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The leukocyte NADPH oxidase is an enzyme in phagocytes and B lymphocytes that when activated catalyzes the production of O-2 from oxygen and NADPH. During oxidase activation, serine residues in the C-terminal quarter of the oxidase component p47(PHOX) become extensively phosphorylated, the protein acquiring as many as 9 phosphate residues. In a study of 11 p47(PHOX) mutants, each containing an alanine instead of a serine at a single potential phosphorylation site, we found that all but S379A corrected the defect in O-2 production in Epstein-Barr virus (EBV)-transformed p47(PHOX)-deficient B cells (Faust, L. P., El Benna, J., Babior, B. M., and Chanock, S. J. (1995) J. Clin. Invest. 96, 1499-1505). In particular, O-2 production was restored to these cells by the mutants S303A and S304A. Therefore, apart from serine 379, whose state of phosphorylation in the activated oxidase is unclear, no single potential phosphorylation site appeared to be essential for oxidase activation. We now report that the double mutant p47(PHOX) S303A/S304A was almost completely inactive when expressed in EBV-transformed p47(PHOX)-deficient B cells, even though it was expressed in normal amounts in the transfected cells and was able to translocate to the plasma membrane when the cells were stimulated. In contrast, the double mutant p47(PHOX) S303E/S304E was able to support high levels of O-2 production by EBV-transformed p47(PHOX)-deficient B cells. The surprising discovery that the double mutant S303K/S304K was also able to support considerable O-2 production suggests either that the effect of phosphorylation is related to the increase in hydrophilicity around serines 303 and 304 or that activation involves the formation of a metal bridge between the phosphorylated serines and another region of the protein.

The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to superoxide anion (O-2) at the expense of NADPH. This multicomponent enzyme is dormant in resting cells but is activated on exposure to an appropriate stimulus. The phosphorylation-dependent mechanisms regulating the activation of the respiratory burst oxidase are unclear, particularly the phosphorylation status of the cytosolic component p67(phox). In this study, we found that activation of human neutrophils with formyl-methionyl-leucyl-phenylalanine (fMLP), a chemotactic peptide, or phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC), resulted in the phosphorylation of p67(phox). Using an anti-p67(phox) antibody or an anti-p47(phox) antibody, we showed that phosphorylated p67(phox) and p47(phox) form a complex. Phosphoamino acid analysis of the phosphorylated p67(phox) revealed only 32P-labeled serine residues. Two-dimensional tryptic peptide mapping analysis showed that p67(phox) is phosphorylated at the same peptide whether fMLP or PMA is used as a stimulus. In addition, PKC induced the phosphorylation of recombinant GST-p67(phox) in vitro, at the same peptide as that phosphorylated in intact cells. PMA-induced phosphorylation of p67(phox) was strongly inhibited by the PKC inhibitor GF109203X. In contrast, fMLP-induced phosphorylation was minimally affected by this PKC inhibitor. Taken together, these results show that p67(phox) is phosphorylated in human neutrophils by different pathways, one of which involves protein kinase C.

The leukocyte NADPH oxidase catalyzes the 1-electron reduction of oxygen to O2- at the expense of NADPH: 2 O2 + NADPH --> 2 O2- + NADP+ + H+. The oxidase is dormant in resting cells but acquires activity when the cells are stimulated with a suitable agent. Activation in whole cells is accompanied by extensive phosphorylation of p47(PHOX), an oxidase subunit located in the cytosol of resting cells that during oxidase activation migrates to the plasma membrane to complex with cytochrome b558, an oxidase-specific flavohemoprotein. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile as activating agent. We now report a cell-free system in which the oxidase can be activated in two stages using phosphorylated p47(PHOX). The first stage, which effects a change in the membrane, requires ATP and GTP and is blocked by the protein kinase inhibitor GF-109203X, suggesting a protein kinase requirement. The second stage requires phosphorylated p47(PHOX) and GTP, but no ATP, and is unaffected by GF-109203X; assembly of the oxidase may take place during this stage. Activation is accomplished by p47(PHOX) phosphorylated by protein kinase C but not protein kinase A or mitogen-activated protein kinase. We believe that activation by phosphorylated p47(PHOX) is more physiological than activation by amphiphiles, because the mutant p47(PHOX) S379A, which is inactive in whole cells, is also inactive in this system but works in systems activated by amphiphiles.

