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 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, zeta protein kinase C (zeta PKC) 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 zeta PKC and Raf-1 lead to reversion of PC-PLC-transformed cells. Interestingly, expression of kinase defective zeta PKC 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 zeta PKC 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.
Bjørkøy
G
G
Department of Biochemistry, University of Tromsø, Norway.
Overvatn
A
A
Diaz-Meco
M T
MT
Moscat
J
J
Johansen
T
T
D016475:3T3 Cells; D000818:Animals; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D004261:DNA Replication; D005799:Genes, Dominant; D006868:Hydrolysis; D051379:Mice; D008934:Mitogens / Q000378:metabolism*; D009154:Mutation; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D010703:Phorbol Esters / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism*; D011494:Protein Kinases / Q000187:drug effects / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019908:Proto-Oncogene Proteins c-raf; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism*
D008934:Mitogens; D010703:Phorbol Esters; D010713:Phosphatidylcholines; D011518:Proto-Oncogene Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D010738:Type C Phospholipases; D015689:Oncogene Protein p21(ras)