Phosphorylation of the Respiratory Burst Oxidase Subunit p67phox during Human Neutrophil Activation
REGULATION BY PROTEIN KINASE C-DEPENDENT AND INDEPENDENT PATHWAYS*

(Received for publication, January 16, 1997, and in revised form, March 28, 1997)

Jamel El Benna Dagger §, Pham My-Chan Dang Dagger , Murielle Gaudry Dagger , Michèle Fay Dagger , Françoise Morel , Jacques Hakim Dagger and Marie-Anne Gougerot-Pocidalo Dagger

From the Dagger  INSERM U294, CHU Xavier Bichat, Service d'Hématologie et d'Immunologie Biologiques, 46 rue Henri Huchard, 75018 Paris and the  Laboratoire d'Enzymologie, Centre Hospitalier Universitaire, 38043 Grenoble, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to superoxide anion (Obardot 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 p67phox. 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 p67phox. Using an anti-p67phox antibody or an anti-p47phox antibody, we showed that phosphorylated p67phox and p47phox form a complex. Phosphoamino acid analysis of the phosphorylated p67phox revealed only 32P-labeled serine residues. Two-dimensional tryptic peptide mapping analysis showed that p67phox is phosphorylated at the same peptide whether fMLP or PMA is used as a stimulus. In addition, PKC induced the phosphorylation of recombinant GST-p67phox in vitro, at the same peptide as that phosphorylated in intact cells. PMA-induced phosphorylation of p67phox 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 p67phox is phosphorylated in human neutrophils by different pathways, one of which involves protein kinase C.


INTRODUCTION

The respiratory burst oxidase, or NADPH1 oxidase, of neutrophils and B lymphocytes is a multicomponent enzyme that catalyzes the NADPH-dependent reduction of oxygen to superoxide anion (Obardot 2), a precursor of microbicidal oxidants (1, 2). The importance of the oxidase in host defenses is demonstrated by the recurrent and life-threatening infections that occur in patients with chronic granulomatous disease (CGD), a hereditary disorder resulting in defective NADPH oxidase activity (3, 4). Components of this oxidase include cytochrome b558, a membrane-bound flavohemoprotein, the cytosolic proteins p47phox, p67phox, and p40phox, and a small GTP-binding protein Rac2 or Rac1. In resting cells the enzyme is inactive, and its components are distributed between the cytosol and membranes. When cells are activated, the cytosolic components migrate to the membranes and their cytoskeleton fraction, where they associate with cytochrome b to form the catalytically active oxidase (5-7).

Activation of neutrophils by formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA) leads to a marked increase in the phosphorylation of multiple proteins on serines, threonines, and tyrosines (8, 9). The functional significance of these phosphorylations and the relevant protein kinases is unclear (2, 10). One of the phosphorylated proteins is the cytosolic oxidase subunit p47phox, which is phosphorylated on several serines (11-14). This phosphorylation is required for the translocation of p47phox to the plasma membrane and for the activation of NADPH oxidase (15, 16). Translocation of p67phox is also essential for the activation of NADPH oxidase, as the oxidase from CGD patients deficient in this protein fails to produce Obardot 2 (3). While the phosphorylation of p47phox has been extensively investigated, conflicting results have been reported on the phosphorylation of p67phox (17, 18). Using an antibody that specifically immunoprecipitates p67phox, we show here that this protein becomes phosphorylated in human neutrophils stimulated with fMLP or PMA. In addition, we show that the PKC inhibitor GF109203X inhibits PMA-induced phosphorylation of p67phox without affecting fMLP-induced phosphorylation. These results suggest that phosphorylation of p67phox participates in the regulation of NADPH oxidase by PKC-dependent and -independent pathways.


