PL
Moolenaar
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WH
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D051380:GRB2 Adaptor Protein; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D008954:Models, Biological; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011506:Proteins; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism; D000077191:Wortmannin; D018631:ras Proteins / Q000378:metabolism*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes; D002352:Carrier Proteins; D051380:GRB2 Adaptor Protein; C495950:Grb2 protein, rat; D010750:Phosphoproteins; D011506:Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D018631:ras Proteins; D002118:Calcium; D000077191:Wortmannin