Ral and Rho-dependent activation of phospholipase D in v-Raf-transformed cells

Phospholipase D (PLD) activity is commonly elevated in response to mitogeni c signals. We reported previously that although the transformed phenotype i nduced by v-Src was dependent upon Raf-l, the PLD activity induced by v-Src was independent of Raf-l, This observation suggested to us that Raf would not likely be an activator of PLD. However, upon examination of PLD activit y in v-Raf-transformed cells, surprisingly, we found that PLD activity is e levated to levels that were even higher than that observed in v-Src-transfo rmed cells. To characterize the mechanism of v-Raf-induced PLD activity, we examined the dependence of v-Raf-induced PLD activity upon protein kinase C (PKC) the small GTPases Ral and Rho, which have all been implicated in th e activation of PLD. The v-Raf-induced PLD activity was inhibited by domina nt negative mutants for both Ral and Rho. The dependence upon Ral was parti cularly surprising since Ral is a downstream target of Ras, which is an ups tream activator of Raf. Depleting cells of PKC by long term phorbol ester t reatment actually increased PLD activity in v-Raf-transformed cells, indica ting that v-Raf-induced PLD activity is not dependent on PKC, These data de scribe a novel mechanism for PLD activation by v-Raf that is independent of PKC, but dependent upon both Ral and Rho GTPases. (C) 1999 Academic Press.