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• W2054526825 abstract "Using a phosphorylation-dependent cell-free system to study NADPH oxidase activation (McPhail, L. C., Qualliotine-Mann, D., and Waite, K. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7931–7935), we previously showed that p47 phox, a cytosolic NADPH oxidase component, is phosphorylated. Now, we show that p22 phox , a subunit of the NADPH oxidase component flavocytochrome b 558, also is phosphorylated. Phosphorylation is selectively activated by phosphatidic acid (PA) versus other lipids and occurs on a threonine residue in p22 phox . We identified two protein kinase families capable of phosphorylating p22 phox : 1) a potentially novel, partially purified PA-activated protein kinase(s) known to phosphorylate p47 phox and postulated to mediate the phosphorylation-dependent activation of NADPH oxidase by PA and 2) conventional, but not novel or atypical, isoforms of protein kinase C (PKC). In contrast, all classes of PKC isoforms could phosphorylate p47 phox . In a gel retardation assay both the phosphatidic acid-dependent kinase and conventional PKC isoforms phosphorylated all molecules of p22 phox . These findings suggest that phosphorylation of p22 phox by conventional PKC and/or a novel PA-activated protein kinase regulates the activation/assembly of NADPH oxidase. Using a phosphorylation-dependent cell-free system to study NADPH oxidase activation (McPhail, L. C., Qualliotine-Mann, D., and Waite, K. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7931–7935), we previously showed that p47 phox, a cytosolic NADPH oxidase component, is phosphorylated. Now, we show that p22 phox , a subunit of the NADPH oxidase component flavocytochrome b 558, also is phosphorylated. Phosphorylation is selectively activated by phosphatidic acid (PA) versus other lipids and occurs on a threonine residue in p22 phox . We identified two protein kinase families capable of phosphorylating p22 phox : 1) a potentially novel, partially purified PA-activated protein kinase(s) known to phosphorylate p47 phox and postulated to mediate the phosphorylation-dependent activation of NADPH oxidase by PA and 2) conventional, but not novel or atypical, isoforms of protein kinase C (PKC). In contrast, all classes of PKC isoforms could phosphorylate p47 phox . In a gel retardation assay both the phosphatidic acid-dependent kinase and conventional PKC isoforms phosphorylated all molecules of p22 phox . These findings suggest that phosphorylation of p22 phox by conventional PKC and/or a novel PA-activated protein kinase regulates the activation/assembly of NADPH oxidase. phosphatidic acid chronic granulomatous disease 1,2-dicapryl-sn-glycero-3-phosphate glutathioneS-transferase protein kinase C phosphatidylserine polyacrylamide gel electrophoresis monoclonal antibody Phagocytic cells are the first line of defense against invading microorganisms (reviewed in Ref. 1McPhail L.C. Harvath L. Abramson J.S. Wheeler J.G. The Neutrophil. Oxford University Press, Oxford1993: 63-107Google Scholar). This defense is achieved, in part, by the respiratory burst, leading to the production of superoxide anion, which along with its metabolic products are toxic to invading microorganisms. The respiratory burst is mediated by the multicomponent enzyme complex, the NADPH oxidase. The two membrane-bound components (p22 phox and gp91 phox) form the heterodimeric flavocytochrome b 558 (2Parkos C.A. Allen R.A. Cochrane C.G. Jesaitis A.J. J. Clin. Invest. 1987; 80: 732-742Crossref PubMed Scopus (314) Google Scholar). The flavocytochrome contains a putative NADPH binding site, FAD, and two hemes; thus, it possesses all of the electron machinery required to transfer two electrons from NADPH to molecular oxygen (3Rotrosen D. Yeung C.L. Leto T.L. Malech H.L. Kwong C.H. Science. 1992; 256: 1459-1462Crossref PubMed Scopus (315) Google Scholar, 4Sumimoto H. Sakamoto N. Nozaki M. Sakaki Y. Takeshige K. Minakami S. Biochem. Biophys. Res. Commun. 