Phosphatidic acid (PA), generated by phospholipase D activation, has been linked to the activation of the neutrophil respiratory burst enzyme, NADPH oxidase; however, the intracellular enzyme targets for PA remain unclear. We have recently shown (McPhail, L. C., Qualliotine-Mann, D., and Waite, K. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7931-7935) that a PA-activated protein kinase is involved in the activation of NADPH oxidase in a cell-free system. This protein kinase phosphorylates numerous endogenous proteins, including p47-phox, a component of the NADPH oxidase complex. Phospholipids other than PA were less effective at inducing endogenous protein phosphorylation. Several of these endogenous substrates were also phosphorylated during stimulation of intact cells by opsonized zymosan, an agonist that induces phospholipase D activation. We sought to identify the PA-activated protein kinase that phosphorylates p47-phox. The PA-dependent protein kinase was shown to be cytosolic. cis-Unsaturated fatty acids were poor inducers of protein kinase activity, suggesting that the PA-activated protein kinase is not a fatty acid-regulated protein kinase (e.g. protein kinase N). Chromatographic techniques separated the PA-activated protein kinase from a number of other protein kinases known to be activated by PA or to phosphorylate p47-phox. These included isoforms of protein kinase C, p21 (Cdc42/Rac)-activated protein kinase, and mitogen-activated protein kinase. Gel filtration chromatography indicated that the protein kinase has an apparent molecular size of 125 kDa. Screening of cytosolic fractions from several cell types and rat brain suggested the enzyme has widespread cell and tissue distribution. The partially purified protein kinase was sensitive to the same protein kinase inhibitors that diminished NADPH oxidase activation and was independent of guanosine 5 -3-O-(thio)triphosphate and Ca 2؉ . Phosphoamino acid analysis showed that serine and tyrosine residues were phosphorylated on p47-phox by this kinase(s). These data indicate that one or more potentially novel protein kinases are targets for PA in neutrophils and other cell types. Furthermore, a PA-activated protein kinase is likely to be an important regulator of the neutrophil respiratory burst by phosphorylation of the NADPH oxidase component p47-phox.Phospholipase D (PLD) 1 is activated in a variety of cells by hormones and growth factors (1-3). This activation results in the generation of phosphatidic acid (PA), which can be further metabolized by PA phosphohydrolase to diacylglycerol (DG). The generation of PA by PLD in neutrophils has been linked, by us and others, to the activation of the respiratory burst enzyme, NADPH oxidase (2-7). The enzymes involved in the activation of the oxidase, which are downstream of the generation of PA, have not been identified. We have developed a cell-free activation system for NADPH oxidase, which utilizes PA and is phosphorylation-dependent (7, 8). The system requires the presence of both cellular membranes and c...

The phosphorylation-dependent mechanisms regulating activation of the human neutrophil respiratory-burst enzyme, NADPH oxidase, have not been elucidated. We have shown that phosphatidic acid (PA) and diacylglycerol (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 system activators. NADPH oxidase activation by PA plus DG is reduced -70% by several protein kinase inhibitors [1-(5-isoquinolinesulfonyl)piperazine, staurosporine, GF-109203X]. Similarly, depletion of ATP by dialysis reduces PA plus DG-mediated NADPH oxidase activation by -70%. Addition of ATP, but not a nonhydrolyzable ATP analog, to the dialyzed system restores activation levels to normal. In contrast, these treatments have little effect on NADPH oxidase activation by arachidonic acid or SDS plus DG. PA plus DG induces the phosphorylation 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-phox 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-free system. Thus, PA induces activation of one or more protein kinases that regulate NADPH oxidase activation in a cell-free system. This cell-free system will be useful for identifying a functionally important PA-activated protein kinase(s) and for dissecting the phosphorylation-dependent mechanisms responsible for NADPH oxidase activation.Phagocytic cells undergo a respiratory burst in response to infection or inflammation, and the oxygen products produced are toxic to microbes and surrounding tissue (1-4). The enzyme responsible is a multicomponent NADPH oxidase, consisting of a membrane-bound heterodimeric flavocytochrome, cytochrome b558, and two cytosolic proteins termed p47-phox (phagocyte NADPH oxidase component migrating at 47 kDa on SDS/PAGE) and p67-phox (1-4). At least three other proteins are also involved: a cytosolic protein termed p40-phox and two small GTP-binding proteins-membraneassociated Rapla and cytosolic Racl or Rac2 (5-9). Activation involves translocation of the cytosolic proteins to assemble with cytochrome b558 in the membrane (2, 4, 10), mediated in part by Src homology 3 regions in p47-phox and p67-phox (10).Physiological stimuli induce activation of NADPH oxidase by binding to receptors on the cell surface, triggering intracellular signaling events (11,12). Key intermediates are phospholipases A2, C, and D and their products: arachidonic acid (AA), inositol trisphosphate, diacylglycerol (DG), and phosphatidic acid (PA) (11,12). These second messengers lead to protein kinase activation (11)(12)(13)(14)(15)(16)(17)(18), and studies suggest that protein phosphorylation regulates NADPH oxidase activation (4, 11). A prominent substrate...

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