Activation of the Leukocyte NADPH Oxidase by Protein Kinase C in a Partially Recombinant Cell-free System*

Lucia Rossetti LopesDagger §, Carolyn R. HoyalDagger , Ulla G. Knausparallel , and Bernard M. BabiorDagger

From the Dagger  Department of Molecular and Experimental Medicine and the parallel  Department of Immunology, The Scripps Research Institute, La Jolla, California 92037

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The leukocyte NADPH oxidase is an enzyme present in phagocytes and B lymphocytes that when activated catalyzes the production of Obardot 2 from oxygen at the expense of NADPH. A correlation between the activation of the oxidase and the phosphorylation of p47PHOX, a cytosolic oxidase component, is well recognized in whole cells, and direct evidence for a relationship between the phosphorylation of this oxidase component and the activation of the oxidase has been obtained in a number of cell-free systems containing neutrophil membrane and cytosol. Using superoxide dismutase-inhibitable cytochrome c reduction to quantify Obardot 2 production, we now show that p47PHOX phosphorylated by protein kinase C activates the NADPH oxidase not only in a cell-free system containing neutrophil membrane and cytosol, but also in a system in which the cytosol is replaced by the recombinant proteins p67PHOX, Rac2, and phosphorylated p47PHOX, suggesting that neutrophil plasma membrane plus those three cytosolic proteins are both necessary and sufficient for oxidase activation. In both the cytosol-containing and recombinant cell-free systems, however, activation by SDS yielded greater rates of Obardot 2 production than activation by protein kinase C-phosphorylated p47PHOX, indicating that a system that employs protein kinase C-phosphorylated p47PHOX as the sole activating agent, although more physiological than the SDS-activated system, is nevertheless incomplete.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The NADPH oxidase is a membrane-associated enzyme that catalyzes the one electron reduction of oxygen to Obardot 2 at the expense of NADPH (1). The oxidase comprises multiple protein components present in both the cytosol and the plasma membrane. The enzyme is dormant in resting cells but becomes activated when the cells are exposed to appropriate stimuli. Upon activation, a cytosolic complex consisting of the oxidase components p47PHOX, p67PHOX, and p40PHOX associates with the membrane-bound cytochrome b558 to assemble the active oxidase (2-7).

The phosphorylation of p47PHOX is a well recognized concomitant of oxidase activation in whole cells, but the mechanism of activation of the oxidase is not fully understood (8-14). One key to understanding the activation of the oxidase emerged with the discovery of the cell-free activation system (15-17) in which it was shown that NADPH oxidase activity could be induced in a mixture of membrane and cytosol by the addition of amphiphiles like arachidonic acid (15, 17) and SDS (16, 18). Recently, increasing attention has been paid to cell-free systems in which the oxidase is activated without using anionic amphiphiles (19-22). Our studies showed that the oxidase can be activated by p47PHOX phosphorylated by protein kinase C in a cell-free system containing neutrophil membrane and cytosol (21). In addition, these studies also revealed a that the phosphorylation of p47PHOX was not the only ATP-dependent step in the activation of the oxidase by protein kinase C. A preceding phosphorylation event occurs in the membranes rendering them capable of supporting oxidase activation. The target of this event has yet to be determined. Although these experiments showed a direct relationship between the phosphorylation of p47PHOX and the activation of the oxidase, the use of whole cytosol made it difficult to recognize whether cytosolic factors other than those necessary for activation by SDS are required for oxidase activation by a kinase. In this paper we report studies of a recombinant cell-free system containing only membrane and cytosolic oxidase components (p47PHOX, p67PHOX, and Rac2). Our findings suggest that the cytosolic components phosphorylated p47PHOX, p67PHOX, and Rac2 are sufficient for partial activation of the oxidase.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- Chemicals and enzymes were obtained from the following sources: dextran and Ficoll-Hypaque from Amersham Pharmacia Biotech; phosphatidylserine, diacylglycerol, isopropyl-beta -D-thiogalactopyranoside, NADPH, ATP, guanosine 5-O-(3-thiotriphosphate) (GTPgamma S),1 guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S), glutathione agarose and cytochrome c from Sigma; rat brain protein kinase C, calyculin A, and GF-109203X from Calbiochem; and the Bradford protein assay reagent from Bio-Rad.

