Activation of c-Raf-1 by Ras and Src through different mechanisms: activationin vivoandin vitro

摘要: The c-Raf-1 protein kinase plays a critical role in intracellular signaling downstream from many tyrosine and G-protein-linked receptors. binds to the proto-oncogene Ras GTP-dependent manner, but exact mechanism of activation by is still unclear. We have established system study vitro. This involves mixing membranes cells expressing oncogenic H-RasG12V, with cytosol epitope-tagged full-length wild-type c-Raf-1. results fraction binding concomitant 10- 20-fold increase specific activity. was only component these required for activation, as purified recombinant farnesylated K-Ras.GTP, not non-farnesylated K-Ras.GTP or K-Ras.GDP, able activate same degree intact H-RasG12V membranes. most potent occurred under conditions which phosphorylation prohibited. Under phosphorylation-permissive conditions, substantially inhibited. Consistent other groups, we find that Src vivo occurs on c-Raf-1, vitro, requires presence ATP. Therefore propose through different mechanisms.