Phospholipid peroxidation induces cytosolic phospholipase A(2) activity: Membrane effects versus enzyme phosphorylation

Cytosolic phospholipase A(2) (cPLA(2)) is a signal-responsive enzyme that is highly selective to the nature of phospholipid substrates, A mechanism for cPLA(2) activity regulation through a signal transduction pathway has been proposed and this signaling appears to be influenced by oxidants, Oxidant-mediated signaling of PLA(2) may serve as an alternative mechanism for enzyme regulation; however, the manner of regulation has yet to be delineated, In this report we demonstrate there is a direct effect of membrane oxidation on cPLA(2) phosphorylation and activity, A simple in vitro system consisting of purified cPLA(2) and phospholipid vesicles was used to facilitate protein kinase C (PKC) activity and provide substrates for cPLA(2). Using these vesicles we found that the activity of cPLA(2) was enhanced twofold when the vesicles contained as little as 5 mol% phosphatidylcholine hydroperoxides (PLPCOOH), The order of hydrolytic preference for fatty acyl species was 20:4 > 18:2 > 18:1 > 16:0, and the presence of PLPCOOH stimulated hydrolysis largely of phosphatidylcholine containing 20:4, The Ca2+ concentrations required for stimulated hydrolytic activity were also twofold lower for oxidized compared to unoxidized vesicles, Using phospholipid micelles as substrates, PKC-mediated phosphorylation of cPLA(2), increased hydrolytic activity 71% compared to preparations lacking PKC. Using phospholipid vesicles as substrates, PKC-mediated phosphorylation resulted in an 85% increase in cPLA(2) activity compared to preparations without PKC, PKC-mediated phosphorylation of cPLA(2), therefore, stimulates catalytic activity toward membrane phospholipids and the extent of activation is enhanced directly by peroxidation of membrane phospholipids and involves a peroxide-induced stimulation of cPLA(2) phosphorylation. (C) 1997 Academic Press