p40 phox Is Phosphorylated on Threonine 154 and Serine 315 during Activation of the Phagocyte NADPH Oxidase

The superoxide-generating NADPH oxidase complex of phagocytic cells is a multicomponent system containing a membrane-bound flavocytochrome b and a small G protein Rac as well as cytosolic factors p67 phox (phagocyte oxidase), p47 phox , and p40 phox , which translocate to the membrane upon activation. In a previous paper, we reported that p40 phox undergoes phosphorylation on multiple sites upon stimulation of the NADPH oxidase by either phorbol 12-myristate 13-acetate or by formyl peptide with a time course that is strongly correlated with that of superoxide production (Fuchs, A., Bouin, A. P., Rabilloud, T., and Vignais, P. V. (1997) Eur. J. Biochem. 249, 531–539). In this study, through phosphoamino acid and tryptic peptide maps of in vivo and in vitro phosphorylated p40 phox , we show that p40 phox is phosphorylated on serine and threonine residues during activation of the NADPH oxidase in dimethyl sulfoxide-differentiated HL60 promyelocytes as well as in isolated human neutrophils. In vitro phosphorylation studies using casein kinase II and protein kinase C (PKC) as well as the effect of various protein kinase inhibitors on the isoelectric focusing pattern of p40 phox in whole cell lysates point to a role of a PKC type kinase in the phosphorylation of p40 phox . Directed mutagenesis of all PKC consensus sites enable us to conclude that Thr154 and Ser315 in p40 phox are phosphorylated during activation of the NADPH oxidase.

The superoxide-generating NADPH oxidase complex of phagocytic cells is a multicomponent system containing a membrane-bound flavocytochrome b and a small G protein Rac as well as cytosolic factors p67 phox (phagocyte oxidase), p47 phox , and p40 phox , which translocate to the membrane upon activation. In a previous paper, we reported that p40 phox undergoes phosphorylation on multiple sites upon stimulation of the NADPH oxidase by either phorbol 12myristate 13-acetate or by formyl peptide with a time course that is strongly correlated with that of superoxide production (Fuchs, A., Bouin, A. P., Rabilloud, T., and Vignais, P. V. (1997) Eur. J. Biochem. 249, 531-539). In this study, through phosphoamino acid and tryptic peptide maps of in vivo and in vitro phosphorylated p40 phox , we show that p40 phox is phosphorylated on serine and threonine residues during activation of the NADPH oxidase in dimethyl sulfoxide-differentiated HL60 promyelocytes as well as in isolated human neutrophils. In vitro phosphorylation studies using casein kinase II and protein kinase C (PKC) as well as the effect of various protein kinase inhibitors on the isoelectric focusing pattern of p40 phox in whole cell lysates point to a role of a PKC type kinase in the phosphorylation of p40 phox . Directed mutagenesis of all PKC consensus sites enable us to conclude that Thr 154 and Ser 315 in p40 phox are phosphorylated during activation of the NADPH oxidase.
In response to invasive microorganisms, neutrophils and other phagocytic cells react violently to produce superoxide anion and other microbicidal toxic oxygen derivatives in the phagocytosis vacuole. This phenomenon is known as the respiratory burst, and the mechanism by which these cells regulate the burst is not yet fully elucidated. The production of superoxide anion is assigned to a multicomponent system, the NADPH oxidase, which consists of a membrane-bound flavocytochrome b and a small G protein Rac as well as cytosolic factors p67 phox1 (phagocyte oxidase), p47 phox , and p40 phox (for review, see Refs. [2][3][4]. Known mechanisms underlying the activation of the respiratory burst include translocation to the membrane of the cytosolic proteins, specific src homology 3 (SH3)/polyproline motif interactions (5)(6)(7)(8)(9)(10), as well as phosphorylation events on p47 phox , p67 phox , and p40 phox (1,11,12). Translocation of the three phox proteins is dependent upon the presence of flavocytochrome b in the membrane (13), and the translocation of p40 phox appears to be p47 phox -dependent and is mediated by p67 phox (14). Interestingly, the SH3 domain of p40 phox down-regulates activation of the NADPH oxidase (15). As shown by two hybrid studies (6,7) and by in vitro binding assays (16 -18), p47 phox interacts with both p40 phox and p67 phox through its proline-rich COOH-terminal region (6,18). It seems plausible that some modification occurs during oxidase activation which could probably change the specificity of the prolinerich domain of p47 phox for either the SH3 domain of p40 phox or the COOH-terminal SH3 domain of p67 phox . This modification could be the phosphorylation of p47 phox on a cluster of serine residues close to the polyproline motif in the COOH-terminal region. However, we have recently stressed the fact that the time course of p40 phox phosphorylation is strongly correlated with that of superoxide production (1), and we have postulated that p40 phox phosphorylation could therefore play a critical role in the rearrangement of the ternary complex consisting of p47 phox , p67 phox , and p40 phox during the respiratory burst. In this report we identify activation-induced phosphorylation sites on p40 phox and describe experimental data supporting a critical role of the state of phosphorylation of these residues on the structure of the cytosolic activating complex.

