Diacylglycerol generated by exogenous phospholipase C activates the mitogen-activated protein kinase pathway independent of Ras- 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 bacterial phosphatidylcholine-specific phospholipase C (PC-PLC). This treatment 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 synthesis, and activates MAPK only transiently (<45 min). Down-regulation of protein kinase C (PKC) -alpha, -delta and -epsilon isotypes has little or no effect on MAPK activation by either PC-PLC or growth factors. However, Ro 31-8220, a highly selective inhibitor of all PKC isotypes, 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 activated by PC-PLC and PDGF, but not by EGF, as shown by a kinase assay in vitro, using biotinylated epsilon-peptide as a substrate. Furthermore, dominant-negative PKC-zeta inhibits, while (wild-type) PKC-zeta overexpression enhances MAPK activation by PDGF and PC-PLC. The results suggest that DG generated by PC-PLC can activate the MAPK pathway independent of Ras and phorbol-ester-sensitive PKC but, instead, via PKC-zeta.
van Dijk
M
M
Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Muriana
F J
FJ
van Der Hoeven
P C
PC
de Widt
J
J
Schaap
D
D
Moolenaar
W H
WH
van Blitterswijk
W J
WJ
D000818:Animals; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D006133:Growth Substances / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1; D008938:Mitosis / Q000187:drug effects; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*; D018631:ras Proteins / Q000502:physiology; D000099221:Protein Kinase C zeta
D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004075:Diglycerides; D004791:Enzyme Inhibitors; D006133:Growth Substances; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D007211:Indoles; D007527:Isoenzymes; D019950:Mitogen-Activated Protein Kinase 1; D010703:Phorbol Esters; D011493:Protein Kinase C; D011505:Protein-Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D010738:Type C Phospholipases; D018631:ras Proteins; D000099221:Protein Kinase C zeta; C105598:phosphatidylcholine-specific phospholipase C; C064758:Ro 31-8220