Activation of c-Raf-1 by Ras and Src through different mechanisms: activation in vivo and in vitro.
The c-Raf-1 protein kinase plays a critical role in intracellular signaling downstream from many tyrosine kinase and G-protein-linked receptors. c-Raf-1 binds to the proto-oncogene Ras in a GTP-dependent manner, but the exact mechanism of activation of c-Raf-1 by Ras is still unclear. We have established a system to study the activation of c-Raf-1 in vitro. This involves mixing membranes from cells expressing oncogenic H-RasG12V, with cytosol from cells expressing epitope-tagged full-length wild-type c-Raf-1. This results in a fraction of the c-Raf-1 binding to the membranes and a concomitant 10- to 20-fold increase in specific activity. Ras was the only component in these membranes required for activation, as purified recombinant farnesylated K-Ras.GTP, but not non-farnesylated K-Ras.GTP or farnesylated K-Ras.GDP, was able to activate c-Raf-1 to the same degree as intact H-RasG12V membranes. The most potent activation occurred under conditions in which phosphorylation was prohibited. Under phosphorylation-permissive conditions, activation of c-Raf-1 by Ras was substantially inhibited. Consistent with the results from other groups, we find that the activation of c-Raf-1 by Src in vivo occurs concomitant with tyrosine phosphorylation on c-Raf-1, and in vitro, activation of c-Raf-1 by Src requires the presence of ATP. Therefore we propose that activation of c-Raf-1 by Ras or by Src occurs through different mechanisms.
Stokoe
D
D
Onyx Pharmaceuticals, Richmond, CA 94806, USA. dhstokoe@onyx-pharm.com
McCormick
F
F
D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D015536:Down-Regulation; D004789:Enzyme Activation; D066298:In Vitro Techniques; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D015854:Up-Regulation; D018631:ras Proteins / Q000378:metabolism*; D018909:src Homology Domains
D011518:Proto-Oncogene Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins