Phosphorylation of the Respiratory Burst Oxidase Subunit p67 phox during Human Neutrophil Activation

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 only32P-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 respiratory burst oxidase, or NADPH 1 oxidase, of neutrophils and B lymphocytes is a multicomponent enzyme that catalyzes the NADPH-dependent reduction of oxygen to superoxide anion (O 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 b 558 , a mem-brane-bound flavohemoprotein, the cytosolic proteins p47 phox , p67 phox , and p40 phox , 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)(6)(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 p47 phox , which is phosphorylated on several serines (11)(12)(13)(14). This phosphorylation is required for the translocation of p47 phox to the plasma membrane and for the activation of NADPH oxidase (15,16). Translocation of p67 phox is also essential for the activation of NADPH oxidase, as the oxidase from CGD patients deficient in this protein fails to produce O 2 . (3). While the phosphorylation of p47 phox has been extensively investigated, conflicting results have been reported on the phosphorylation of p67 phox (17,18). Using an antibody that specifically immunoprecipitates p67 phox , 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 p67 phox without affecting fMLP-induced phosphorylation. These results suggest that phosphorylation of p67 phox 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. [ 32 P]Orthophosphate and [␥-32 P]ATP were from DuPont NEN Life Science Products. Rabbit anti-p67 phox 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-p47 phox antibody and EBV-transformed B lymphocytes from p67 phox -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 ϫ 10 8 /ml in phosphate-free buffer (10 mM Hepes, 137 mM NaCl, 5.4 mM KCl, 5.6 mM D-glucose, 0.8 mM MgCl 2 , and 0.025% bovine serum albumin) and treated with 2.5 mM diisopropyl fluorophosphate (DFP) on ice for 20 min. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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 p67 phox antibody (1/150 dilution) or p47 phox antibody (1/200 dilution) or their respective IgG controls in the presence of 50 l 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 (p67 phox antibody ϭ 1/1000 dilution, p47 phox antibody ϭ 1/10,000 dilution), using ECL or colorimetric detection.
Recombinant p67 phox and p47 phox -P67 phox 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-p47 phox (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-␤-D-thiogalactoside and grown for an additional 3 h at room temperature (p67 phox ) or 37°C (p47 phox ). The bacteria were recovered by centrifugation at 5000 ϫ 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 MgCl 2 , 1 mM dithiothreitol, 20 mM leupeptin, 15 mM pepstatin, and 0.5 mM DFP) and disrupted by sonication (3 ϫ 10 s). The sonicate was clarified by centrifugation at 20,000 ϫ 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 p47 phox and p67 phox -Five micrograms of recombinant GST-p67 phox or p47 phox was incubated with 0.5 g of protein kinase C in 40 mM Hepes, 10 mM MgCl 2 , 2 mM MnCl 2 , 0.5 mM CaCl 2 , 1 mM dithiothreitol, 50 M (1 Ci) [␥-32 P]ATP, 5 g/ml diolein, and 50 g/ml phosphatidylserine in a final volume of 100 l. The reaction was run for 30 min at 30°C and terminated by adding 5 ϫ Laemmli sample buffer. The proteins were analyzed by SDS-PAGE.
Two-Dimensional Tryptic Phosphopeptide Mapping-Tryptic digestion of p67 phox and p47 phox 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 ϭ (3:17)), and applied to one corner of a 20 ϫ 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 p67 phox was performed using one-dimensional analysis by the method of Boyle et al. (23). The polyvinylidene difluoride membrane containing the radiolabeled p67 phox 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 l 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
p67 phox Is Phosphorylated in Activated Human Neutrophils-To determine if p67 phox is phosphorylated, neutrophils were loaded with [ 32 P]inorganic phosphate and activated with fMLP or PMA; p67 phox was immunoprecipitated with a specific antibody as described under "Experimental Procedures." Fig.  1A shows the autoradiography of the corresponding gel. While p67 phox was weakly phosphorylated in resting cells, its state of phosphorylation clearly increased after stimulation of human neutrophils with PMA (1 g/ml for 8 min) or fMLP (1 M 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 p67 phox . Furthermore, as conflicting results have been reported on the phosphorylation of p67 phox , possibly due to the use of different and not very specific antibodies, we checked that our antibody did not recognize other proteins than p67 phox . Fig. 2A shows that in EBV-transformed B cells from a CGD patient deficient in p67 phox , the anti-p67 phox antibody did not cross-react with another protein around the 67-kDa area. However p47 phox is normally expressed in these cells and both phox (phagocyte oxidase) proteins are expressed in normal lymphoblasts. In addition, the 32 P-labeled protein was not immunoprecipitated from p67 phox -deficient cells but was immunoprecipitated from normal lymphoblasts (Fig. 2, B and C). These results clearly show that p67 phox is phosphorylated in activated neutrophils and EBV-transformed B lymphocytes.
Phosphorylated p67 phox and p47 phox Form a Complex-As in resting cells p47 phox and p67 phox form a complex, we wondered if these proteins remain in the complex after being phosphorylated. Immunoprecipitation with either p67 phox antibody or p47 phox antibody resulted in the isolation of both phosphorylated p67 phox and p47 phox (Fig. 3). However, more of each phosphorylated protein was precipitated when the corresponding antibody was used.
P67 phox Is Phosphorylated on Serine Residues-Phosphoamino acid analysis (Fig. 4) showed that p67 phox was phosphorylated on serine residues; no phosphothreonine or phosphotyrosine was detected with appropriate markers. In addition, no detectable staining was observed with an antiphosphotyrosine antibody (data not shown). These results suggest that a Ser/Thr protein kinase, not a tyrosine kinase, phosphorylates p67 phox .
FMLP and PMA Induce the Phosphorylation of p67 phox 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 p67 phox , 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 32 P-labeled p67 phox from PMA-activated neutrophils was identical to that of labeled p67 phox 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.
PKC Is Involved in p67 phox Phosphorylation in Intact Neutrophils-The phosphorylation of p67 phox is induced by PMA, a direct activator of PKC, suggesting the involvement of this kinase in the phosphorylation of p67 phox . To assess this possibility and to determine whether or not PKC is involved in fMLP-induced phosphorylation of p67 phox , neutrophils were incubated with the PKC antagonist GF109203X. As seen in Fig.   6, p67 phox phosphorylation induced by PMA was strongly inhibited by GF109203X; in contrast, fMLP-induced p67 phox 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 p67 phox phosphorylation induced by fMLP. In comparison with p67 phox , the p47 phox phosphorylation induced by PMA or fMLP was inhibited by GF109203X.
PKC Phosphorylates p67 phox in Vitro-To determine if PKC phosphorylates p67 phox directly, recombinant GST-p67 phox was incubated with [␥-32 P]ATP and purified protein kinase C. As shown in Fig. 7, PKC phosphorylated GST-p67 phox , although to a lesser extent than p47 phox . 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 p67 phox phosphorylation.

DISCUSSION
The link between respiratory burst oxidase activation and protein phosphorylation is believed to be exclusively mediated by p47 phox phosphorylation. Recent and conflicting results (17,18) have raised the possibility of p67 phox phosphorylation: Dusi and Rossi (17) have reported that p67 phox is phosphorylated during neutrophil activation, while Heyworth et al. (18) observed no such phosphorylation. The difficulties in p67 phox phosphorylation analysis are due mainly to the very low level of the protein in cells (24), the fact that p67 phox is very sensitive to proteolysis (25), and the lack of a strongly specific antibody for immunoprecipitation studies. We used an antibody that specifically immunoprecipitates p67 phox , 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-p67 phox antibody. Using this antibody, we observed that the p67 phox 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, p47 phox and p67 phox exist in a complex (22) that translocates to associate with cytochrome b 558 during oxidase activation. In this report we show that p47 phox and p67 phox 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 p67 phox and p47 phox , a protein named p40 phox that participates in oxidase activation (26). We found that p40 phox copurified with the phosphorylated p47 phox / p67 phox complex but was not phosphorylated. 2 Our results show that p67 phox 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 p67 phox . 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 p67 phox . Indeed, human neutrophils express in addition to the ␣ and ␤ PKC isoforms the PKC (27). The PKC inhibitor GF109203X could be more effective against the ␣ and ␤ isoforms than the one. However, we found that PKC is not able to phosphorylate p67 phox in vitro. 3 Whether or not GF109203X inhibits PKC, this result suggests that PKC is not involved in p67 phox phosphorylation. Two-dimensional phosphopeptide mapping showed that the same p67 phox 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 p67 phox 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-p67 phox in vitro by purified PKC, on this same peptide, suggests that PKC participates in the phosphorylation of p67 phox . After neutrophil activation with PMA or fMLP, p47 phox is phosphorylated on several serines (13,14), the result obtained by tryptic peptide mapping of p67 phox suggests that p67 phox has less phosphorylated sites than p47 phox . 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 FIG. 6. Effect of the protein kinase C inhibitor on p67 phox phosphorylation. 32 P-Labeled neutrophils were incubated with 2.5 M of the PKC inhibitor GF109203X for 10 min, before stimulation with 1 M fMLP for 2 min or 1 g/ml PMA for 8 min. Immunoprecipitation of p67 phox and p47 phox was performed as described under "Experimental Procedures." Data are representative of three experiments.
FIG. 7. GST-p67 phox is phosphorylated in vitro by protein kinase C. GST-p67 phox expressed in and purified from bacteria was incubated with purified PKC and analyzed by SDS-PAGE and autoradiography (top panel). GST-p47 phox was used as comparator. Phosphorylated p67 phox was analyzed by tryptic phosphopeptide mapping (bottom panel). Data are representative of three experiments. oxidase activation. PMA, an activator of PKC, is a strong stimulus of O 2 . production in whole cells (28). Purified p47 phox is a good substrate for PKC in vitro (29). Staurosporine, a powerful inhibitor of PKC, inhibits superoxide production and p47 phox phosphorylation (15,30). The data presented here provide clear evidence that, in addition to p47 phox , p67 phox 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 p47 phox . Our findings suggest that protein kinases other than PKC may participate in p67 phox phosphorylation. The kinases involved in this process are currently under investigation.