EXPERIMENTAL PROCEDURES
Materials-33 P i at 4,000Ci/mmol was purchased from NEN Life Science Products. Protein A-Sepharose beads, the enhanced chemiluminescence detection kit, and [␥-32 P]ATP were from Amersham Pharmacia Biotech. Protein kinase C (PKC) from rat brain was from Boehringer Mannheim. RPMI medium and fetal calf serum were from Life Technologies, Inc. Protein A-horseradish peroxidase conjugate was from Bio-Rad. Polyvinylidene difluoride (PVDF) protein transfer membranes (ProBlott) were from Applied Biosystems. Nitrocellulose membranes were from Schleicher & Schuell. SeeBlue protein molecular weight standards were from Novex. Thrombin was from Sigma, and endoproteinase-Lys-C from Boehringer Mannheim.
Antisera-Anti-p40 phox antisera were raised in rabbits using a synthetic peptide corresponding to the NH 2 -terminal 18 amino acid residues of p40 phox (1). Anti-p67 phox antiserum was obtained using purified recombinant p67 phox protein expressed in the baculovirus/insect cell system as described (19). After four antigen injection boosts at 4-week intervals, the serum was tested against a total cell lysate by Western blot. All antisera recognized a unique band at the appropriate molecular weight. Specificity was confirmed by testing the preimmune sera in parallel. A COOH-terminal anti-p40 phox antiserum was a kind gift from * This work was supported, in part, by research grants from the Direction des systèmes de forces et de la prospective (Direction Générale pour l'Armement), the Center National de la Recherche Scientifique (CNRS), the Commissariat à l'Energie Atomique (CEA), the Université Joseph Fourier-Faculté de Médecine, the Association pour la Recherche contre le Cancer, and the Association Recherche et Partage. 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.
Neutrophil Preparation and Metabolic Labeling-Neutrophils were isolated from buffy coats as described (20). Neutrophils were suspended at 5 ϫ 10 7 cells/ml in 10 mM HEPES, pH 7.4, containing 137 mM NaCl, 0.8 mM MgCl 2 , 5.4 mM KCl, and 5.6 mM glucose and treated with 5 mM diisopropyl fluorophosphate for 30 min at room temperature. The cells were washed once and suspended at 10 8 cells/ml in the same buffer containing 0.5mCi/ml 33 P i and incubated for 90 min at 30°C. The cell suspension was then supplemented with 1 M okadaic acid, 0.5 mM CaCl 2 , and 1 mM MgCl 2 and warmed to 37°C for 3 min before the addition of 1 g/ml phorbol 12-myristate 13-acetate (PMA). After 3 min at 37°C, cells were pelleted by centrifugation and lysed directly in the indicated lysis buffer.
Differentiation of HL60 Cells, Metabolic Labeling, and Activation of the NADPH Oxidase-HL60 cells were subcultured, differentiated in 1.25% dimethyl sulfoxide, and activated as described (1). Cells were washed twice in phosphate-free buffer consisting of 10 mM HEPES, pH 7.4, 137 mM NaCl, 3 mM KCl and cultured in phosphate-free RPMI medium supplemented with 20 mM glucose, 20 mM HEPES, pH 7.4, 1 mM glutamine, and 0.5 mCi/ml 33 P i . After 3 h at 37°C, cells were pretreated with 1 M okadaic acid for 10 min before activation with PMA at a final concentration of 1 g/ml for 3 min at 37°C. In PKC inhibition studies, bisindolylmaleimide (GFX) was added at a final concentration of 5 M to the cells 10 min before activation.
