PKCα Regulates Phosphorylation and Enzymatic Activity of cPLA2 In Vitro and in Activated Human Monocytes

Phospholipases A (PLA ) are potent regulators of the inflammatory response. We have observed that Group IV cPLA activity is required for the production of superoxide anion (O ) in human monocytes [Li Q., Cathcart M.K. J. Biol. Chem. 272 (4) (1997) 2404-2411.]. We have previously identified PKCα as a kinase pathway required for monocyte O production [Li Q., Cathcart M.K. J. Biol. Chem. 269 (26) (1994) 17508-17515.]. We therefore investigated the potential interaction between PKCα and cPLA by evaluating the requirement for specific PKC isoenzymes in the process of activating cPLA enzymatic activity and protein phosphorylation upon monocyte activation. We first showed that general PKC inhibitors and antisense oligodeoxyribonucleotides (ODN) to the cPKC group of PKC enzymes inhibited cPLA activity. To distinguish between PKCα and PKCβ isoenzymes in regulating cPLA protein phosphorylation and enzymatic activity, we employed our previously characterized PKCα or PKCβ isoenzyme-specific antisense ODN [Li Q., Subbulakshmi V., Fields A.P., Murray, N.R., Cathcart M.K., J. Biol. Chem. 274 (6) (1999) 3764-3771]. Suppression of PKCα expression, but not PKCβ expression, inhibited cPLA protein phosphorylation and enzymatic activity. Additional studies ruled out a contribution by Erk1/2 to cPLA phosphorylation and activation. We also found that cPLA co-immunoprecipitated with PKCα and vice versa. In vitro studies demonstrated that PKCα could directly phosphorylate cPLA .and enhance enzymatic activity. Finally, we showed that addition of arachidonic acid restored the production of O in monocytes defective in either PKCα or cPLA expression. Taken together, our data suggest that PKCα, but not PKCβ, is the predominant cPKC isoenzyme required for cPLA protein phosphorylation and maximal induction of cPLA enzymatic activity upon activation of human monocytes. Our data also support the concept that the requirements for PKCα and cPLA in O generation are solely due to their seminal role in generating arachidonic acid. © 2006 Elsevier Inc. All rights reserved. 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 - - -