Phosphorylation of p22 Is Mediated by Phospholipase D-dependent and -independent Mechanisms

Abstract

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.

Rho Is Involved in Superoxide Formation during Phagocytosis of Opsonized Zymosans

“…In addition, PMA stimulates translocation of p47 PHOX , p67 PHOX , and Rac1 to membranes (19). fMLP, on the other hand, was reported to activate phospholipase D (PLD) through receptor to form phosphatidic acid (PA), which in turn stimulates PA-activated protein kinase, resulting in activation of p22 PHOX by phosphorylation (54). PLD induced superoxide formation in J774 cells, and Tat-C3 did not inhibit it (data not shown), suggesting that Rho is not involved in the signaling pathway via PLD to activate NADPH oxidase.…”

Phagocytosis induces superoxide formation and apoptosis in macrophages

“…It has been shown that p22 PHOX can be phosphorylated through phospholipase D (PLD), suggesting that PLD activity is required for superoxide formation (Regier et al, 2000). Involvement of PLD was further evidenced by that the activation of PLD is tightly coupled to the phagocytosis of opsonized zymosan by human macrophages (Kusner et al, 1996) and RhoA is an activator of PLD activity (Malcolm et al, 1994).…”

Downstream components of RhoA required for signal pathway of superoxide formation during phagocytosis of serum opsonized zymosans in macrophages

“…We found that the addition of exogenous PLD to the macrophages induced the enhancement of the superoxide formation (results not shown), suggesting that PLD activity is required for the superoxide formation. It has been shown that p22 PHOX can be phosphorylated through PLD, suggesting that PLD activity is required for the superoxide formation (Regier et al, 2000). Moreover, the activation of PLD is tightly coupled to the phagocytosis of opsonized zymosan by human macrophages (Fallman et al, 1993;Kusner et al, 1996).…”