Preparation of Immunoaffinity Sepharose Beads-For immunoprecipitation experiments, antibodies were cross-linked beforehand to protein A-Sepharose beads as described (1) and were stored in NaCl/P i with 0.01% NaN 3 at 4°C until needed.
Immunoprecipitation Assays Using the Anti-p67 phox Antiserum-For each immunoprecipitation assay, cells were harvested, metabolically labeled, and then activated as described above. After the activation step, the cell pellets were resuspended in 600 l of ice-cold Nonidet P-40 lysis buffer consisting of 50 mM HEPES, pH 7.5, 250 mM NaCl, 0.1% Nonidet P-40, 10% glycerol, 1 mM EDTA, 0.5 mM dithiothreitol supplemented with 1 mM diisopropyl fluorophosphate, 10 g/ml leupeptin, 10 mM NaF, 125 nM okadaic acid, 250 M Na 3 VO 4 , and 1 mM p-nitrophenyl phosphate. After a 15-min incubation on ice, the homogenate was centrifuged for 15 min at 20,000 ϫ g. The supernatant was incubated with 30 l of cross-linked p67 phox antibody-protein A-Sepharose beads for no more than 45 min at 4°C. Longer incubation times resulted in partial dephosphorylation of p40 phox . Even with careful control of incubation temperature and the addition of freshly prepared antiphosphatases, no phosphorylation could be visualized with incubation times longer than 2 h. After four 1-ml washes in Nonidet P-40 buffer, the immunocomplexes were solubilized in Laemmli depolymerization buffer and subjected to SDS-PAGE on an 11% acrylamide gel followed by electrotransfer onto a PVDF or nitrocellulose membrane. The membrane was dried and radioactivity detected as above.
In Vitro Proteolytic Digestion-After immunoprecipitation was carried out on metabolically labeled cells, the immunocomplex still linked to the protein A-Sepharose beads was incubated with 2 units of thrombin in 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM CaCl 2 for 1 h at room temperature. Laemmli solubilization buffer was added directly to the bead suspension. Proteins were separated by SDS-PAGE, and this was followed by immunodetection with an anti-p40 phox NH 2 -terminal antiserum and exposure to a PhosphorImager screen.
Two-dimensional Gel Electrophoresis and Western Blot Analysis-33 P i -labeled immunocomplexes were solubilized directly in IEF buffer (9 M urea, 4% CHAPS, 50 mM dithiothreitol, 10 mM spermine base, pH 11). Isoelectric focusing was carried out as described by Rabilloud et al. (21) using Immobiline DryStrips from Amersham Pharmacia Biotech. The second dimension was carried out on a 15-cm long 9% acrylamide gel as described by Laemmli and Favre (22). The two-dimensional gel was electrotransferred to a nitrocellulose membrane. The membrane was blocked for 2 h at room temperature or overnight at 4°C in NaCl/P i buffer containing 0.05% Tween 20 and 1% polyvinylpyrrolidone with an average molecular weight of 40,000 (23) and probed with anti-p40 phox antiserum at a dilution of 1/1,000 in the previous buffer. The presence of primary antibody was detected with protein A coupled to horseradish peroxidase using the enhanced chemiluminescence detection kit.
p40 phox cDNA Constructions-The full-length clone of p40 phox starting at 18 base pairs before the start codon was obtained as described (7) and cloned in the pET-32a plasmid (Invitrogen) downstream of the thioredoxin (Trx) coding sequence and poly-His tag. Directed mutagenesis was carried out using the QuikChange mutagenesis kit from Stratagene. Residues Thr 154 , Thr 211 , Thr 251 , Thr 274 , Ser 315 , and Thr 327 were mutated to alanine using sense and antisense primers containing one mismatch and designed according the manufacturer's specifications.
The double mutant (T154A/S315A) was obtained after a second round of directed mutagenesis on the T154A mutant cDNA. After confirmation by sequencing, the pET-32a plasmids carrying the mutated forms of p40 phox were transfected into competent bacteria, and the fusion protein was expressed and purified as described below.
