Phosphatidic acid activation of protein kinase C-zeta overexpressed in COS cells: comparison with other protein kinase C isotypes and other acidic lipids

• Published in: Biochemical journal 1994-12, Vol.304 ( Pt 3) (3), p.1001-1008
• Main Authors: Limatola, C, Schaap, D, Moolenaar, W H, van Blitterswijk, W J
• Format: Article
• Language: English
• Subjects: Animals; Calcium - metabolism; Calcium - pharmacology; Cell Line; Diglycerides - pharmacology; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Hydrogen-Ion Concentration; Isoenzymes - drug effects; Isoenzymes - metabolism; Lipids - pharmacology; Phosphatidic Acids - metabolism; Phosphatidic Acids - pharmacology; Phosphatidic Acids - physiology; Phosphatidylserines - pharmacology; Phosphorus Radioisotopes; Phosphorylation; Protein Kinase C - drug effects; Protein Kinase C - metabolism; Sodium Dodecyl Sulfate; Transfection
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3041001

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.