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• W2064811541 abstract "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 p67phox(phagocyte oxidase), p47phox, and p40phox, which translocate to the membrane upon activation. In a previous paper, we reported that p40phox 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 p40phox, we show that p40phox 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 p40phox in whole cell lysates point to a role of a PKC type kinase in the phosphorylation of p40phox. Directed mutagenesis of all PKC consensus sites enable us to conclude that Thr154 and Ser315 in p40phox 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 p67phox(phagocyte oxidase), p47phox, and p40phox, which translocate to the membrane upon activation. In a previous paper, we reported that p40phox 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 p40phox, we show that p40phox 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 p40phox in whole cell lysates point to a role of a PKC type kinase in the phosphorylation of p40phox. Directed mutagenesis of all PKC consensus sites enable us to conclude that Thr154 and Ser315 in p40phox are phosphorylated during activation of the NADPH oxidase. phagocyte oxidase src homology 3 protein kinase C polyvinylidene difluoride phorbol 12-myristate 13-acetate 3-[N-(dimethylamino)propyl-3-indolyl]-4-[3-indolyl]maleimide polyacrylamide gel electrophoresis 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid casein kinase II. 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 flavocytochromeb and a small G protein Rac as well as cytosolic factors p67phox1(phagocyte oxidase), p47phox, and p40phox (for review, see Refs. 2Morel F. Doussiere J. Vignais P.V. Eur. J. Biochem. 1991; 201: 523-546Crossref PubMed Scopus (525) Google Scholar, 3Thrasher A.J. Keep N.H. Wientjes F. Segal A.W. Biochim. Biophys. Acta. 1994; 1227: 1-24Crossref PubMed Scopus (212) Google Scholar, 4Henderson L.M. Chappel J.B. Biochim. Biophys. Acta. 1996; 1273: 87-107Crossref PubMed Scopus (187) Google Scholar). 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 (5Finan P. Shimizu Y. Gout I. Hsuan J. Truong O. Butcher C. Bennett P. Waterfield M.D. Kellie S. J. Biol. Chem. 1994; 269: 13752-13755Abstract Full Text PDF PubMed Google Scholar, 6Fuchs A. Dagher M.C. Faure J. Vignais P.V. Biochim. Biophys. Acta. 1996; 1312: 39-47Crossref PubMed Scopus (49) Google Scholar, 7Fuchs A. Dagher M.C. Vignais P.V. J. Biol. Chem. 1995; 270: 5695-5697Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 8Leto T.L. Adams A.G. de Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (243) Google Scholar, 9Leusen J.H. Fluiter K. Hilarius P.M. Roos D. Verhoeven A.J. Bolscher B.G. J. Biol. Chem. 1995; 270: 11216-11221Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 10Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (255) Google Scholar), as well as phosphorylation events on p47phox, p67phox, and p40phox (1Fuchs A. Bouin A.P. Rabilloud T. Vignais P.V. Eur. J. Biochem. 1997; 249: 531-539Crossref PubMed Scopus (36) Google Scholar, 11Heyworth P.G. Shrimpton C.F. Segal A.W. Biochem. J. 1989; 260: 243-248Crossref PubMed Scopus (68) Google Scholar, 12Dusi S. Della Bianca V. Grzeskowiak M. Rossi F. Biochem. J. 1993; 290: 173-178Crossref PubMed Scopus (107) Google Scholar). Translocation of the three phox proteins is dependent upon the presence of flavocytochromeb in the membrane (13Wientjes F.B. Hsuan J.J. Totty N.F. Segal A.W. Biochem. J. 1993; 296: 557-561Crossref PubMed Scopus (258) Google Scholar), and the translocation of p40phox appears to be p47phox-dependent and is mediated by p67phox (14Dusi S. Donini M. Rossi F. Biochem. J. 1996; 314: 409-412Crossref PubMed Scopus (110) Google Scholar). Interestingly, the SH3 domain of p40phox down-regulates activation of the NADPH oxidase (15Sathyamoorthy M. de Mendez I. Adams A.G. Leto T.L. J. Biol. Chem. 1997; 272: 9141-9146Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). As shown by two hybrid studies (6Fuchs A. Dagher M.C. Faure J. Vignais P.V. Biochim. Biophys. Acta. 1996; 1312: 39-47Crossref PubMed Scopus (49) Google Scholar, 7Fuchs A. Dagher M.C. Vignais P.V. J. Biol. Chem. 1995; 270: 5695-5697Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) and by in vitro binding assays (16Wientjes F.B. Panayotou G. Reeves E. Segal A.W. Biochem. J. 1996; 317: 919-924Crossref PubMed Scopus (75) Google Scholar, 17Tsunawaki S. Kagara S. Yoshikawa K. Yoshida L.S. Kuratsuji T. Namiki H. J. Exp. Med. 