Expression and Purification of Recombinant p40 phox Trx Fusion Protein-The protease-deficient BL21-DE3(pLysS) Escherichia coli strain was transformed with the pET-32a plasmid carrying the wild-type or mutated forms of p40 phox . An overnight preculture was diluted 10-fold in fresh LB medium containing 100 g/ml ampicillin and incubated at 37°C with agitation for 1 h before induction with 1 mM isopropyl-␤-Dthiogalactopyranoside for 3 h at 30°C. Bacteria were harvested by centrifugation and lysed by sonication in 20 mM HEPES, pH 7.9, 0.5 M NaCl, 10 mM imidazole, supplemented with 1 mM diisopropyl fluorophosphate, and 10 g/ml leupeptin. The homogenate was centrifuged at 300,000 ϫ g for 15 min, and the supernatant was incubated with ProBond resin (Invitrogen) for 1 h at 4°C. The resin was packed into a fast protein liquid chromatography column and washed in 20 mM HEPES, pH 7.9, 0.5 M NaCl, 30 mM imidazole. The fusion protein was eluted with 75 mM imidazole and digested with 20 units of enterokinase/mg of protein in enterokinase buffer (Invitrogen) at 4°C overnight. Digestion products and enterokinase could be separated further on a Mono S column (Amersham Pharmacia Biotech) with an NaCl gradient in 20 mM HEPES, pH 7.9.
In Vitro Phosphorylation Assays-The assays consisted of 1 g of recombinant protein, 20 M ATP, 2 Ci of [␥-32 P]ATP, 10 mM MgCl 2 , either 8-microunits of PKC or 60 ng of casein kinase II (CKII) ␣ subunit (kind gift of Dr. O. Filhol, Laboratory BRCE, CEA-Grenoble, France) in 10 l of 20 mM HEPES, pH 7.5, 100 mM NaCl. In PKC assays, the buffer was supplemented with 0.2 mM CaCl 2 and 100 g/ml phosphatidylserine. Phosphorylation was carried out for 1 h at room temperature. After SDS-PAGE and electrotransfer to a nitrocellulose membrane, incorporation of phosphate was visualized using the PhosphorImager apparatus.
Phosphoamino Acid Analysis and Tryptic Peptide Maps-p40 phox labeled either in vitro by 32 P i or in vivo by 33 P i was detected on the membrane by a specific anti-p40 phox antiserum, and radioactivity was detected using a PhosphorImager apparatus. For phosphoamino acid analysis, the band corresponding to p40 phox was excised from a PVDF membrane, and p40 phox was hydrolyzed in 6 N HCl for 1 h at 110°C. After drying, the hydrolysate was supplemented with phosphoamino acid standards and spotted onto a cellulose thin layer plate. Phosphoamino acids were separated by high voltage electrophoresis in pH 3.5 buffer (24). Standards were stained with ninhydrin, and radioactivity was detected using the PhosphorImager apparatus. For tryptic peptide maps, the nitrocellulose band carrying p40 phox was digested with trypsin, and the resulting peptides were separated by high voltage electrophoresis and chromatography on a cellulose thin layer plate as described in Ref. 24. Phosphopeptides were detected using the Phos-phorImager apparatus (exposure of 24 -72 h). When indicated, phosphopeptide spots were scraped from the cellulose, and phosphoamino acid analysis was carried out as described (24).

RESULTS
Phosphoamino Acid Analysis and Tryptic Peptide Mapping of in Vivo Phosphorylated p40 phox -We had shown previously that p40 phox is phosphorylated during the course of NADPH oxidase activation (1). To identify the amino acids that are modified in tight correlation with the level of superoxide production, we first undertook a phosphoamino acid analysis of in vivo phosphorylated p40 phox . Dimethyl sulfoxide-differentiated HL60 cells were metabolically labeled with 33 P i and activated by PMA. The p67 phox -p47 phox -p40 phox complex was immunoprecipitated using an anti-p67 phox antiserum. The immunoprecipitate was subjected to SDS-PAGE, and the resolved proteins were transferred to a PVDF membrane. p40 phox was identified by Western blotting, and the band carrying p40 phox was excised from the PVDF membrane. Hydrolysis of peptide bonds was achieved by concentrated hydrochloric acid, and phosphoamino acids were separated by high voltage electrophoresis. By comparing the positions of the phosphoamino acid standards stained with ninhydrin with the profile obtained after Phos-phorImager exposure, both phosphoserine and phosphothreonine residues are clearly present in activated p40 phox (Fig. 1).