EXPERIMENTAL PROCEDURES

Materials

PMA, fMLP, phosphatases inhibitors, proteases inhibitors, phosphoserine, phosphothreonine, and phosphotyrosine were from Sigma. Protein kinase C was from Calbiochem or Promega (Madison, WI). GF109203X was from Calbiochem. Sequencing grade trypsin was from Boehringer Mannheim (Germany). SDS-PAGE reagents were from Bio-Rad. [32P]Orthophosphate and [gamma -32P]ATP were from DuPont NEN Life Science Products. Rabbit anti-p67phox polyclonal antibody raised against the synthetic peptide extending from amino acid 512 to the C-terminal residue was prepared as described elsewhere (19). The anti-p47phox antibody and EBV-transformed B lymphocytes from p67phox-deficient CGD patient, and normal subjects were kindly provided by Dr. Bernard M. Babior (The Scripps Research Institute, La Jolla, CA).

Neutrophil Preparation

Neutrophils were obtained from normal subjects by dextran sedimentation and Ficoll-Hypaque fractionation of freshly drawn citrated blood (6). The cells were resuspended at 1 × 108/ml in phosphate-free buffer (10 mM Hepes, 137 mM NaCl, 5.4 mM KCl, 5.6 mM D-glucose, 0.8 mM MgCl2, and 0.025% bovine serum albumin) and treated with 2.5 mM diisopropyl fluorophosphate (DFP) on ice for 20 min.

32P Labeling of Neutrophils and Lymphoblasts

Neutrophils were incubated in phosphate-free buffer containing 1 mCi of [32P]orthophosphate/108 cells/ml for 1 h at 30 °C. The cells were then washed and activated with PMA (1 µg/ml/108 cells) for 8 min or with fMLP (1 µM/108 cells) for 2 min in the presence of 1 mM MgCl2 and 1 mM CaCl2. Activation was terminated with 10 volumes of ice-cold buffer. The cells were pelleted by centrifugation (400 × g for 10 min at 4 °C) and resuspended at 1 × 108 cells/ml in ice-cold lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EGTA, 5 mM EDTA, 15 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 1.5 mM phenylmethylsulfonyl fluoride, 1 mM DFP, 0.5% Triton X-100, 25 mM NaF, 5 mM NaVO4, 5 mM beta -glycerophosphate, 1 mM p-nitrophenyl phosphate, 0.25 M sucrose, and 1 mg/ml DNase I), sonicated (3 × 10 s), and centrifuged (100,000 × g, 30 min at 4 °C). Lymphoblasts were labeled with 32P as described previously (16). Briefly, the cells were incubated overnight in phosphate-free medium, then transferred to fresh medium containing 32P (0.2 mCi/ml) and incubated for 4 h at 37 °C. The cells were then activated for 12 min with PMA (1 µg/ml/108 cells) then lysed like the neutrophils.

Immunoprecipitation Electrophoresis and Immunoblotting Experiments

Gamma Bind G-Sepharose beads (Pharmacia Biotech Inc.) were equilibrated with lysis buffer containing 1 mg/ml bovine serum albumin for 1 h at 4 °C. The cleared lysate was incubated with p67phox antibody (1/150 dilution) or p47phox antibody (1/200 dilution) or their respective IgG controls in the presence of 50 µl of Sepharose beads overnight at 4 °C with gentle mixing. Then, the beads were washed extensively with lysis buffer without DFP or DNase I. The immunoprecipitated proteins were eluted by boiling in electrophoresis sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2.3% SDS, 2% 2-mercaptoethanol). The beads were pelleted by brief centrifugation, and the supernatant was subjected to 10% SDS-PAGE according to Laemmli (20). Proteins were blotted onto nitrocellulose using Towbin buffer (21) and detected by autoradiography or antibody labeling (p67phox antibody = 1/1000 dilution, p47phox antibody = 1/10,000 dilution), using ECL or colorimetric detection.

