A novel protein kinase target for the lipid second messenger phosphatidic acid.

Activation of phospholipase D occurs in response to a wide variety of hormones, growth factors, and other extracellular signals. The initial product of phospholipase D, phosphatidic acid (PA), is thought to serve a signaling function, but the intracellular targets for this lipid second messenger are not clearly identified. The production of PA in human neutrophils is closely correlated with the activation of NADPH oxidase, the enzyme responsible for the respiratory burst. We have developed a cell-free system, in which the activation of NADPH oxidase is induced by the addition of PA. Characterization of this system revealed that a multi-functional cytosolic protein kinase was a target for PA, and that two NADPH oxidase components were substrates for the enzyme. Partial purification of the PA-activated protein kinase separated the enzyme from known protein kinase targets of PA. The partially purified enzyme was selectively activated by PA, compared to other phospholipids, and phosphorylated the oxidase component p47-phox on both serine and tyrosine residues. PA-activated protein kinase activity was present in a variety of hematopoietic cells and cell lines and in rat brain, suggesting it has widespread distribution. We conclude that this protein kinase may be a novel target for the second messenger function of PA.

Phosphatidic acid-mediated phosphorylation of the NADPH oxidase component p47-phox. Evidence that phosphatidic acid may activate a novel protein kinase.

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 Ca2+. 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.

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 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 approximately 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 approximately 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.

Phosphatidic acid and diacylglycerol synergize in a cell-free system for activation of NADPH oxidase from human neutrophils.

NADPH oxidase, the respiratory burst enzyme of human neutrophils, is a multi-component complex that is assembled and activated during stimulation of the cells by inflammatory or phagocytic stimuli. The signal mechanisms leading to activation of the enzyme are unclear, but it is likely that phospholipases are involved. Recent work has shown that phosphatidic acid, the initial product of phospholipase D activation, is a weak activator of NADPH oxidase in a cell-free system. We now show that diacylglycerol enhances the cell-free activation of NADPH oxidase activation by phosphatidic acid. 1,2-Didecanoyl phosphatidic acid (10:0-PA) and 1,2-dioctanoylglycerol (8:0-DG) each increased levels of NADPH oxidase activity in mixtures of membrane and cytosolic fractions about 2-fold. The combination of both lipids increased NADPH oxidase activity approximately 12-fold, indicative of a synergistic response. Fatty acid and neutral lipid metabolites of 10:0-PA or 8:0-DG were ineffective, suggesting activation is directly mediated by phosphatidic acid and diacylglycerol. Activation was time- and concentration-dependent with maximum activation at 30-60 min and a sharp peak of maximal activity at 10 microM 10:0-PA and 30 microM 8:0-DG. In lipid specificity studies, activity of PA or DG decreased with increasing acyl chain length but was restored by introducing unsaturation in the acyl chain. Natural forms of PA stimulated levels of activity comparable to that seen with 10:0-PA. Synthetic and natural phosphatidylserines, but not other phospholipids, could replace phosphatidic acid in the synergistic response. These studies provide direct evidence for a synergistic interaction between phosphatidic acid and diacylglycerol in mediating a cellular function: the assembly and activation of NADPH oxidase. Our results support the concept that the generation of second messenger lipids by phospholipase D is a key step in activation of the respiratory burst enzyme.

Phospholipases and activation of the NADPH oxidase.

The signal transductional mechanisms regulating the activation of NADPH oxidase, the respiratory burst enzyme in phagocytic cells, are not completely understood. Receptors for most physiologic stimuli trigger the activation of various phospholipases, including phospholipases A2, C, and D. The lipid mediators formed (arachidonic acid, 1,2-diacylglycerol, and phosphatidic acid) have been implicated as second messengers in the induction of the respiratory burst. In intact cells, we have correlated phospholipase D activation and the production of phosphatidic acid with the activation of NADPH oxidase, using the drug propranolol. Phosphatidic acid activated NADPH oxidase in a cell-free system, but the level of activation was low. 1,2-Diacylglycerol markedly enhanced NADPH oxidase activation by phosphatidic acid. The synergistic effect required the diacyl species, since mono- or tri-acylglycerols were ineffective. Phosphatidic acid could be replaced by either lysophosphatidic acid or phosphatidylserine, but not by phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol, suggesting specificity for an anionic phospholipid. Since other cell-free activators of NADPH oxidase (arachidonic acid, sodium dodecyl sulfate) are also anionic amphiphiles, phosphatidic acid may directly interact with an enzyme component(s). The targets for phosphatidic acid and diacylglycerol in the cell-free system are currently under investigation. These results emphasize the critical importance of phospholipases, particularly phospholipase D, in the regulation of the respiratory burst.

Effects of promethazine-hydrochloride on human polymorphonuclear leukocytes.

Promethazine hydrochloride at a concentration of 0.033 mg/ml has pronounced effects on leukocyte metabolism and function. The drug inhibits the phagocytosis-induced increases in O(2) consumption and hexose monophosphate shunt activity. Associated with these effects is an inhibition of the iodination of zymosan particles and an inhibition of bacterial killing by the cell. At least two mechanisms appear to be involved. Many of the effects can be explained by an inhibition of phagocytosis, but promethazine also inhibits the decarboxylation of amino acids and iodide fixation in a cell-free system, indicating a specific effect on metabolism. These results may partially account for the action of the drug in ameliorating the effects of erythroblastosis.