DIACYLGLYCEROL GENERATED BY EXOGENOUS PHOSPHOLIPASE-C ACTIVATES THE MITOGEN-ACTIVATED PROTEIN-KINASE PATHWAY INDEPENDENT OF RAS-SENSITIVE AND PHORBOL ESTER-SENSITIVE PROTEIN-KINASE-C - DEPENDENCE ON PROTEIN-KINASE C-ZETA

The role of diacylglycerol (DG) formation from phosphatidylcholine in mitogenic signal transduction is poorly understood. We have generated this lipid at the plasma membrane by treating Rat-1 fibroblasts with b acterial phosphatidylcholine-specific phospholipase C (PC-PLC). This t reatment leads to activation of mitogen-activated protein kinase (MAPK ). However, unlike platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), PC-PLC fails to activate Ras and to induce DNA sy nthesis, and activates MAPK only transiently (< 45 min). Down-regulati on of protein kinase C (PKC)-alpha, -delta and -epsilon isotypes has l ittle or no effect on MAPK activation by either PC-PLC or growth facto rs. However, Ro 31-8220, a highly selective inhibitor of all PKC isoty pes, including atypical PKC-zeta but not Raf-1, blocks MAPK activation by PDGF and PC-PLC, but not that by EGF, suggesting that atypical PKC mediates the PDGF and PC-PLC signal. In line with this, PKC-zeta is a ctivated by PC-PLC and PDGF, but not by EGF, as shown by a kinase assa y in vitro, using biotinylated epsilon-peptide as a substrate. Further more, dominant-negative PKC-zeta inhibits, while (wild-type) PKC-zeta overexpression enhances MAPK activation by PDGF and PC-PLC. The result s suggest that DG generated by PC-PLC can activate the MAPK pathway in dependent of Ras and phorbolester-sensitive PKC but, instead, via PKC- zeta.