Preparation of Neutrophil Fractions-- Neutrophil cytosol and membrane were prepared as described by Borregaard et al. (23). Neutrophils were prepared from normal subjects by dextran sedimentation and Ficoll-Hypaque fractionation of freshly drawn citrate-anticoagulated blood. The neutrophils were suspended at 108 cells/ml in a modified relaxation buffer (0.1 M KCl, 3 mM NaCl, 3.5 mM MgCl2, 10 mM PIPES buffer, pH 7.3). Plasma membrane and cytosol were prepared by nitrogen cavitation followed by centrifugation through a Percoll gradient. Both cytosol and membrane were divided into aliquots and stored at -70 °C until use.

Production and Purification of Recombinant p67PHOX from Baculovirus-infected Sf9 Cells-- Purified recombinant p67PHOX was produced by means of the baculovirus system described by Leto et al. (1991), using a p67PHOX-expressing recombinant virus generously provided by T. L. Leto. Large scale production of pure recombinant p67PHOX was achieved by infecting monolayer cultures of Sf9 cells in 150 cm2 flasks at a density of 1-2 × 106 cells/ml (24). Cells were harvested 72 h postinfection, washed twice in phosphate-buffered saline by centrifugation at 400 × g for 10 min, and then resuspended to 5 × 107/ml in lysis buffer (50 mM KCl, 3 mM NaCl, 2 mM MgCl2, 0.1 mM dithiothreitol, 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, 5.4 mM PIPES, pH 7.5). All subsequent work was conducted at 4 °C. Cells were disrupted by sonication (4 × 10 s) and centrifuged at 400 × g for 10 min. The supernatant fraction containing p67PHOX was brought to 45% saturation with solid ammonium sulfate. The resulting precipitate was isolated by centrifugation (1200 × g at 4 °C for 30 min), then dissolved in 10 ml of buffer A (20 mM Tris, pH 7.5, 0.1 mM dithiothreitol, 1 mM EDTA, 2 mM EGTA, 0.15 mM phenylmethylsulfonyl fluoride) and dialyzed overnight against the same buffer. The dialyzed solution was applied to a Mono Q-Sepharose column (Amersham Pharmacia Biotech) previously equilibrated with buffer A and washed with 5 volumes of the same buffer. Proteins were eluted from the column by fast protein liquid chromatography with a 0.1-0.3 M NaCl gradient in the same buffer at a flow rate of 0.8 ml/min. The fractions containing purified p67PHOX were pooled and stored at -70 °C.

Preparation of Recombinant GST-p47PHOX and Rac2 Fusion Proteins-- Recombinant fusion proteins composed of glutathione S-transferase (GST) linked downstream to p47PHOX or Rac2 were isolated from Escherichia coli transformed with pGEX-1lambda T plasmids containing cDNA inserts encoding the downstream proteins as described by Park et al. (3). The fusion proteins were purified by affinity chromatography on glutathione-agarose beads. Initially the culture was grown overnight at 37 °C in 100 ml of "Terrific Broth" containing 0.1% ampicillin, then diluted into 1 liter of fresh Terrific Broth/ampicillin. The diluted cultures were grown for an additional hour at 37 °C (for GST-p47PHOX expression) or an additional 2.5 h at 37 °C (for GST-Rac2 expression). Isopropyl-beta -D-thiogalactopyranoside (0.1 mM) was then added, and the cultures were grown with vigorous agitation for an additional 3 h at 37 °C for GST-p47PHOX expression or 30 °C for GST-Rac2 expression. At the conclusion of the incubations, the cells were recovered by centrifugation at 2000 × g for 10 min at 4 °C. The GST-p47PHOX pellet was suspended in 10 ml of ice-cold phosphate-buffered saline containing a 1 × mixture of protease inhibitors (Roche Molecular Biochemicals), while the GST-Rac2 pellet was resuspended in a lysis buffer (50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). The cells were then disrupted by sonication. The sonicates were clarified by centrifugation at 14,000 × g for 15 min at 4 °C. The fusion proteins were isolated from the supernatant by purification over glutathione-agarose as described by Smith and Johnson (25). Before use, excess glutathione was removed from the solution of purified recombinant protein by dialysis against relaxation buffer. The concentrations of all proteins (95-99% pure) were determined with a Bio-Rad assay kit using bovine serum albumin as a standard.