For tryptic peptide mapping of p40 phox , the band corresponding to p40 phox was excised from the nitrocellulose membrane, and the protein was digested by trypsin in situ. Tryptic peptides were separated by high voltage electrophoresis followed by chromatography. The two-dimensional profile of p40 phox obtained from PMA-activated differentiated HL60 cells is shown in Fig. 2A. Phosphoamino acid analysis of the two major spots (peptides 1 and 2) revealed only phosphothreonine (panel B). Because of the limited amount of radioactivity, the minor spots were not analyzed further in this manner (peptides 3-6).
Proteolytic Analysis of p40 phox Phosphorylated in Vivo-To localize the phosphorylation sites in p40 phox we tested various proteases for their efficiency to digest p40 phox partially. Thrombin proved to be a suitable protease for this study because it breaks p40 phox down into only two products, a 16-kDa NH 2terminal polypeptide and a COOH-terminal 23-kDa polypeptide. After transfer of the two polypeptides to a PVDF membrane, the proteolysis site was identified by NH 2 -terminal sequencing of the 23-kDa product 2 as being Arg 153 in the -Pro-Arg 153 -Thr-site upstream of the p40 phox SH3 domain (data not shown). After metabolic labeling and immunoprecipitation of the p67 phox -p47 phox -p40 phox complex using the anti-p67 phox antiserum, we added thrombin to both the resting complex and the activated complex. The digest was analyzed by Western blot followed by exposure of the blot to a PhosphorImager screen. Panel A in Fig. 3 is the enhanced chemiluminescence image of the blot immunodetected by the NH 2 -terminal anti-p40 phox antiserum. Before digestion, p40 phox was present in equal amounts in both the resting complex (lane 1) and the activated complex (lane 2). The resting and activated complexes were then incubated with thrombin (lanes 3 and 4,  respectively). An extensive digestion of p40 phox is seen in the resting complex (lane 3) with the appearance of the NH 2 -terminal 16-kDa moiety of p40 phox . The presence of the COOHterminal 23-kDa moiety can also be visualized with a COOHterminal anti-p40 phox antibody (not shown), with a greater amount of the COOH terminus in lane 3 than in lane 4. Interestingly, the effect of thrombin on the activated complex is not as extensive (lane 4). For a large part, in the activated complex p40 phox has resisted digestion by thrombin: the NH 2 -terminal 16-kDa moiety is far less immunodetected, whereas the native 40-kDa protein is more abundant than in the resting complex digest (lane 3). The blot was then exposed to a PhosphorImager screen. Panel B shows the incorporation of 33 P i in the resting and activated complexes, before and after digestion with thrombin. Surprisingly, the profile of phosphate incorporation is not modified by thrombin digestion (lane 4 versus lane 2). The NH 2 -terminal 16-kDa and COOH-terminal 23-kDa products immunodetected with the NH 2 -terminal and COOH-terminal anti-p40 phox antisera carry no labeled phosphate. Thus, only nonphosphorylated p40 phox has been digested by thrombin in the resting and activated complexes. This result also explains the extensive digestion of the resting complex versus the activated complex because much more nonphosphorylated p40 phox is present in the resting complex than in the activated complex. We also noted that in vitro phosphorylation of rp40 phox by PKC protected the recombinant protein from digestion by thrombin (not shown). Thus, protection from proteolytic cleavage by thrombin is brought by the addition of phosphate groups on p40 phox alone and does not result from the activation of the other components of the activated complex.
Tryptic Peptide Mapping and Phosphoamino Acid Analysis of p40 phox Phosphorylated in Vitro by CKII and PKC-We had shown previously that both CKII␣ and PKC are able to phosphorylate p40 phox in vitro. This is not surprising because p40 phox holds consensus sites bearing either threonine or serine for both PKC and CKII. We therefore undertook a tryptic peptide mapping of rp40 phox phosphorylated in vitro by either PKC or CKII␣. The aim of this experiment was to assign to the peptide spots from the in vivo phosphorylation map described in a previous section to either a phosphorylation by PKC or by CKII␣, bearing in mind that Thr 251 , Thr 274 , and Thr 327 are located in consensus sites recognized by both PKC and CKII␣.
