Regulation of the Ras signaling pathway by GTPase-activating protein in PC12 cells.
We have investigated the role of Ras GTPase-activating protein (GAP) in NGF-induced neuronal differentiation by overexpressing both wild-type and membrane-targeted GAP in PC12 cells. Extension of neurites in response to NGF was completely blocked in cells expressing the highest level of membrane-targeted GAP and significantly inhibited in cells expressing 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, indicating that GAP inhibits differentiation of PC12 cells by downregulating Ras function. GAP overexpression also inhibited stimulation of mitogen-activated protein (MAP) kinase and induction of immediate-early genes in response to NGF. In cells expressing wild-type GAP or lower levels of membrane-targeted GAP, the initial activation of MAP kinase and immediate-early gene expression were only partially inhibited. However, GAP expression in these cells resulted in substantial inhibition of sustained MAP kinase activity following NGF treatment, consistent with the inhibition of neurite extension in these cell lines. These results indicate that GAP acts as a negative regulation, rather than an effector, of Ras signaling in PC12 cells.
D000818:Animals; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004790:Enzyme Induction; D020690:GTPase-Activating Proteins; D017781:Genes, Immediate-Early; D016762:Genes, fos; D011905:Genes, ras; D016391:Genes, src; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000494:pharmacology; D009474:Neurons / Q000166:cytology; D016716:PC12 Cells; D011506:Proteins / Q000502:physiology*; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology*; D051381:Rats; D015398:Signal Transduction; D014162:Transfection; D020703:ras GTPase-Activating Proteins
D017931:DNA Primers; D020690:GTPase-Activating Proteins; D009414:Nerve Growth Factors; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D016283:Proto-Oncogene Proteins p21(ras)