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Evidence for a Bifurcation of the Mitogenic Signaling Pathway Activated by Ras and Phosphatidylcholine-hydrolyzing Phospholipase C

1995; Elsevier BV; Volume: 270; Issue: 36 Linguagem: Inglês

10.1074/jbc.270.36.21299

1083-351X

Geir Bj, Aud, Marı́a T. Diaz-Meco, Jorge Moscat, Terje Johansen,

Endoplasmic Reticulum Stress and Disease

NIH 3T3 cells stably transfected with the gene encoding phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) from Bacillus cereus display a chronic elevation of intracellular diacylglycerol levels and a transformed phenotype. We have used such PC-PLC-transformed cells to evaluate the roles of the cytoplasmic serine/threonine kinases Raf-1, ζ protein kinase C (ζPKC) and protein kinase A (PKA) in oncogenesis and mitogenic signal transduction elicited by phosphatidylcholine hydrolysis. We demonstrate here that stable expression of dominant negative mutants of both ζPKC and Raf-1 lead to reversion of PC-PLC-transformed cells. Interestingly, expression of kinase defective ζPKC also reverted NIH 3T3 cells transformed by the v-Ha-ras oncogene. Activation of PKA in response to elevation of cAMP levels also lead to reversion of PC-PLC-induced transformation, implicating PKA as a negative regulator acting downstream of PC-PLC. On the other hand, inhibition or depletion of phorbol ester responsive PKCs attenuated but did not block the ability of PC-PLC-transformed cells to induce DNA synthesis in the absence of growth factors. These results clearly implicate both Raf-1 and ζPKC as necessary downstream components for transduction of the mitogenic/oncogenic signal generated by PLC-mediated hydrolysis of phosphatidylcholine and suggest, together with other recent evidence, a bifurcation in the signaling pathway downstream of PC-PLC. NIH 3T3 cells stably transfected with the gene encoding phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) from Bacillus cereus display a chronic elevation of intracellular diacylglycerol levels and a transformed phenotype. We have used such PC-PLC-transformed cells to evaluate the roles of the cytoplasmic serine/threonine kinases Raf-1, ζ protein kinase C (ζPKC) and protein kinase A (PKA) in oncogenesis and mitogenic signal transduction elicited by phosphatidylcholine hydrolysis. We demonstrate here that stable expression of dominant negative mutants of both ζPKC and Raf-1 lead to reversion of PC-PLC-transformed cells. Interestingly, expression of kinase defective ζPKC also reverted NIH 3T3 cells transformed by the v-Ha-ras oncogene. Activation of PKA in response to elevation of cAMP levels also lead to reversion of PC-PLC-induced transformation, implicating PKA as a negative regulator acting downstream of PC-PLC. On the other hand, inhibition or depletion of phorbol ester responsive PKCs attenuated but did not block the ability of PC-PLC-transformed cells to induce DNA synthesis in the absence of growth factors. These results clearly implicate both Raf-1 and ζPKC as necessary downstream components for transduction of the mitogenic/oncogenic signal generated by PLC-mediated hydrolysis of phosphatidylcholine and suggest, together with other recent evidence, a bifurcation in the signaling pathway downstream of PC-PLC.