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

Phospholipase D (PLD) activity is commonly elevated in response to mitogenic signals. We reported previously that although the transformed phenotype induced by v-Src was dependent upon Raf-1, the PLD activity induced by v-Src was independent of Raf-1. This observation suggested to us that Raf would not likely be an activator of PLD. However, upon examination of PLD activity in v-Raf-transformed cells, surprisingly, we found that PLD activity is elevated to levels that were even higher than that observed in v-Src-transformed 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 the activation of PLD. The v-Raf-induced PLD activity was inhibited by dominant negative mutants for both Ral and Rho. The dependence upon Ral was particularly surprising since Ral is a downstream target of Ras, which is an upstream activator of Raf. Depleting cells of PKC by long term phorbol ester treatment actually increased PLD activity in v-Raf-transformed cells, indicating that v-Raf-induced PLD activity is not dependent on PKC. These data describe a novel mechanism for PLD activation by v-Raf that is independent of PKC, but dependent upon both Ral and Rho GTPases.