The tryptic maps illustrated in Figs. 4A and 5A were obtained after phosphorylation by CKII␣ and PKC, respectively. All phosphorylated spots were eluted from the cellulose plates as described in Ref. 24 and subjected to phosphoamino acid analysis. Only serine residues were phosphorylated in vitro by CKII␣, whereas both threonine and serine residues were phosphorylated by PKC (Figs. 4B and 5B). This was confirmed by a direct phosphoamino acid analysis of phosphorylated rp40 phox in both cases. By depositing both samples on the same TLC plate, each phosphorylated spot was well individualized, proving that in vitro PKC and CKII do not phosphorylate the same sites on p40 phox (data not shown). All of these results point to a likely role of PKC in the in vivo phosphorylation of p40 phox during superoxide production.
FIG. 1. Phosphoamino acid analysis of p40 phox immunoprecipitated from differentiated HL60 cells that were activated by PMA. After metabolic labeling and immunoprecipitation of a p67 phox -p47 phox -p40 phox complex as described under "Experimental Procedures," the immunocomplex was separated by SDS-PAGE and transferred to a PVDF membrane. The band carrying p40 phox was immunodetected and excised from the membrane. After hydrolysis of the protein in concentrated hydrochloric acid, phosphoamino acids were separated by high voltage electrophoresis on a TLC cellulose plate. Positions of phosphoamino acid standards are indicated by dotted ovals and are named in the margin (pS, phosphoserine, pT, phosphothreonine; pY, phosphotyrosine).

Two-dimensional Analysis of the Effect of a PKC Inhibitor on
the Isoelectric Focusing Pattern of p40 phox in Vivo in Differentiated HL60 Cells as Well as in Isolated Human Neutrophils-To ascertain that PKC is involved in p40 phox phospho-rylation, we studied the effect of a potent and selective inhibitor of PKCs, GFX, on the two-dimensional profile of p40 phox from PMA-activated differentiated HL60 cells. This study was also carried out with human neutrophils isolated FIG. 2. Tryptic peptide map of p40 phox immunoprecipitated from differentiated HL60 cells that were activated by PMA. p40 phox immobilized on a nitrocellulose membrane was digested by trypsin 24 h at 37°C. Eluted peptides were separated on a TLC cellulose plate by high voltage electrophoresis and chromatography, and the TLC plate was exposed to a PhosphorImager screen (panel A). Peptides 1 and 2 were eluted from the cellulose as described (24), and phosphoamino acid analysis was performed (panel B). The positions of phosphoamino acid standards are indicated.
FIG. 3. Thrombin digestion of p40 phox in the cytosolic phox complex isolated from differentiated HL60 cells. After metabolic labeling, the phox cytosolic complex was immunoprecipitated from 2 ϫ 10 6 resting or PMA-activated cells using the anti-p67 phox antiserum. Half of each immunoprecipitate was incubated with 2 units of thrombin for 1 h at room temperature. The control immunoprecipitate was left in thrombin buffer without the protease. The immunocomplex was then analyzed by Western blot and exposure to a PhosphorImager screen. Panel A shows the immunodetected p40 phox products using an NH 2 -terminal anti-p40 phox antibody. Panel B shows the PhosphorImager image of the membrane. Both panels are images from the same nitrocellulose membrane. Prior incubation with thrombin is indicated with a plus sign in the lower part of the figure.