Phosphorylation of GST-p47PHOX-- Phosphorylation of recombinant GST-p47PHOX was carried out using 200 µg of fusion protein in final volume of 200 µl. The reaction mixture contained 1 mM ATP, 10 mM magnesium acetate, 1.0 mM CaCl2, 10 µg phosphatidylserine, 1 µg diolein, and 0.5 unit of protein kinase C in 200 µl of relaxation buffer (0.1 M KCl, 3 mM NaCl, 3.5 mM MgCl2, 10 mM PIPES buffer, pH 7.3). The lipids were added as mixed liposomes prepared by dissolving 2.5 mg/ml phosphatidylserine and 1 mg/ml diacylglycerol in chloroform, removing the chloroform under a stream of nitrogen, and then sonicating the dried lipids for 2 min on ice in 0.8 ml of 20 mM Tris buffer, pH 7.4. Incubations were carried out for 30 min at 37 °C. The phosphorylated protein, designated p47PHOXP6,2 was separated from the reaction mixture as described elsewhere (21).

Cell-free Activation of the NADPH Oxidase with p47PHOXP6-- Activation of the NADPH oxidase in the cell-free system was directly measured by following the superoxide dismutase-inhibitable reduction of cytochrome c at 550 nm in a dual beam recording spectrophotometer. The complete reaction mixture contained 5 × 106 cell equivalents of membrane (12 ± 1.4 pmol of cytochrome b558) incubated for 10 min at 30 °C with 50 µM GTPgamma S or GDPbeta S, 1 mM ATP, 250 nM calyculin A, and 105 pmol of GST-Rac2, 75 pmol of p67PHOX and 70 pmol of GST p47PHOX, phosphorylated or unphosphosphorylated, in a final volume of 200 µl. GST-Rac2 was reconstituted with 130 µM GTPgamma S by preincubation of 105 pmol of the protein with 2.6 mM EDTA for 10 min at room temperature followed by the addition of 4 mM MgCl2 (26). Reactions were started by adding the detection mixture (0.1 mM cytochrome c and 0.16 mM NADPH, final concentrations). Reduction was followed in a Uvikon 941 dual beam recording spectrophotometer (Kontron Instruments, Milan, Italy), reading against a reference containing the same components plus 150 units of superoxide dismutase. Unless otherwise indicated, the rate of Obardot 2 production is expressed as moles of Obardot 2/moles of cytochrome b558/minute. Where indicated, cytosol (107 cell equivalents) was added to the system instead of the recombinant proteins GST-p47PHOX, p67PHOX, and GST-Rac2. Experiments were also performed in which the recombinant proteins (p67PHOX, GST-p47PHOX, and GST-Rac2) were individually omitted to determine which of these proteins was required for the production of Obardot 2 by the oxidase.

Cell-free Activation of the NADPH Oxidase with SDS-- In these experiments, 5 × 106 cell equivalents of membrane were incubated for 10 min at 30 °C with 105 pmol of GST-Rac2, 75 pmol of p67PHOX and 70 pmol of GST p47PHOX plus 50 µM GTPgamma S as described previously, with the exception that unphosphorylated p47PHOX was used in place of p47PHOXP6. After the preincubation, SDS (90 µM final concentration) was added to the reaction mixture and incubated for 1 min. In some experiments, cytosol (107 cell eq) was used in place of the recombinant proteins as described above.

Cytochrome b558 Determination-- Neutrophil membranes (1 × 107 cell eq) were resuspended in 400 µl of Triton buffer (0.1 M potassium phosphate buffer, pH 7.25, containing 2% Triton (v/v). Cytochrome b558 content was measured as the dithionite-reduced minus oxidized absorption assuming Delta E559-540 = 21.6 mM-1 cm-1 (27). In some spectra the height of the peak was estimated by interpolation to correct for base-line drift.

