The overall goal of the project is elucidation of the roles of ras proto- oncogenes in signal transduction pathways leading to cell growth and differentiation. Although Ras proteins clearly play a central role in the transduction of signals from receptor tyrosine kinases, the effector(s) of Ras in mammalian cells have eluded identification. However, recent studies utilizing a dominant inhibitory ras mutant (ras N-17) developed in our laboratory have opened new approaches to this problem and have led to the identification of several potential targets for Ras action: the Raf protein-serine/threonine kinase, MAP kinase, translation factor eIF-4E, and phosphatidylcholine (PC) turnover. PC hydrolysis is of particular interest since it is a common response of cells to growth factor stimulation and is thought to serve as a sustained source of diacylglycerol which plays a critical role in long-term response such as cell growth and differentiation. In addition, current data suggests that PC hydrolysis acts upstream of Raf, implying that activation of a PC phospholipase plays a central role in the Ras signaling pathway. We therefore plan to focus our studies on Ras- dependent PC hydrolysis, according to the following specific aims: 1. Analysis of the relationship between PC turnover and Raf activation during mitogenic signal transduction. 2. Investigation of the role of GAP in Ras-dependent activation of PC-PLC and in neuronal differentiation of PC12 cells. 3. Characterization of the PC-specific phospholipase activated by Ras during mitogenic signaling. 4. Identification of protein kinase C isozyme that serve as targets for activation by PC-derived diacylglycerol.
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