FIG. 4. Tryptic peptide map and phosphoamino acid analysis of p40 phox phosphorylated in vitro by CKII␣. Recombinant p40 phox was phosphorylated in vitro by CKII␣, resolved by SDS-PAGE, and transferred to a nitrocellulose membrane. After immunodetection with the anti-p40 phox antiserum, the band carrying p40 phox was excised and incubated with trypsin as described. The tryptic peptide map is given in panel A. The arrowed peptides were subjected to phosphoamino acid analysis shown in panel B. Positions of phosphoamino acid standards are indicated on the right. from whole blood and activated by PMA. Identical results were obtained with differentiated HL60 cells and human neutrophils. Only the results obtained with neutrophils are presented here. Fig. 6A gives the 33 P i incorporation profiles of p40 phox isolated from the neutrophil lysate by immunoprecipitation. Fig. 6B gives the corresponding Western blot profiles of p40 phox . In resting neutrophils, p40 phox was present mainly as three species characterized by pI values of 6.6, 6.3, and 6.1, as was already shown in resting differentiated HL60 cells (1). Only the 6.1 species had incorporated labeled phosphate. This suggests that either the 6.3 species has a very low phosphorylation turnover or that its more acidic migration results from a post-translational modification of p40 phox different from phosphorylation yet to be defined. Upon activation by PMA, two additional spots with more acidic pI values of 5.8 and 5.9 were revealed by radiolabeling (panel A) and immunodetection (panel B). PMA-induced phosphorylation of p40 phox was precluded by pretreatment with GFX and concomitantly oxidase activation was abolished (not shown).
The existence of a strong link between the in vivo phosphorylation of p40 phox and the activity of a PKC type kinase can be inferred from the following: 1) p40 phox is phosphorylated upon the addition of PMA, a potent activator of PKC; 2) the phosphorylation of p40 phox is strongly inhibited by GFX; and 3) the tryptic map of p40 phox phosphorylated in vitro by PKC is very similar to that obtained after activation-induced in vivo phosphorylation. This very strong correlation prompted us to mutate all consensus PKC sites in p40 phox and study the in vitro phosphorylation of these mutants by tryptic peptide mapping.
In Vitro Analysis of Mutant Forms of p40 phox -Threonine residues at positions 154, 211, 251, 274, and 327 and Ser 315 were mutated into alanine residues to generate the mutants p40-T154A, p40-T211A, p40-T251A, p40-T274A, p40-T315A, and p40-S315A. The directed mutagenesis was performed directly onto the p40 phox cDNA cloned in a bacterial expression plasmid. Mutants and wild-type p40 phox were expressed as proteins fused to thioredoxin and bearing a polyhistidine tag for easy purification on a nickel affinity column. Fusion proteins were digested with enterokinase to yield the p40 phox protein moiety. After in vitro phosphorylation by PKC in the presence of [␥-32 P]ATP, proteins were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. A tryptic map was carried out on each phosphorylated band. Tryptic peptide maps of the p40-T154A, p40-S315A, and p40-T154A/S315A FIG. 6. Effect of GFX on the isoelectric focusing pattern of immunoprecipitated neutrophil p40 phox . The cytosolic phox complex was immunoprecipitated from isolated 33 P i -radiolabeled human neutrophils as described under "Experimental Procedures." The immunocomplex was subjected to two-dimensional gel electrophoresis and transferred to a nitrocellulose membrane. 33 P i incorporation was visualized by a Phospho-rImager apparatus (panel A), and p40 phox was immunodetected with the NH 2 -terminal anti-p40 phox antiserum (panel B). In panel A is shown the isoelectric focusing pattern of 33 P i -labeled p40 phox in the resting state (0) mutants are shown in Fig. 7. The maps of the other threonine mutant forms of p40 phox (p40-T211A, p40-T251A, p40-T274A, and p40-T327A) are identical to the wild-type p40 phox tryptic map and are not presented. The p40-T154A map shows the most dramatic change of pattern. In particular, the two major spots, namely peptides 1 and 2 indicated by arrows, are absent from this map. It was therefore likely that both major spots containing phosphothreonine were in fact peptides carrying threonine 154. The sequence surrounding Thr 154 , NH 2 -. . . Arg-Thr 154 -Arg-Lys . . . -COOH is rich in arginine and lysine residues. The random attack by trypsin at any of these peptide bonds is expected to produce intermediate small peptides (24) such as TR and TRK. The di-and tripeptides TR and TRK synthesized in a phosphorylated form were used as standards in tryptic peptide maps. Comparison of the ninhydrin-stained TLC plates and PhosphorImager images of the same plates showed that the synthetic peptides comigrated with the major radiolabeled spots (Fig. 8), confirming that peptides 1 and 2 correspond to phospho-TRK and phospho-TR respectively.