Recombinant p67phox and p47phox

P67phox cDNA was prepared from EBV-transformed B lymphocytes mRNA as described previously (19). The cDNA was cloned in pGEX-3X plasmid at the EcoRI site then transformed into competent XL-Blue I Escherichia coli bacteria. E. coli expressing GST-p47phox (22) was a kind gift from Dr. Bernard M. Babior (The Scripps Research Institute, La Jolla, CA). Recombinant proteins were obtained as glutathione S-transferase GST-fusion proteins expressed in bacteria and purified on glutathione-agarose beads (22). Briefly, E. coli was grown at 37 °C overnight in 50 ml of ampicillin broth medium. The overnight culture was diluted into 450 ml of fresh medium and grown for an additional hour at 37 °C. The culture was then made up to 0.1 mM in isopropyl-beta -D-thiogalactoside and grown for an additional 3 h at room temperature (p67phox) or 37 °C (p47phox). The bacteria were recovered by centrifugation at 5000 × g for 20 min at 4 °C. The pellet was resuspended in 5 ml of ice-cold bacteria lysis buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 20 mM leupeptin, 15 mM pepstatin, and 0.5 mM DFP) and disrupted by sonication (3 × 10 s). The sonicate was clarified by centrifugation at 20,000 × g for 20 min at 4 °C. The fusion proteins were isolated from the clarified sonicate by purification over glutathione-agarose beads as described previously (22).

In Vitro Phosphorylation of p47phox and p67phox

Five micrograms of recombinant GST-p67phox or p47phox was incubated with 0.5 µg of protein kinase C in 40 mM Hepes, 10 mM MgCl2, 2 mM MnCl2, 0.5 mM CaCl2, 1 mM dithiothreitol, 50 µM (1 µCi) [gamma -32P]ATP, 5 µg/ml diolein, and 50 µg/ml phosphatidylserine in a final volume of 100 µl. The reaction was run for 30 min at 30 °C and terminated by adding 5 × Laemmli sample buffer. The proteins were analyzed by SDS-PAGE.

Two-Dimensional Tryptic Phosphopeptide Mapping

Tryptic digestion of p67phox and p47phox blotted onto nitrocellulose was performed as described elsewhere (13, 23). The dried peptides were dissolved in water and lyophilized three times, redissolved in electrophoresis buffer (88% formic acid/water = (3:17)), and applied to one corner of a 20 × 20-cm cellulose thin-layer plate. Electrophoresis was carried out at 6 °C for 25 min at 1100 V on an LKB-flat gel apparatus. Chromatography was performed as described previously (13, 23).

Phosphoamino Acid Analysis

Phosphoamino acid analysis of p67phox was performed using one-dimensional analysis by the method of Boyle et al. (23). The polyvinylidene difluoride membrane containing the radiolabeled p67phox band was located by autoradiography, excised, and transferred to a microcentrifuge tube. Protein was then hydrolyzed in 0.25 ml of 5.7 N HCl for 1 h at 110 °C. The sample was dried using a Speed-Vac concentrator and then resuspended in 10 µl of a buffer composed of 100:10:1890 (v/v) acetic acid:pyridine:water. The sample was spotted on a thin-layer cellulose plate (Merck) and subjected to electrophoresis at 1100 V for 45 min with water cooling. Phosphoserine, phosphothreonine, and phosphotyrosine (1 mg/ml) were used as markers. The plate was then dried, sprayed with ninhydrin to localize the phosphoamino acid standards, and used for autoradiography.


RESULTS

p67phox Is Phosphorylated in Activated Human Neutrophils

To determine if p67phox is phosphorylated, neutrophils were loaded with [32P]inorganic phosphate and activated with fMLP or PMA; p67phox was immunoprecipitated with a specific antibody as described under "Experimental Procedures." Fig. 1A shows the autoradiography of the corresponding gel. While p67phox was weakly phosphorylated in resting cells, its state of phosphorylation clearly increased after stimulation of human neutrophils with PMA (1 µg/ml for 8 min) or fMLP (1 µM for 2 min). The absence of the phosphorylated protein after immunoprecipitating with the control IgG showed that the presence of this phosphorylated protein was not the result of nonspecific binding to the beads. Corresponding Western blot analysis (Fig. 1B) identified this phosphoprotein as p67phox. Furthermore, as conflicting results have been reported on the phosphorylation of p67phox, possibly due to the use of different and not very specific antibodies, we checked that our antibody did not recognize other proteins than p67phox. Fig. 2A shows that in EBV-transformed B cells from a CGD patient deficient in p67phox, the anti-p67phox antibody did not cross-react with another protein around the 67-kDa area. However p47phox is normally expressed in these cells and both phox (phagocyte oxidase) proteins are expressed in normal lymphoblasts. In addition, the 32P-labeled protein was not immunoprecipitated from p67phox-deficient cells but was immunoprecipitated from normal lymphoblasts (Fig. 2, B and C). These results clearly show that p67phox is phosphorylated in activated neutrophils and EBV-transformed B lymphocytes.


