A critical role of protein kinase Cδ activation loop phosphorylation in formyl-methionyl-leucyl-phenylalanine-induced phosphorylation of p47 ^phox and rapid activation of nicotinamide adenine dinucleotide phosphate oxidase

Generation of superoxide by professional phagocytes is an important mechanism of host defense against bacterial infection. Several protein kinase C (PKC) isoforms have been found to phosphorylate p47^phox, resulting in its membrane translocation and activation of the NADPH oxidase. However, the mechanism by which specific PKC isoforms regulate NADPH oxidase activation remains to be elucidated. In this study, we report that PKCδ phosphorylation in its activation loop is rapidly induced by fMLF and is essential for its ability to catalyze p47^phox phosphorylation. Using transfected COS-7 cells expressing gp91^phox, p22^phox, p67^phox, and p47^phox (COS-phox cells), we found that a functionally active PKCδ is required for p47^phox phosphorylation and reconstitution of NADPH oxidase. PKCβII cannot replace PKCδ for this function. Characterization of PKCδ/PKCβII chimeras has led to the identification of the catalytic domain of PKCδ as a target of regulation by fMLF, which induces a biphasic (30 and 180 s) phosphorylation of Thr⁵⁰⁵ in the activation loop of mouse PKCδ. Mutation of Thr⁵⁰⁵ to alanine abolishes the ability of PKCδ to catalyze p47^phox phosphorylation in vitro and to reconstitute NADPH oxidase in the transfected COS-phox cells. A correlation between fMLF-induced activation loop phosphorylation and superoxide production is also established in the differentiated PLB-985 human myelomonoblastic cells. We conclude that agonist-induced PKCδ phosphorylation is a novel mechanism for NADPH oxidase activation. The ability to induce PKCδ phosphorylation may distinguish a full agonist from a partial agonist for superoxide production.