1996; 184: 893-902Crossref PubMed Scopus (79) Google Scholar, 18Ito T. Nakamura R. Sumimoto H. Takeshige K. Sakaki Y. FEBS Lett. 1996; 385: 229-232Crossref PubMed Scopus (51) Google Scholar), p47phox interacts with both p40phox and p67phox through its proline-rich COOH-terminal region (6Fuchs A. Dagher M.C. Faure J. Vignais P.V. Biochim. Biophys. Acta. 1996; 1312: 39-47Crossref PubMed Scopus (49) Google Scholar, 18Ito T. Nakamura R. Sumimoto H. Takeshige K. Sakaki Y. FEBS Lett. 1996; 385: 229-232Crossref PubMed Scopus (51) Google Scholar). It seems plausible that some modification occurs during oxidase activation which could probably change the specificity of the proline-rich domain of p47phox for either the SH3 domain of p40phox or the COOH-terminal SH3 domain of p67phox. This modification could be the phosphorylation of p47phox 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 p40phox phosphorylation is strongly correlated with that of superoxide production (1Fuchs A. Bouin A.P. Rabilloud T. Vignais P.V. Eur. J. Biochem. 1997; 249: 531-539Crossref PubMed Scopus (36) Google Scholar), and we have postulated that p40phox phosphorylation could therefore play a critical role in the rearrangement of the ternary complex consisting of p47phox, p67phox, and p40phox during the respiratory burst. In this report we identify activation-induced phosphorylation sites on p40phox and describe experimental data supporting a critical role of the state of phosphorylation of these residues on the structure of the cytosolic activating complex. 33Pi at 4,000Ci/mmol was purchased from NEN Life Science Products. Protein A-Sepharose beads, the enhanced chemiluminescence detection kit, and [γ-32P]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. Anti-p40phox antisera were raised in rabbits using a synthetic peptide corresponding to the NH2-terminal 18 amino acid residues of p40phox (1Fuchs A. Bouin A.P. Rabilloud T. Vignais P.V. Eur. J. Biochem. 1997; 249: 531-539Crossref PubMed Scopus (36) Google Scholar). Anti-p67phox antiserum was obtained using purified recombinant p67phox protein expressed in the baculovirus/insect cell system as described (19Fuchs A. Dagher M.C. Jouan A. Vignais P.V. Eur. J. Biochem. 1994; 226: 587-595Crossref PubMed Scopus (38) Google Scholar). 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-p40phox antiserum was a kind gift from Prof. Segal (University College, London). Neutrophils were isolated from buffy coats as described (20Morel F. Cholley L.C. Dianoux A.C. Renversez J.C. Anthony D. Revol C. Boulay F. Gagnon J. Vignais P.V. Inflammation. 1992; 16: 325-341Crossref PubMed Scopus (9) Google Scholar). Neutrophils were suspended at 5 × 107 cells/ml in 10 mmHEPES, pH 7.4, containing 137 mm NaCl, 0.8 mmMgCl2, 5.4 mm KCl, and 5.6 mmglucose and treated with 5 mm diisopropyl fluorophosphate for 30 min at room temperature. The cells were washed once and suspended at 108 cells/ml in the same buffer containing 0.5mCi/ml 33Pi and incubated for 90 min at 30 °C. The cell suspension was then supplemented with 1 μm okadaic acid, 0.5 mm CaCl2, and 1 mm MgCl2 and warmed to 37 °C for 3 min before the addition of 1 μg/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. HL60 cells were subcultured, differentiated in 1.25% dimethyl sulfoxide, and activated as described (1Fuchs A. Bouin A.P. Rabilloud T. Vignais P.V. Eur. J. Biochem. 1997; 249: 531-539Crossref PubMed Scopus (36) Google Scholar). Cells were washed twice in phosphate-free buffer consisting of 10 mm HEPES, pH 7.4, 137 mm NaCl, 3 mmKCl 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 33Pi. After 3 h at 37 °C, cells were pretreated with 1 μm okadaic acid for 10 min before activation with PMA at a final concentration of 1 μg/ml for 3 min at 37 °C. In PKC inhibition studies, bisindolylmaleimide (GFX) was added at a final concentration of 5 μm to the cells 10 min before activation. For immunoprecipitation experiments, antibodies were cross-linked beforehand to protein A-Sepharose beads as described (1Fuchs A. Bouin A.P. Rabilloud T. Vignais P.V. Eur. J. Biochem. 1997; 249: 531-539Crossref PubMed Scopus (36) Google Scholar) and were stored in NaCl/Pi with 0.01% NaN3 at 4 °C until needed. 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 μl 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 mmdithiothreitol supplemented with 1 mm diisopropyl fluorophosphate, 10 μg/ml leupeptin, 10 mm NaF, 125 nm okadaic acid, 250 μmNa3VO4, and 1 mm p-nitrophenyl phosphate. After a 15-min incubation on ice, the homogenate was centrifuged for 15 min at 20,000 ×g. The supernatant was incubated with 30 μl of cross-linked p67phox antibody-protein A-Sepharose beads for no more than 45 min at 4 °C. Longer incubation times resulted in partial dephosphorylation of p40phox. 