Phosphatidic acid activation of protein kinase C-zeta overexpressed in COS cells: comparison with other protein kinase C isotypes and other acidic lipids.
Phosphatidic acid (PA) is produced rapidly in agonist-stimulated cells, but the physiological function of this PA is unknown. We have examined the effects of PA on distinct isoforms of protein kinase C (PKC) using a new cell-free assay system. Addition of PA to cytosol from COS cells overexpressing PKC-alpha, -epsilon or -zeta differentially-activated all three isotypes, as shown by PKC autophosphorylation, and prominent phosphorylation of multiple endogenous substrates. In the absence of Ca2+, the diacylglycerol-insensitive zeta-isotype of PKC was most strongly activated by both PA and bisPA, a newly identified product of activated phospholipase D, with each lipid inducing its own profile of protein phosphorylation. BisPA was also a strong activator of PKC-epsilon, but a weak activator of PKC-alpha. Ca2+, at > or = 0.1 microM, inhibited PA and bisPA activation of PKC-zeta, but did not affect PKC-epsilon activation. In contrast, PKC-alpha was strongly activated by PA only in the presence of Ca2+. BisPA-induced phosphorylations mediated by PKC-zeta could be mimicked in part by other acidic phospholipids and unsaturated fatty acids. PA activation of PKC-zeta was unique in that PA not only stimulated PKC-zeta-mediated phosphorylation of distinctive substrates, but also caused an upward shift in electrophoretic mobility of PKC-zeta, which was not observed with other acidic lipids or with PKC-alpha or -epsilon. We have presented evidence that this mobility shift is not caused by PKC-zeta autophosphorylation, but it coincides with physical binding of PA to PKC-zeta. These results suggest that in cells stimulated under conditions where intracellular Ca2+ is at (or has returned to) basal level, PA may be a physiological activator of PKC-zeta.
Limatola
C
C
Division of Cellular Biochemistry, Netherlands Cancer Institute, Amsterdam.
Schaap
D
D
Moolenaar
W H
WH
van Blitterswijk
W J
WJ
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D004075:Diglycerides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000187:drug effects* / Q000378:metabolism*; D008055:Lipids / Q000494:pharmacology*; D010712:Phosphatidic Acids / Q000378:metabolism / Q000494:pharmacology* / Q000502:physiology; D010718:Phosphatidylserines / Q000494:pharmacology; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011493:Protein Kinase C / Q000187:drug effects* / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D014162:Transfection
D004075:Diglycerides; D007527:Isoenzymes; D008055:Lipids; D010712:Phosphatidic Acids; D010718:Phosphatidylserines; D010761:Phosphorus Radioisotopes; D012967:Sodium Dodecyl Sulfate; D011493:Protein Kinase C; D002118:Calcium