Human neutrophils participate in the host innate immune response, partly mediated by the multicomponent superoxide-generating enzyme NADPH oxidase. A correlation between phosphorylation of cytosolic NADPH oxidase components and enzyme activation has been identified but is not well understood. We previously showed that p22 phox , the small subunit of the membranebound oxidase component flavocytochrome b 558 , is an in vitro substrate for both a phosphatidic acid-activated kinase and conventional protein kinase C isoforms ( In intact neutrophils, several signaling mechanisms, initiated by receptor-ligand interactions, are involved in the regulation of NADPH oxidase (reviewed in Refs. 1 and 15). These events include activation of phospholipases, production of lipid second messengers, activation of protein kinases, and phosphorylation of neutrophil proteins and NADPH oxidase components. Activation of phospholipase A 2 and production of arachidonic acid is required for NADPH oxidase activity (16,17), although the role of arachidonic acid has not been identified. Phospholipase C also may be involved in NADPH oxidase activation (18 -20). Production of diacylglycerol by phospholipase C can induce activation of protein kinase C (PKC), which may mediate the phosphorylation of several NADPH oxidase components (see below) and is clearly an important regulator of NADPH oxidase activation (21, 22). Activation of phospholipase D (PLD), which cleaves phospholipids to form phosphatidic acid (PA), correlates with NADPH oxidase activation (23-30). The mechanism by which PA regulates NADPH oxidase has not been determined; however, a PA-activated protein kinase has been partially purified from neutrophil cytosol (31). phox , and p67 phox , 22-, 47, and 67-kDa phox component, respectively; dCB, dihydrocytochalasin B; fMLP, N-formyl-methionyl-leucyl-phenylalanine; OPZ, opsonized zymosan; PA, phosphatidic acid; PKC, protein kinase C; PLD, phospholipase D; PMA, phorbol 12-myristate 13-acetate; PVDF, polyvinylidene difluoride membrane; PAGE, polyacrylamide gel electrophoresis; GTP␥S, guanosine 5Ј-O-(3-thiotriphosphate); mAb, monoclonal antibody; Pipes, 1,4-piperazinediethanesulfonic acid.