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. 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 CaCl2 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-p40phoxNH2-terminal antiserum and exposure to a PhosphorImager screen. 33Pi-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. (21Rabilloud T. Valette C. Lawrence J.J. Electrophoresis. 1994; 15: 1552-1558Crossref PubMed Scopus (319) Google Scholar) 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 (22Laemmli U.K. Favre M. J. Mol. Biol. 1973; 80: 575-599Crossref PubMed Scopus (3024) Google Scholar). 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/Pi buffer containing 0.05% Tween 20 and 1% polyvinylpyrrolidone with an average molecular weight of 40,000 (23Haycock J.W. Anal. Biochem. 1993; 208: 397-399Crossref PubMed Scopus (82) Google Scholar) and probed with anti-p40phoxantiserum 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. The full-length clone of p40phox starting at 18 base pairs before the start codon was obtained as described (7Fuchs A. Dagher M.C. Vignais P.V. J. Biol. Chem. 1995; 270: 5695-5697Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) 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 Thr154, Thr211, Thr251, Thr274, Ser315, and Thr327 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 p40phox were transfected into competent bacteria, and the fusion protein was expressed and purified as described below. The protease-deficient BL21-DE3(pLysS)Escherichia coli strain was transformed with the pET-32a plasmid carrying the wild-type or mutated forms of p40phox. An overnight preculture was diluted 10-fold in fresh LB medium containing 100 μg/ml ampicillin and incubated at 37 °C with agitation for 1 h before induction with 1 mm isopropyl-β-d-thiogalactopyranoside for 3 h at 30 °C. Bacteria were harvested by centrifugation and lysed by sonication in 20 mm HEPES, pH 7.9, 0.5m NaCl, 10 mm imidazole, supplemented with 1 mm diisopropyl fluorophosphate, and 10 μg/ml leupeptin. The homogenate was centrifuged at 300,000 × 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. The assays consisted of 1 μg of recombinant protein, 20 μm ATP, 2 μCi of [γ-32P]ATP, 10 mm MgCl2, either 8-microunits of PKC or 60 ng of casein kinase II (CKII) α subunit (kind gift of Dr. O. Filhol, Laboratory BRCE, CEA-Grenoble, France) in 10 μl of 20 mm HEPES, pH 7.5, 100 mm NaCl. In PKC assays, the buffer was supplemented with 0.2 mmCaCl2 and 100 μg/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. p40phox labeled either in vitro by 32Pi or in vivo by33Pi was detected on the membrane by a specific anti-p40phox antiserum, and radioactivity was detected using a PhosphorImager apparatus. For phosphoamino acid analysis, the band corresponding to p40phoxwas excised from a PVDF membrane, and p40phox was hydrolyzed in 6 nHCl 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 (24Van der Geer P. Luo K. Sefton B.M. Hunter T. Protein Phosphorylation: A Practical Approach. IRL Press, New York1993: 31-59Google Scholar). Standards were stained with ninhydrin, and radioactivity was detected using the PhosphorImager apparatus. For tryptic peptide maps, the nitrocellulose band carrying p40phox 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. 24Van der Geer P. Luo K. Sefton B.M. Hunter T. Protein Phosphorylation: A Practical Approach. IRL Press, New York1993: 31-59Google Scholar. Phosphopeptides were detected using the PhosphorImager apparatus (exposure of 24–72 h). When indicated, phosphopeptide spots were scraped from the cellulose, and phosphoamino acid analysis was carried out as described (24Van der Geer P. Luo K. Sefton B.M. Hunter T. Protein Phosphorylation: A Practical Approach. IRL Press, New York1993: 31-59Google Scholar). Peptides phospho-TRK and phospho-TR were synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) precursors on an Applied Biosystems 432A peptide synthesizer in J. Garin's laboratory (Chimie des Protéines, CEA-Grenoble). Cleavage from the resin and removal of side chains were performed by treatment with 90% trifluoroacetic acid, 5% thioanisole, and 5% ethanedithiol. We had shown previously that p40phox is phosphorylated during the course of NADPH oxidase activation (1Fuchs A. Bouin A.P. Rabilloud T. Vignais P.V. Eur. J. Biochem. 