Phospholipid peroxidation induces cytosolic phospholipase A2 activity: membrane effects versus enzyme phosphorylation.
Cytosolic phospholipase A2 (cPLA2) is a signal-responsive enzyme that is highly selective to the nature of phospholipid substrates. A mechanism for cPLA2 activity regulation through a signal transduction pathway has been proposed and this signaling appears to be influenced by oxidants. Oxidant-mediated signaling of PLA2 may serve as an alternative mechanism for enzyme regulation; however, the manner of regulation has yet to be delineated. In this report we demonstrate that there is a direct effect of membrane oxidation on cPLA2 phosphorylation and activity. A simple in vitro system consisting of purified cPLA2 and phospholipid vesicles was used to facilitate protein kinase C (PKC) activity and provide substrates for cPLA2. Using these vesicles we found that the activity of cPLA2 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 cPLA2 increased hydrolytic activity 71% compared to preparations lacking PKC. Using phospholipid vesicles as substrates, PKC-mediated phosphorylation resulted in an 85% increase in cPLA2 activity compared to preparations without PKC. PKC-mediated phosphorylation of cPLA2, 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 cPLA2 phosphorylation.
Rashba-Step
J
J
Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles 90033, USA.
Tatoyan
A
A
Duncan
R
R
Ann
D
D
Pushpa-Rehka
T R
TR
Sevanian
A
A
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation; D004790:Enzyme Induction; D006801:Humans; D006868:Hydrolysis; D015227:Lipid Peroxidation; D010741:Phospholipases A / Q000096:biosynthesis* / Q000378:metabolism; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D010766:Phosphorylation; D013379:Substrate Specificity
D010743:Phospholipids; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium