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 examin ed the effects of PA on distinct isoforms of protein kinase C (PKC) us ing a new cell-free assay system. Addition of PA to cytosol from COS c ells overexpressing PKC-alpha, -epsilon or -zeta differentially activa ted all three isotypes, as shown by PKC autophosphorylation, and promi nent 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 o f protein phosphorylation. BisPA was also a strong activator of PKC-ep silon, but a weak activator of PKC-alpha. Ca2+, at greater than or equ al to 0.1 mu M, 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 phosphory lations 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 phosphoryl ation of distinctive substrates, but also caused an upward shift in el ectrophoretic mobility of PKC-zeta, which was not observed with other acidic lipids or with PKC-alpha or -epsilon. We have presented evidenc e that this mobility shift is not caused by PKC-zeta autophosphorylati on, but it coincides with physical binding of PA to PKC-zeta These res ults suggest that in cells stimulated under conditions where intracell ular Ca2+ is at (or has returned to) basal level, PA may be a physiolo gical activator of PKC-zeta.