Phosphorylation of p22phox is mediated by phospholipase D-dependent and -independent mechanisms - Correlation of NADPH oxidase activity and p22phox phosphorylation

Human neutrophils participate in the host innate immune response, partly me diated by the multicomponent superoxide-generating enzyme NADPH oxidase. A correlation between phosphorylation of cytosolic NADPH oxidase components a nd enzyme activation has been identified but is not well understood. We pre viously showed that p22(phox), the small subunit of the membrane-bound oxid ase component flavocytochrome b(558), is an in vitro substrate for both a p hosphatidic acid-activated kinase and conventional protein kinase C isoform s (Regier, D. S., Waite, K. A, Wallin, R., and McPhail, L. C. (1999) J. Bio l. Chem. 274, 36601-36608). Here we show that several neutrophil agonists ( phorbol myristate acetate, opsonized zymosan, and N-formyl-methionyl-leucyl -phenylalanine) induce p22(phox) phosphorylation in intact neutrophils. To determine if phospholipase D (PLD) is needed for p22(phox) phosphorylation, cells were pretreated with ethanol, which reduces phosphatidic acid produc tion by PLD in stimulated cells. Phorbol myristate acetate-induced phosphor ylation of p22(phox) and NADPH oxidase activity were not reduced by ethanol , In contrast, ethanol reduced both activities when cells were stimulated b y N-formyl-methionyl-leucyl-phenylalanine or opsonized zymosan. Varying the time of stimulation with opsonized zymosan showed that the phosphorylation of p22(phox) coincides with NADPH oxidase activation. GF109203X, an inhibi tor of protein kinase C and the phosphatidic acid-activated protein kinase, decreased both p22(phox) phosphorylation and NADPH oxidase activity in par allel in opsonized zymosan-stimulated cells. Stimulus-induced phosphorylati on of p22(phox) was on Thr residue(s), in agreement with in vitro results. Overall, these data show that NADPH oxidase activity and p22(phox) phosphor ylation are correlated and suggest two mechanisms (PLD-dependent and -indep endent) by which p22(phox) phosphorylation occurs.