The leukocyte NADPH oxidase catalyzes the reduction of oxygen to superoxide (O-2) at the expense of NADPH in phagocytes and B lymphocytes. The enzyme is dormant in resting cells but becomes active when the cells are exposed to appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47(PHOX) becomes phosphorylated on several serines and migrates to the plasma membrane. We report here that p47(PHOX)-deficient B lymphoblasts expressing the p47(PHOX) S359A/S370A or p47(PHOX) S359K/S370K double mutation show dramatically reduced levels of enzyme activity and phosphorylation of p47(PHOX) as compared with the same cells expressing wild type p47(PHOX). In addition, these mutant p47(PHOX) proteins fails to translocate to the plasma membrane when the cells are stimulated. In contrast, normal phosphorylation and translocation are seen in mutants containing aspartate or glutamate at positions 359 and 370, but oxidase activity is still greatly reduced. These results imply that a negative charge at position 359 and/or 370 is sufficient to allow the phosphorylation and translocation of p47(PHOX) to take place but that features unique to a phosphorylated hydroxyamino acid are required to support O-2 production. These findings, plus those from an earlier study (Inanami, O., Johnson, J. L., McAdara, J. K., El Benna, J., Faust, L. P., Newburger, P. E., and Babior, B. M. (1998) J. Biol. Chem. 273, 9539-9543), suggest that oxidase activation requires 1) the sequential phosphorylation of at least two serines on p47(PHOX): Ser-359 or Ser-370, followed by Ser-303 or Ser-304; and 2) the translocation of p47(PHOX) to the membrane at some point after the first phosphorylation takes place.

Vitamin E (alpha-tocopherol), one of the most important natural antioxidants, is assumed to be beneficial in the prevention of cardiovascular diseases. alpha-Tocopherol exhibits acyl-peroxyl-radical scavenger properties and exerts cell-mediated actions in the hemovascular compartment, such as inhibition of superoxide anion (O-2) production by leukocytes. The aim of this study was to examine the mechanism underlying the inhibitory effect of alpha-tocopherol on O-2 production by human monocytes. In activated monocytes O-2 is produced by the NADPH-oxidase enzyme complex. The oxidase activation elicited by phorbol myristate acetate (PMA) requires membrane translocation of several cytosolic factors. We found that in human PMA-stimulated adherent monocytes, alpha-tocopherol (but not beta-tocopherol) inhibited O-2 production in intact cells but had no effect on a membrane preparation containing activated NADPH-oxidase, suggesting that alpha-tocopherol impairs the assembly process of the enzyme complex. We showed that translocation and phosphorylation of the cytosolic factor p47(phox) were reduced in monocytes preincubated with alpha-tocopherol. We verified that the tryptic phosphopeptide map of monocyte p47(phox) was similar to that of neutrophil p47(phox), indicating that several serine residues were phosphorylated. Peptides whose phosphorylation is dependent on protein kinase C (PKC) were phosphorylated to a lesser degree when p47(phox) was immunoprecipitated from alpha-tocopherol-treated monocytes. In vitro, the activity of PKC from monocytes was inhibited by alpha-tocopherol in a specific manner compared with that of beta-tocopherol or Trolox(R). Membrane translocation of PKC was not affected. These results show that alpha-tocopherol inhibits O-2 production by human adherent monocytes by impairing the assembly of the NADPH-oxidase and suggest that the inhibition of phosphorylation and translocation of the cytosolic factor p47(phox) results from a decrease in PKC activity.