Fig. 1. P67phox is phosphorylated in PMA and fMLP-treated human neutrophils. 32P-Labeled neutrophils were activated with PMA (1 µg/ml/108 cells for 8 min) or fMLP (1 µM/108/ml for 2 min). P67phox was immunoprecipitated and then analyzed by SDS-PAGE and blotting onto nitrocellulose membranes. The protein was detected by autoradiography (A) and by use of an anti-p67phox antibody (B). The preparation shown was obtained from 50 × 106 cells/lane. "Hch" is the immunoglobulin heavy chain."Ab" is antibody. Data are representative of three experiments.
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Fig. 2. The phosphorylated protein is absent from p67phox-deficient B lymphoblasts. Normal and p67phox-deficient lymphoblasts were lysed (A) or labeled with 32P, activated with PMA (1 µg/ml for 12 min), lysed, and incubated with anti-p67phox antibody as described under "Experimental Procedures" (B and C). The proteins were analyzed by SDS-PAGE and detected by the anti-p67phox antibody (in A and C) or the anti-p47phox antibody (A) and autoradiography (B). Each track contains protein from 50 × 106 cells for the immunoprecipitated p67phox. "Hch" is the immunoglobulin heavy chain. Data are representative of three experiments.
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Phosphorylated p67phox and p47phox Form a Complex

As in resting cells p47phox and p67phox form a complex, we wondered if these proteins remain in the complex after being phosphorylated. Immunoprecipitation with either p67phox antibody or p47phox antibody resulted in the isolation of both phosphorylated p67phox and p47phox (Fig. 3). However, more of each phosphorylated protein was precipitated when the corresponding antibody was used.


Fig. 3. Phosphorylated p67phox forms a complex with phosphorylated p47phox. 32P-Labeled neutrophils were stimulated with PMA (1 µg/ml/108 cells for 8 min), lysed, and incubated with anti-p47phox antibody or anti-p67phox antibody. After SDS-PAGE, proteins were detected by autoradiography and Western blot using anti-p47phox and anti-p67phox antibodies. Data are representative of three experiments. IP, immunoprecipitation.
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P67phox Is Phosphorylated on Serine Residues

Phosphoamino acid analysis (Fig. 4) showed that p67phox was phosphorylated on serine residues; no phosphothreonine or phosphotyrosine was detected with appropriate markers. In addition, no detectable staining was observed with an anti-phosphotyrosine antibody (data not shown). These results suggest that a Ser/Thr protein kinase, not a tyrosine kinase, phosphorylates p67phox.


Fig. 4. P67phox is phosphorylated on serine residues. Purified 32P-labeled p67phox from neutrophils was blotted onto polyvinylidene difluoride membranes and hydrolyzed in HCl. The resulting phosphoamino acids were analyzed by one-dimensional high-voltage electrophoresis as described under "Experimental Procedures." The position of phosphoserine (PS), phosphothreonine (PT), and phosphotyrosine (PY), determined by ninhydrin staining of standards, is indicated. Data are representative of three experiments.
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FMLP and PMA Induce the Phosphorylation of p67phox on Serines Located in the Same Peptide

The chemotactic peptide fMLP activates neutrophils via a membrane receptor that triggers a multitude of signaling pathways involving phospholipases and protein kinases. However, PMA, which bypasses the receptor, is believed to be more specific for PKC activation. To determine if fMLP and PMA induced the phosphorylation of the same or different phosphopeptides in p67phox, we used two-dimensional tryptic peptide mapping. Fig. 5 shows the presence of one major phosphorylated peptide after neutrophil activation and that the phosphopeptide map of 32P-labeled p67phox from PMA-activated neutrophils was identical to that of labeled p67phox from fMLP-activated neutrophils. In resting cells the same peptide was weakly phosphorylated (data not shown). It is not clear if this peptide contains one or several phosphoserines that could be phosphorylated by different protein kinases.


