Phospholipase D Activation by v-Src and v-Ras: In response to the oncogenic stimuli of v-Src, there is an activation of phospholipase D (PLD) that is dependent upon a GTPase cascade of Ras and Ral. Transformation by v-Src is also dependent upon both Ras and Ral, suggesting a role for PLD in cell transformation. PLD hydrolyzes phosphatidylcholine to phosphatidic acid (PA) and choline. The best understood effects of PA are mediated by the PA metabolite diacylglycerol, which leads to the activation of protein kinase C. However, PA is also biologically active and has been implicated in regulating a variety of signaling molecules including Raf-l, phosphatidylinositol kinases, and the GTPase activating proteins (GAPs) for Ras, Rac and Arf. PLD activity and PA have also been implicated in vesicle transport. Although RalA is required for v-Src-induced PLD activity and PLD exists in a complex with Ral, an activated RalA is not sufficient for PLD activation. Biochemical and genetic evidence suggest at least two factors in addition to RalA are required for PLD activation by v-Src. The objective of the proposed studies is to characterize the interaction between RalA and PLD and to identify factors contributing to the activation of PLD via the newly emerging Ras/Ral signaling pathway. Candidate proteins implicated in the activation of PLD by v-Src are the Rho family GTPases Rho, Rac, Cdc42 and the RalA binding protein Ral-BP1, which is a GAP protein for Rho family GTPases. Arf (ADP ribosylation factor), which activates the PLD associated with RalA is another candidate. Proposed studies will test whether these and other implicated factors contribute to activation of PLD by v-Src. Because of the many extracellular stimuli that activate PLD via tyrosine kinases and the many intracellular responses to the PLD-generated PA, understanding of the mechanism of PLD activation by tyrosine kinases will provide several new targets for therapeutic intervention in diseases such as human breast cancer where altered regulation of tyrosine kinase activity has been implicated.
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