Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.

The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the «isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC« inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC« stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC«also stimulated Raf kinase in baculovirusinfected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKCafunctioned similarly to PKC«in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKCa and PKC« overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerolregulated PKCs function as redundant activators of Raf-1 in vivo.