1997; 249: 531-539Crossref PubMed Scopus (36) Google Scholar). 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 vivophosphorylated p40phox. Dimethyl sulfoxide-differentiated HL60 cells were metabolically labeled with33Pi and activated by PMA. The p67phox-p47phox-p40phoxcomplex was immunoprecipitated using an anti-p67phox antiserum. The immunoprecipitate was subjected to SDS-PAGE, and the resolved proteins were transferred to a PVDF membrane. p40phoxwas identified by Western blotting, and the band carrying p40phox 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 PhosphorImager 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. 2 A. 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). 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 NH2-terminal polypeptide and a COOH-terminal 23-kDa polypeptide. After transfer of the two polypeptides to a PVDF membrane, the proteolysis site was identified by NH2-terminal sequencing of the 23-kDa product 2J. Garin, unpublished observation. as being Arg153 in the -Pro-Arg153-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 NH2-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 NH2-terminal 16-kDa moiety of p40 phox . The presence of the COOH-terminal 23-kDa moiety can also be visualized with a COOH-terminal anti-p40 phox antibody (not shown), with a greater amount of the COOH terminus in lane 3than 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 NH2-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 33Pi 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 NH2-terminal 16-kDa and COOH-terminal 23-kDa products immunodetected with the NH2-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 vitrophosphorylation 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. We had shown previously that both CKIIα 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 vitroby either PKC or CKIIα. 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 CKIIα, bearing in mind that Thr251, Thr274, and Thr327 are located in consensus sites recognized by both PKC and CKIIα. The tryptic maps illustrated in Figs. 4 A and 5 A were obtained after phosphorylation by CKIIα and PKC, respectively. All phosphorylated spots were eluted from the cellulose plates as described in Ref. 24Van der Geer P. Luo K. Sefton B.M. Hunter T. Protein Phosphorylation: A Practical Approach. IRL Press, New York1993: 31-59Google Scholar and subjected to phosphoamino acid analysis. Only serine residues were phosphorylated in vitro by CKIIα, whereas both threonine and serine residues were phosphorylated by PKC (Figs. 4 B and 5 B). 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 vivophosphorylation of p40 phox during superoxide production.Figure 5Tryptic peptide map and phosphoamino acid analysis of p40 phox phosphorylated in vitro by PKC. Recombinant p40 phox was phosphorylatedin vitro by PKC, 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 as shown in panel B. The positions of phosphoamino acid standards are indicated on theright.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To ascertain that PKC is involved in p40 phox phosphorylation, we studied the effect of a potent and selective inhibitor of PKCs, GFX, on the two-dimensional profile of p40phox from PMA-activated differentiated HL60 cells. This study was also carried out with human neutrophils isolated 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. 6 A gives the 33Pi incorporation profiles of p40phox isolated from the neutrophil lysate by immunoprecipitation. Fig. 6 B gives the corresponding Western blot profiles of p40phox. In resting neutrophils, p40phox 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 (1Fuchs A. Bouin A.P. Rabilloud T. Vignais P.V. Eur. J. Biochem. 1997; 249: 531-539Crossref PubMed Scopus (36) Google Scholar). 