Fig. 5. Tryptic phosphopeptide maps of 32P-labeled p67phox from PMA- and fMLP-activated neutrophils. 32P-Labeled neutrophils were activated with PMA (1 µg/ml for 8 min) or fMLP (1 µM for 2 min) as described in the text. 32P-Labeled p67phox was purified by immunoprecipitation followed by SDS-PAGE, then blotted onto nitrocellulose membranes and analyzed by tryptic phosphopeptide mapping. TLC, thin-layer chromatography. TLE, thin-layer electrophoresis. The point of application of the sample is indicated by the dot in the lower left corner of each panel. Data are representative of three experiments.
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PKC Is Involved in p67phox Phosphorylation in Intact Neutrophils

The phosphorylation of p67phox is induced by PMA, a direct activator of PKC, suggesting the involvement of this kinase in the phosphorylation of p67phox. To assess this possibility and to determine whether or not PKC is involved in fMLP-induced phosphorylation of p67phox, neutrophils were incubated with the PKC antagonist GF109203X. As seen in Fig. 6, p67phox phosphorylation induced by PMA was strongly inhibited by GF109203X; in contrast, fMLP-induced p67phox phosphorylation was minimally affected by this inhibitor. This suggests that, in addition to the GF109203X-sensitive PKC isoforms, other protein kinases (insensitive to GF109203X) are involved in p67phox phosphorylation induced by fMLP. In comparison with p67phox, the p47phox phosphorylation induced by PMA or fMLP was inhibited by GF109203X.


Fig. 6. Effect of the protein kinase C inhibitor on p67phox phosphorylation. 32P-Labeled neutrophils were incubated with 2.5 µM of the PKC inhibitor GF109203X for 10 min, before stimulation with 1 µM fMLP for 2 min or 1 µg/ml PMA for 8 min. Immunoprecipitation of p67phox and p47phox was performed as described under "Experimental Procedures." Data are representative of three experiments.
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PKC Phosphorylates p67phox in Vitro

To determine if PKC phosphorylates p67phox directly, recombinant GST-p67phox was incubated with [gamma -32P]ATP and purified protein kinase C. As shown in Fig. 7, PKC phosphorylated GST-p67phox, although to a lesser extent than p47phox. Furthermore, tryptic peptide mapping showed that the serine(s) phosphorylated in vitro by PKC was/were located in the same peptide as that phosphorylated in intact cells. These results strongly support the participation of PKC in p67phox phosphorylation.


Fig. 7. GST-p67phox is phosphorylated in vitro by protein kinase C. GST-p67phox expressed in and purified from bacteria was incubated with purified PKC and analyzed by SDS-PAGE and autoradiography (top panel). GST-p47phox was used as comparator. Phosphorylated p67phox was analyzed by tryptic phosphopeptide mapping (bottom panel). Data are representative of three experiments.
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DISCUSSION

The link between respiratory burst oxidase activation and protein phosphorylation is believed to be exclusively mediated by p47phox phosphorylation. Recent and conflicting results (17, 18) have raised the possibility of p67phox phosphorylation: Dusi and Rossi (17) have reported that p67phox is phosphorylated during neutrophil activation, while Heyworth et al. (18) observed no such phosphorylation. The difficulties in p67phox phosphorylation analysis are due mainly to the very low level of the protein in cells (24), the fact that p67phox is very sensitive to proteolysis (25), and the lack of a strongly specific antibody for immunoprecipitation studies. We used an antibody that specifically immunoprecipitates p67phox, as shown by the lack of an immunoprecipitant p67 protein in EBV-transformed B cells from a CGD patient deficient in this protein, as well as the single band obtained by Western blotting of the immunoprecipitate with another anti-p67phox antibody. Using this antibody, we observed that the p67phox component of NADPH oxidase clearly became phosphorylated on stimulation of neutrophils with either fMLP, a receptor-dependent chemotactic activator, or PMA, a PKC activator. The weak phosphorylation observed in nonstimulated cells could correspond to basal phosphorylation or to slight activation during neutrophil isolation.

