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 Pas guanine nucleotide- binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Pas recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been establishe d. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived d iacylglycerol, as a Raf activator. A dominant negative mutant of PKC e psilon inhibited both proliferation of MH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression Of active PKC epsilon sti mulated Raf kinase activity in COS cells and overcame the inhibitory e ffects of dominant negative Pas in NIH 3T3 cells, PKC epsilon also sti mulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 c ells and was able to directly activate Raf in vitro, Consistent with i ts previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assa ys. In addition, constitutively active mutants of both PKC alpha and P KC epsilon overcame the inhibitory effects of dominant negative mutant s of the other PKC isotype, indicating that these diacylglycerol-regul ated PKCs function as redundant activators of Raf-l in vivo.