CELL-FREE ACTIVATION OF NEUTROPHIL NADPH OXIDASE BY A PHOSPHATIDIC ACID-REGULATED PROTEIN-KINASE

The phosphorylation-dependent mechanisms regulating activation of the human neutrophil respiratory-burst enzyme, NADPH oxidase, have not bee n elucidated. We have shown that phosphatidic acid (PA) and diacylglyc erol (DG), products of phospholipase activation, synergize to activate NADPH oxidase in a cell-free system. We now report that activation by PA plus DG involves protein kinase activity, unlike other cell-free s ystem activators. NADPH oxidase activation by PA plus DG is reduced ap proximate to 70% by several protein kinase inhibitors [1-(5-isoquinoli nesulfonyl)piperazine, staurosporine, GF-109203X]. Similarly, depletio n of ATP by dialysis reduces PA plus DG-mediated NADPH oxidase activat ion by approximate to 70%. Addition of ATP, but not a nonhydrolyzable ATP analog, to the dialyzed system restores activation levels to norma l. In contrast, these treatments have little effect on NADPH oxidase a ctivation by arachidonic acid or SDS plus DG. PA plus DG induces the p hosphorylation of a number of endogenous proteins. Phosphorylation is largely mediated by PA, not DG. A predominant substrate is p47-phox, a phosphoprotein component of NADPH oxidase. Phosphorylation of p47-pho x precedes activation of NADPH oxidase and is markedly reduced by the protein kinase inhibitors. In contrast, arachidonic acid alone or SDS plus DG is a poor activator of protein phosphorylation in the cell-fre e system. Thus, PA induces activation of one or more protein kinases t hat regulate NADPH oxidase activation in a cell-free system. This cell -free system will be useful for identifying a functionally important P A-activated protein kinase(s) and for dissecting the phosphorylation-d ependent mechanisms responsible for NADPH oxidase activation.