In resting cells, p47phox and p67phox exist in a complex (22) that translocates to associate with cytochrome b558 during oxidase activation. In this report we show that p47phox and p67phox remain, at least partially, complexed after their phosphorylation. Partial dissociation cannot be ruled out, as more phosphorylated protein was precipitated by the corresponding specific antibody than by the antibody directed against the other member of the complex. In addition, the observed dissociation could occur naturally or be induced by the antibodies. It has recently been shown that the cytosolic oxidase complex contains, in addition to p67phox and p47phox, a protein named p40phox that participates in oxidase activation (26). We found that p40phox copurified with the phosphorylated p47phox/p67phox complex but was not phosphorylated.2

Our results show that p67phox undergoes phosphorylation on serine residues during neutrophil activation, without threonine or tyrosine phosphorylation. These results suggest that a Ser/Thr protein kinase, not a tyrosine kinase, induces the phosphorylation of p67phox. Our observation that GF109203X, a PKC inhibitor, strongly inhibited PMA-induced phosphorylation and barely modified fMLP-induced phosphorylation, points to both PKC-dependent (isoforms sensitive to GF109203X) and PKC-independent (or GF109203X-insensitive) pathways in the phosphorylation of p67phox. Indeed, human neutrophils express in addition to the alpha  and beta PKC isoforms the PKCzeta (27). The PKC inhibitor GF109203X could be more effective against the alpha  and beta  isoforms than the zeta  one. However, we found that PKCzeta is not able to phosphorylate p67phox in vitro.3 Whether or not GF109203X inhibits PKCzeta , this result suggests that PKCzeta is not involved in p67phox phosphorylation. Two-dimensional phosphopeptide mapping showed that the same p67phox peptide was phosphorylated after fMLP and PMA stimulation. It is conceivable that this peptide contains several serines that are phosphorylated by different protein kinases: the different sensitivity of p67phox phosphorylation to the PKC inhibitor (GF109203X) when induced by fMLP or PMA supports this hypothesis. Whatever the other kinases involved, the phosphorylation of recombinant GST-p67phox in vitro by purified PKC, on this same peptide, suggests that PKC participates in the phosphorylation of p67phox. After neutrophil activation with PMA or fMLP, p47phox is phosphorylated on several serines (13, 14), the result obtained by tryptic peptide mapping of p67phox suggests that p67phox has less phosphorylated sites than p47phox. However, the phosphorylation of both proteins could have a crucial importance in the regulation of NADPH-oxidase activation.

Several lines of evidence support a role of PKC in NADPH oxidase activation. PMA, an activator of PKC, is a strong stimulus of Obardot 2 production in whole cells (28). Purified p47phox is a good substrate for PKC in vitro (29). Staurosporine, a powerful inhibitor of PKC, inhibits superoxide production and p47phox phosphorylation (15, 30). The data presented here provide clear evidence that, in addition to p47phox, p67phox itself could play a role in the regulation of NADPH oxidase by phosphorylation/dephosphorylation reactions and that the phosphorylation events involve a PKC-dependent pathway. Little is known of the possible role of other protein kinases in the regulation of NADPH oxidase. It has recently been suggested that cyclic AMP-dependent protein kinase, mitogen-activated protein kinase (14, 31), and p21-activated kinase (32) could regulate NADPH oxidase by phosphorylating p47phox. Our findings suggest that protein kinases other than PKC may participate in p67phox phosphorylation. The kinases involved in this process are currently under investigation.


FOOTNOTES

*   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.
§   To whom correspondence should be addressed. Tel.: 33-1-40-25-85-21; Fax: 33-01 40-25-88-53; E-mail: benna{at}bichat.inserm.fr.
1   The abbreviations used are: NADPH, nicotinamide adenine diphosphate reduced form; CGD, chronic granulomatous disease; fMLP, formyl-methionyl-leucyl-phenylalanine; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; PAGE, polyacrylamide gel electrophoresis.
2   J. El Benna and M. Gougerot-Pocidalo, unpublished observations.
3   P M.-C. Dang, M.-A. Gougerot-Pocidalo, and J. El Benna, manuscript in preparation.

ACKNOWLEDGEMENTS

We are grateful to Dr. Bernard M. Babior from the Scripps Research Institute for the antibodies, the E. coli expressing p47phox and lymphoblasts.


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