REGULATION OF THE RAS SIGNALING PATHWAY BY GTPASE-ACTIVATING PROTEIN IN PC12 CELLS

We have investigated the role of Pas GTPase-activating protein (GAP) i n NGF-induced neuronal differentiation by overexpressing both wild-typ e and membrane-targeted GAP in PC12 cells. Extension of neurites in re sponse to NGF was completely blocked in cells expressing the highest l evel of membrane-targeted GAP and significantly inhibited in cells exp ressing either wild-type GAP or lower levels of membrane-targeted GAP. Overexpression of membrane-targeted GAP similarly inhibited induction of differentiation by src, but not by ras or raf oncogenes, indicatin g that GAP inhibits differentiation of PC12 cells by downregulating Ra s function. GAP overexpression also inhibited stimulation of mitogen-a ctivated protein (MAP) kinase and induction of immediate-early genes i n response to NGF. In cells expressing wild-type GAP or lower levels o f membrane-targeted GAP, the initial activation of MAP kinase and imme diate-early gene expression were only partially inhibited. However, GA P expression in these cells resulted in substantial inhibition of sust ained MAP kinase activity following NGF treatment, consistent with the inhibition of neurite extension in these cell lines. These results in dicate that GAP acts as a negative regulation, rather than an effector , of Ras signaling in PC12 cells.