A critical role of protein kinase Cδ activation loop phosphorylation in formyl-methionyl-leucyl-phenylalanine-induced phosphorylation of p47phox 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 PKC delta 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 PKC delta is required for p47(phox) phosphorylation and reconstitution of NADPH oxidase. PKC beta II cannot replace PKC delta for this function. Characterization of PKC delta/PKC beta II chimeras has led to the identification of the catalytic domain of PKC delta as a target of regulation by fMLF, which induces a biphasic (30 and 180 s) phosphorylation of Thr(505) in the activation loop of mouse PKC delta. Mutation of Thr(505) to alanine abolishes the ability of PKC delta 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 PKC delta phosphorylation is a novel mechanism for NADPH oxidase activation. The ability to induce PKC delta phosphorylation may distinguish a full agonist from a partial agonist for superoxide production.