The p40-S315A mutant map compared with the p40 phox wildtype map (Fig. 7) shows that a minor spot has disappeared. This minor spot corresponds to peptide 3 in Fig. 5 and is present in all threonine mutant forms of p40 phox . This spot had been identified previously as a phosphoserine-containing peptide (Fig. 5B). This confirms that PKC phosphorylates p40 phox on serine 315. This spot was also present in the tryptic peptide map of in vivo phosphorylated p40 phox (Fig. 3, peptide 3).
The presence of minor spots in the double mutant p40-T154A/S315A map points to other possible phosphorylated residues on p40 phox yet to be identified. In particular, phosphoserine-containing peptides 4 and 6 point to a phosphorylation site on a serine distinct from serine 315 because these spots do not disappear in the p40-S315A tryptic map. DISCUSSION This report provides strong evidence that p40 phox is phosphorylated by a PKC type kinase during NADPH oxidase activation. In a previous report we had put forward a strong correlation between the phosphorylation state of p40 phox and the level of superoxide production in differentiated HL60 cells stimulated by PMA or fMet-Leu-Phe-Lys. Here we show that p40 phox is phosphorylated in an identical manner in human neutrophils stimulated by PMA and that p40 phox phosphorylation and superoxide production are inhibited by GFX, a potent inhibitor of PKCs. Finally, tryptic peptide mapping of in vivo and in vitro phosphorylated p40 phox points to a role of a PKC type kinase in the activation-induced phosphorylation of p40 phox . The tryptic phosphorylation pattern obtained with differentiated HL60 cells stimulated by PMA is identical to the patterns obtained either with isolated neutrophils stimulated by PMA or with differentiated HL60 cells stimulated by formyl peptide at micromolar concentrations (not shown), suggesting that the activation of the signaling pathway through cell surface receptors activates a PKC-type kinase responsible for p40 phox phosphorylation.
Two in vivo phosphorylation sites have been identified on p40 phox as being threonine 154 localized 20 residues upstream of the SH3 domain and serine 315 at the COOH terminus of p40 phox .
Thr 154 is in a basic region of p40 phox NH 2 -Arg-Arg-Leu-Arg-Pro-Arg-Thr 154 -Arg-Lys-Val-Lys-COOH which is excessively sensitive to proteolytic cleavage. 7 residues out of the 11 in the above sequence are basic residues. Digestion of p40 phox by thrombin cuts the protein at arginine 153 immediately adjacent to the major phosphorylation site at threonine 154. Digestion by endoproteinase-Lys-C cuts the protein in the same region (not shown). This region is adjacent to the SH3 domain of p40 phox (residues 175-224). This stretch could therefore act as a very exposed hinge between the NH 2 terminus and the SH3-containing COOH terminus. The presence of phosphate on Thr 154 inhibits the digestion by thrombin at residue Arg 153 , possibly by steric hindrance. The negative charges brought by the phosphate residue could also trigger a change of conformation of p40 phox which could participate in the transition of the cytosolic phox complex from an inactive to an active state.
The second phosphorylation site identified on p40 phox is serine 315, localized in the COOH terminus of the protein which has been shown to interact with the inter-SH3 domain of p67 phox (6,7). This second site might therefore play a strategic function in the p40 phox /p67 phox interaction. Preliminary results from two hybrid studies appear to indicate that mutagenesis of the serine 315 or threonine 154 to alanine or aspartate did not change the interaction of p40 phox with either p47 phox or p67 phox (not shown). A more subtle regulation of the spatial and temporal organization of the three phox partners is probably brought by a hierarchy of phosphorylation on Thr 154 and Ser 315 as well as on other phosphorylation sites yet to be determined. The in vivo and in vitro tryptic phosphorylation maps present phosphorylated peptides that are still present in the tryptic map of the double mutant T154A/S315A. Two of these sites were shown to be phosphorylated in vitro by PKC on serine (peptides 4 and 6). We are now searching for a nonconsensus serine site for PKC on p40 phox . FIG. 8. Identification of the major spots as phospho-TR and phospho-TRK. A tryptic peptide map of wild-type rp40 phox phosphorylated in vitro by PKC was run with 1 g of the phospho-TR or phospho-TRK synthetic peptide. Ninhydrin staining of the TLC plates is shown underneath the corresponding Phosphor-Imager images.