EVIDENCE FOR A BIFURCATION OF THE MITOGENIC SIGNALING PATHWAY ACTIVATED BY RAS AND PHOSPHATIDYLCHOLINE-HYDROLYZING PHOSPHOLIPASE-C

NIH 3T3 cells stably transfected with the gene encoding phosphatidylch oline-hydrolyzing phospholipase C (PC-PLC) from Bacillus cereus displa y a chronic elevation of intracellular diacylglycerol levels and a tra nsformed phenotype, We have used such PC-PLC-transformed cells to eval uate the roles of the cytoplasmic serine/threonine kinases Raf-1, zeta protein kinase C (zeta PKC) and protein kinase A (PKA) in oncogenesis and mitogenic signal transduction elicited by phosphatidylcholine hyd rolysis, We demonstrate here that stable expression of dominant negati ve mutants of both zeta PKC and Raf-1 lead to reversion of PC-PLC-tran sformed cells, Interestingly, expression of kinase defective zeta PKC also reverted NIH 3T3 cells transformed by the v-Ha-ras oncogene, Acti vation of PKA in response to elevation of cAMP levels also lead to rev ersion 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 b lock the ability of PC-PLC-transformed cells to induce DNA synthesis i n the absence of growth factors, These results clearly implicate both Raf-1 and zeta PKC as necessary downstream components for transduction of the mitogenic/oncogenic signal generated by PLC-mediated hydrolysi s of phosphatidylcholine and suggest, together with other recent evide nce, a bifurcation in the signaling pathway downstream of PC-PLC.