Electrophoresis and Immunoblotting-- Protein samples were subjected to SDS-PAGE using the Laemmli buffer system (28). The gels were stained with Coomassie Blue. Alternatively, the separated proteins were electrophoretically transferred onto a nitrocellulose sheet (29) and probed with partially purified rabbit polyclonal antibodies raised against full-length Rac2 or the C-terminal decapeptides from p47PHOX and p67PHOX. These antibodies were used at 1:1000, 1:2000, and 1:5000 dilution for GST-Rac2, GST-p47PHOX, and p67PHOX, respectively. The proteins were visualized with a 1:2000 dilution of horseradish peroxidase-labeled goat anti-rabbit Ig antibody (Caltag) and the ECL enzymatic chemoluminescence detection system (Renaissance, DuPont).

    RESULTS
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Absence of Cytosolic Factors from the Neutrophil Membrane-- In the experiments described below, neutrophil membranes were mixed with recombinant cytosolic oxidase proteins in various combinations. SDS-PAGE gels of the recombinant proteins used for these experiments are shown in Fig. 1. Since the experiments involved the addition of cytosolic components to the assay mixtures, it was important to determine whether any of these components was present in the neutrophil membrane that was to be used in the experiments. For this purpose, membrane and cytosol in equal amounts (expressed as cell equivalents) were subjected to SDS-PAGE and immunoblotting, developing with antibodies against the cytosolic components that were to be added in recombinant form. The components were readily visible in the cytosol, but could not be detected in the membrane.


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Fig. 1.   Recombinant proteins used in these experiments. SDS-PAGE of the purified proteins was performed as described under "Experimental Procedures," using a 10% running gel. Coomassie Blue staining was used to show GST-p47PHOX (lane 1), p67PHOX (lane 2), and GST-Rac2 (lane 3).

Activation of the Cell-free Recombinant Leukocyte NADPH Oxidase by p47PHOXP6-- In the cell-free system, the leukocyte NADPH oxidase has customarily been activated using certain anionic amphiphiles including SDS. In an earlier study in which we supplemented the cell-free system with p47PHOX (added as the GST fusion protein), we found that the enzyme could be activated without detergent, provided the p47PHOX was first phosphorylated by protein kinase C (21). We believe that the activation of the cell-free oxidase by protein kinase C may represent a more physiological process than activation by anionic amphiphiles, because in intact cells, as in the kinase-activated cell-free system, oxidase activation is associated with the phosphorylation of p47PHOX.

It has been shown by others that in a cell-free system in which neutrophil cytosol has been replaced by the two recombinant cytosolic oxidase subunits p47PHOX and p67PHOX together with the small GTPase Rac2, Obardot 2 is produced upon the addition of SDS (30). In order to see if the same system could be activated by a kinase, we conducted experiments in which Obardot 2 production was measured in a recombinant system that contained phosphorylated p47PHOX (i.e. p47PHOXP6) instead of the unphosphorylated protein. The results (Fig. 2) showed that Obardot 2 was produced in the complete system, but that the omission of all or any one of the three recombinant cytosolic proteins or the omission of membrane (not shown) essentially eliminated oxidase activity. The use in the recombinant system of Rac2 preloaded with GDPbeta S led to a marked reduction in Obardot 2 formation as compared with a system that employed Rac2 preloaded with GTPgamma S, suggesting that Rac2 had to be in its active form for the oxidase to be activated (Fig. 3). These findings indicate that all four components (membrane, p67PHOX, p47PHOXP6, and Rac2) were required for activation of the NADPH oxidase, indicating that they are both necessary and sufficient for kinase-dependent cell-free oxidase activation.


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Fig. 2.   Obardot 2 production by GST-p47PHOXP6-activated leukocyte NADPH oxidase as a function of time. Incubations were carried out as described in the text, using the cytochrome c assay. Components were omitted from the assays as indicated. The results shown are representative of three or more separate experiments. The means ± S.E. for the final points (22 min) are 388 ± 46 (complete), -67 ± 4.2 (no GST-p47PHOXP6), -4 ± 8 (no p67PHOX), -50 ± 4.1 (no GST-Rac2), and -12.5 ± 4.2 (no recombinant proteins) mol of Obardot 2/mol of cytochrome b558/min. Differences between Obardot 2 production in the complete assay mixture and Obardot 2 production in each of the omission experiments were significant at the level of p < 0.005.


