PKCα regulates phosphorylation and enzymatic activity of cPLA2 in vitro and in activated human monocytes

Phospholipases A(2) (PLA(2)) are potent regulators of the inflammatory response. We have observed that Group IV cPLA(2) activity is required for the production of superoxide anion (O-2(-)) in human monocytes [Li Q., Cathcart M.K. J. Biol. Chem. 272 (4) (1997) 2404-2411.]. We have previously identified PKC alpha as a kinase pathway required for monocyte O-2(-) production [Li Q., Cathcart M.K. J. Biol. Chem. 269 (26) (1994) 17508-17515.]. We therefore investigated the potential interaction between PKC alpha and cPLA(2) by evaluating the requirement for specific PKC isoenzymes in the process of activating cPLA2 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(2) activity. To distinguish between PKC alpha and PKC beta isoenzymes in regulating cPLA(2) protein phosphorylation and enzymatic activity, we employed our previously characterized PKC alpha or PKC beta 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 PKCa expression, but not PKC beta expression, inhibited cPLA2 protein phosphorylation and enzymatic activity. Additional studies ruled out a contribution by Erkl/2 to cPLA(2) phosphorylation and activation. We also found that cPLA(2) co-immunoprecipitated with PKC alpha and vice versa. In vitro studies demonstrated that PKC alpha could directly phosphorylate cPLA(2) and enhance enzymatic activity. Finally, we showed that addition of arachidonic acid restored the production of O-2(-) in monocytes defective in either PKCa or cPLA(2) expression. Taken together, our data suggest that PKC alpha, but not PKC beta, is the predominant cPKC isoenzyme required for cPLA(2) protein phosphorylation and maximal induction of cPLA(2) enzymatic activity upon activation of human monocytes. Our data also support the concept that the requirements for PKC alpha and cPLA(2) in O-2(-) generation are solely due to their seminal role in generating arachidonic acid. (c) 2006 Elsevier Inc. All rights reserved.