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 p40phox 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 p40phox was precluded by pretreatment with GFX and concomitantly oxidase activation was abolished (not shown). The existence of a strong link between the in vivophosphorylation of p40phox and the activity of a PKC type kinase can be inferred from the following: 1) p40phox is phosphorylated upon the addition of PMA, a potent activator of PKC; 2) the phosphorylation of p40phox is strongly inhibited by GFX; and 3) the tryptic map of p40phox phosphorylatedin 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 p40phox and study the in vitrophosphorylation of these mutants by tryptic peptide mapping. Threonine residues at positions 154, 211, 251, 274, and 327 and Ser315 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 p40phox cDNA cloned in a bacterial expression plasmid. Mutants and wild-type p40phox 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 p40phox protein moiety. Afterin vitro phosphorylation by PKC in the presence of [γ-32P]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 mutants are shown in Fig. 7. The maps of the other threonine mutant forms of p40phox (p40-T211A, p40-T251A, p40-T274A, and p40-T327A) are identical to the wild-type p40phox 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 byarrows, 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 Thr154, NH2- … Arg-Thr154-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 (24Van der Geer P. Luo K. Sefton B.M. Hunter T. Protein Phosphorylation: A Practical Approach. IRL Press, New York1993: 31-59Google Scholar) 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.Figure 8Identification of the major spots as phospho-TR and phospho-TRK. A tryptic peptide map of wild-type rp40phox phosphorylated in vitroby PKC was run with 1 μg of the phospho-TR or phospho-TRK synthetic peptide. Ninhydrin staining of the TLC plates is shown underneath the corresponding PhosphorImager images.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The p40-S315A mutant map compared with the p40phox wild-type 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 p40phox. This spot had been identified previously as a phosphoserine-containing peptide (Fig. 5 B). This confirms that PKC phosphorylates p40phox on serine 315. This spot was also present in the tryptic peptide map of in vivo phosphorylated p40phox (Fig. 3, peptide 3). The presence of minor spots in the double mutant p40-T154A/S315A map points to other possible phosphorylated residues on p40phox 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. This report provides strong evidence that p40phox 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 p40phox and the level of superoxide production in differentiated HL60 cells stimulated by PMA or fMet-Leu-Phe-Lys. Here we show that p40phoxis phosphorylated in an identical manner in human neutrophils stimulated by PMA and that p40phoxphosphorylation and superoxide production are inhibited by GFX, a potent inhibitor of PKCs. Finally, tryptic peptide mapping of in vivo and in vitro phosphorylated p40phox points to a role of a PKC type kinase in the activation-induced phosphorylation of p40phox. 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 p40phoxphosphorylation. Two in vivo phosphorylation sites have been identified on p40phox as being threonine 154 localized 20 residues upstream of the SH3 domain and serine 315 at the COOH terminus of p40phox. Thr154 is in a basic region of p40phoxNH2-Arg-Arg-Leu-Arg-Pro-Arg-Thr154 -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 p40phox 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 p40phox (residues 175–224). This stretch could therefore act as a very exposed hinge between the NH2terminus and the SH3-containing COOH terminus. The presence of phosphate on Thr154 inhibits the digestion by thrombin at residue Arg153, possibly by steric hindrance. The negative charges brought by the phosphate residue could also trigger a change of conformation of p40phox which could participate in the transition of the cytosolic phox complex from an inactive to an active state. The second phosphorylation site identified on p40phox 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 (6Fuchs A. Dagher M.C. Faure J. Vignais P.V. Biochim. Biophys. Acta. 1996; 1312: 39-47Crossref PubMed Scopus (49) Google Scholar, 7Fuchs A. Dagher M.C. Vignais P.V. J. Biol. Chem. 1995; 270: 5695-5697Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). This second site might therefore play a strategic function in the p40phox/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 p40phox with either p47phox 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 Thr154 and Ser315 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 p40phox. We thank Dr. O. Filhol for recombinant CKIIα and Drs. J. Garin and A. Chapel for the synthesis of the phosphodi- and tripeptides." @default.
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• W2064811541 title "p40 Is Phosphorylated on Threonine 154 and Serine 315 during Activation of the Phagocyte NADPH Oxidase" @default.
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