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Fig. 3.   Requirement for guanine nucleotides in the activation of the NADPH oxidase by p47PHOXP6 in the recombinant cell-free system. Experiments were conducted as described in the text; Rac2 was loaded with GTPgamma S or GDPbeta S as indicated. The results shown represent the mean ± S.E. of four separate experiments.

Experiments with a cell-free system containing membrane and cytosol indicated that the phosphorylation of p47PHOX was an essential prerequisite for Obardot 2 production by the system (31). To see if the same situation prevailed in the recombinant system, the rate of Obardot 2 production using p47PHOXP6 was compared with the rate of Obardot 2 production using unphosphorylated p47PHOX. The results (Fig. 4) show that Obardot 2 production in this system required the phosphorylation of p47PHOX. It is possible that further phosphorylation of p47PHOX by a membrane-associated kinase might also be necessary for the Obardot 2-forming activity in this system. The addition of the protein kinase inhibitor GF-109203X to the reaction mixture, however, had no effect on Obardot 2 production, indicating that phosphorylation at least by GF-109203X-inhibitable membrane-associated kinases such as activated protein kinase C was not a factor in these experiments.


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Fig. 4.   NADPH oxidase activation requires phosphorylated p47PHOX in the recombinant cell-free system. The cytochrome c assay was conducted as described in the text, except that GST-p47PHOXP6 or unphosphorylated GST-p47PHOX was added to the assay mixtures as indicated. The protein kinase inhibitor GF-109203X (5 µM) was also added to some of the incubations. Results are expressed as mean ± S.E. of three or more experiments.

The effect of protein concentration on Obardot 2 production in the recombinant system was next examined. In these experiments activity was monitored at varying concentrations of the three recombinant proteins in the presence of a constant amount of membrane (Fig. 5A) and at varying concentrations of membrane in the presence of a constant amount of recombinant proteins (Fig. 5B). The maximal rate of Obardot 2 generation by the p47PHOXP6-activated system was 4.6 ± 0.5 nmol Obardot 2/min/107 cell eq of membrane (mean ± S.E., n = 6), equivalent to 190 mol of Obardot 2/mol of cytochrome b558/min, a value achieved using 5 × 106 cell equivalents of membrane (12 pmol of cytochrome b558), 105 pmol of GST-Rac2, 75 pmol of p67PHOX, and 70 pmol of GST p47PHOXP6, the rate falling sharply at concentrations on either side of the optimum. In order to investigate if this effect also occurred in the cytosolic cell-free system, production of Obardot 2 using the same concentration of Rac2 and p67PHOX at different concentrations of p47PHOXP6 was determined. At the concentration of p47PHOXP6 used in this study, the activity of the recombinant and cytosolic cell-free systems were the same (Fig. 6). Curiously, the addition of more than 70 pmol of p47PHOXP6 reduced Obardot 2 production in the recombinant system but not in the cytosolic cell-free system. When the oxidase is activated, p47PHOX, p67PHOX, and Rac2 translocate to the membrane in equimolar quantities (32). Therefore we employed approximately equimolar concentrations of the three recombinant cytosolic components (actual stoichiometry 1.5/1/1 for Rac2/p47PHOX/p67PHOX). Altogether, these results indicate that an excess of either membrane or cytosolic components inhibits Obardot 2 production in the recombinant cell-free system. Why this same relationship doesn't prevail in the cytosol-containing system is a mystery.


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Fig. 5.   Oxidase activity as a function of the concentrations of the recombinant proteins and membranes. The incubations were carried out as described under "Experimental Procedures" using the cytochrome c assay. The reaction mixtures contained 5 × 106 cell eq of membrane (12 ± 1.4 pmol of cytochrome b558), plus increasing concentrations of GST-p47PHOXP6, p67PHOX, and GST-Rac2 (A) or 70 pmol of GST-p47PHOXP6, 105 pmol of GST-Rac2, and 75 pmol of p67PHOX, plus membrane at the concentration shown (B). The values shown represent the initial rates of Obardot 2 production (means ± S.E.) for three or more separate experiments.


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Fig. 6.   Oxidase activity as a function of GST-p47PHOXP6 concentration in the recombinant and cytosolic cell-free systems. The incubations were carried out as described under "Experimental Procedures," assaying Obardot 2 production by cytochrome c reduction. The recombinant reaction mixtures contained 75 pmol of p67PHOX, 105 pmol of GST-Rac2, and GST-p47PHOXP6 at the concentrations shown. Results are expressed as mean ± S.E. of three separate experiments.

Activation of the NADPH Oxidase by SDS Versus Activation by p47PHOXP6-- p47PHOXP6 produced similar rates of production of Obardot 2 in the recombinant and cytosolic cell-free systems (Fig. 7A). In contrast when SDS was used as the stimulus, the rate of Obardot 2 production in the recombinant cell-free system was approximately 50% of the rate seen with cytosol (Fig. 7B), and both rates were considerably greater than the rates obtained in the p47PHOXP6-activated systems. These results strongly suggest that while p47PHOXP6 is sufficient to activate the oxidase at a low level, something from the cytosol that is missing in the recombinant cell-free system is required for maximal activation of the oxidase.


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Fig. 7.   Activation of the NADPH oxidase in the recombinant and cytosolic cell-free system by GST-p47PHOXP6 (A) and SDS (B). The incubations were carried out as described under "Experimental Procedures" using the cytochrome c assay. Results are expressed as mean ± S.E. of three or more experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has been known for many years that p47PHOX becomes heavily phosphorylated on serine residues when the oxidase is activated (8-14, 33). Here we present evidence that p47PHOX phosphorylated by protein kinase C is capable of activating the leukocyte NADPH oxidase in a recombinant cell-free system consisting of neutrophil membrane, p67PHOX and Rac2, therefore identifying the minimum cytosolic components necessary for kinase-dependent activation of the oxidase. These findings strongly suggest that the phosphorylation of p47PHOX that occurs in whole cells during the activation of the leukocyte oxidase is functionally significant and that protein kinase C is a kinase capable of activating p47PHOX by phosphorylation. In addition, our results with GTPgamma S and GDPbeta S confirm that the activation of the enzyme by protein kinase C also requires the activation of Rac2, as shown previously by others (34, 35).

The mechanism of activation of the NADPH oxidase by anionic amphiphiles is still not clear, but our results confirm previous studies demonstrating that the activation of the NADPH oxidase by SDS is not kinase dependent (20). Furthermore, the finding that oxidase activation is associated with the phosphorylation of p47PHOX both in intact cells and in the kinase-dependent cell-free system suggests that as compared with amphiphiles, the activation of the oxidase by protein kinase C may represent a more physiological process. The fact that p47PHOXS379A is nonfunctional both in whole cells and in the kinase-dependent cell-free system, yet is capable of participating in Obardot 2 production in the amphiphile-dependent cell-free system (21, 33), further supports the physiological role played by protein kinase C-dependent activation of the oxidase.

The foregoing experiments also showed that the addition of p47PHOXP6 to a cell-free oxidase activating system is not enough to activate the oxidase to its full extent. A number of cytosolic components can be postulated as candidate factor(s) that allow the oxidase to become fully activated. These include lipids (e.g. arachidonic acid (36)), proteins (e.g. p40PHOX (37)), possibly other kinases, or perhaps a hitherto undiscovered oxidase component. Nevertheless, our findings show that neutrophil membrane, p47PHOX, p67PHOX, and Rac2 are sufficient for protein kinase C-mediated activation of the oxidase, albeit at a relatively low level. The participation of other components in the kinase-dependent activation of NADPH oxidase is the subject of an ongoing investigation.

    FOOTNOTES

* This work was supported in part by United States Public Health Service Grants AI-24227, AI-28479, and RR-00833.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Postdoctoral fellow of the Fundação de Amparo à Pesquisa do Estado de São Paulo (Brazil).

Postdoctoral fellow of the Arthritis Foundation.

2 Protein kinase C-phosphorylated P47PHOX is designated p47PHOXP6, because it contains 6 mol of phosphate/mol of p47PHOX.

    ABBREVIATIONS

The abbreviations used are: GTPgamma S, guanosine 5-O-(3-thiotriphosphate); PIPES, 1,4-piperazinediethanesulfonic acid; GST, glutathione S-transferase; GDPbeta S, guanosine 5'-O-(2-thiodiphosphate); PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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