1992; 186: 1368-1375Crossref PubMed Scopus (111) Google Scholar, 5Segal A.W. West I. Wientjes F. Nugent J.H.A. Chavan A.J. Haley B. Garcia R.C. Rosen H. Scrace G. Biochem. J. 1992; 284: 781-788Crossref PubMed Scopus (291) Google Scholar, 6Quinn M.T. Mullen M.L. Jesaitis A.J. J. Biol. Chem. 1992; 267: 7303-7309Abstract Full Text PDF PubMed Google Scholar, 7Taylor W.R. Jones D.T. Segal A.W. Protein Sci. 1993; 2: 1675-1685Crossref PubMed Scopus (108) Google Scholar, 8Koshkin V. Biochim. Biophys. Acta. 1995; 1232: 225-229Crossref PubMed Scopus (14) Google Scholar, 9Cross A.R. Rae J. Curnutte J.T. J. Biol. Chem. 1995; 270: 17075-17077Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 10Escriou V. Laporte F. Vignais P.V. Biochem. Biophys. Res. Commun. 1996; 219: 930-935Crossref PubMed Scopus (5) Google Scholar). The three required cytosolic components are Rac-GTP and the phosphoproteins p47 phox and p67 phox (11El Benna J. Dang P.M.-C. Gaudry M. Fay M. Morel F. Hakim J. Gougerot-Pocidalo M.-A. J. Biol. Chem. 1997; 272: 17204-17208Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) (reviewed in Ref. 12DeLeo F.R. Quinn M.T. J. Leukocyte Biol. 1996; 60: 677-691Crossref PubMed Scopus (456) Google Scholar). Activation of phagocytic cells leads to the translocation of the cytosolic components to the membrane, where they interact with flavocytochrome b 558 (reviewed in Ref. 12DeLeo F.R. Quinn M.T. J. Leukocyte Biol. 1996; 60: 677-691Crossref PubMed Scopus (456) Google Scholar). Assembly of the NADPH oxidase components is required for activation of electron flow, possibly by inducing a conformational change within the flavocytochrome b 558component. The signaling mechanisms leading to assembly and activation of NADPH oxidase are not well understood. When a ligand binds its receptor on the neutrophil membrane, a cascade of events is initiated (reviewed in Ref. 1McPhail L.C. Harvath L. Abramson J.S. Wheeler J.G. The Neutrophil. Oxford University Press, Oxford1993: 63-107Google Scholar) that includes activation of phospholipases, generation of lipid second messengers, and the activation of protein kinases. One of the phospholipases activated in response to many physiological agonists of neutrophils is phospholipase D (13Cockcroft S. Biochim. Biophys. Acta. 1984; 795: 37-46Crossref PubMed Scopus (100) Google Scholar, 14Agwu D.E. McPhail L.C. Chabot M.C. Daniel L.W. Wykle R.L. McCall C.E. J. Biol. Chem. 1989; 264: 1405-1413Abstract Full Text PDF PubMed Google Scholar, 15Billah N.M. Eckel S. Mullmann T.J. Egan R.W. Siegel M.I. J. Biol. Chem. 1989; 264: 17069-17077Abstract Full Text PDF PubMed Google Scholar, 16Olson S.C. Tyagi S.R. Lambeth J.D. FEBS Lett. 1990; 272: 209-213Crossref PubMed Scopus (31) Google Scholar, 17Bourgoin S. Borgeat P. Poubelle P.E. Agents Actions. 1991; 34: 32-34Crossref PubMed Scopus (10) Google Scholar, 18Kanaho Y. Kanoh H. Saitoh K. Nozawa Y. J. Immunol. 1991; 146: 3536-3541PubMed Google Scholar, 19Bauldry S.A. Bass D.A. Cousart S.L. McCall C.E. J. Biol. Chem. 1991; 266: 4173-4179Abstract Full Text PDF PubMed Google Scholar, 20Della Bianca V. Grzeskowiak M. Lissandrini D. Rossi F. Biochem. Biophys. Res. Commun. 1991; 177: 948-955Crossref PubMed Scopus (28) Google Scholar, 21Fallman M. Gullberg M. Hellberg C. Anderson T. J. Biol. Chem. 1992; 267: 2656-2663Abstract Full Text PDF PubMed Google Scholar, 22Cockcroft S. Biochim. Biophys. Acta. 1992; 1113: 135-160Crossref PubMed Scopus (243) Google Scholar, 23Naccache P.H. Bourgoin S. Plante E. Roberge C.J. DeMedicis R. Lussier A. Poubelle P.E. Arthritis Rheum. 1993; 36: 117-125Crossref PubMed Scopus (33) Google Scholar, 24Sozzani S. Agwu D.E. Ellenburg M.D. Locati M. Rieppi M. Rojas A. Mantovani A. McPhail L.C. Blood. 1994; 84: 3895-3901Crossref PubMed Google Scholar, 25Suchard S.J. Nakamura T. Abe A. Shayman J.A. Boxer L.A. J. Biol. Chem. 1994; 269: 8063-8068Abstract Full Text PDF PubMed Google Scholar, 26Meshulam T. Billah M.M. Eckel S. Griendling K.K. Diamond R.D. J. Leukocyte Biol. 1995; 57: 842-850Crossref PubMed Scopus (11) Google Scholar, 27L'Heureux G.P. Bourgoin S. Jean N. McColl S.R. Naccache P.H. Blood. 1995; 85: 522-531Crossref PubMed Google Scholar). Phospholipase D cleaves neutrophil phospholipids to form phosphatidic acid (PA),1 which can then be converted to diacylglycerol by PA phosphohydrolase. Numerous studies have correlated phospholipase D activation/PA production and NADPH oxidase activation (19Bauldry S.A. Bass D.A. Cousart S.L. McCall C.E. J. Biol. Chem. 1991; 266: 4173-4179Abstract Full Text PDF PubMed Google Scholar, 28Korchak H.M. Vosshall L.B. Haines K.A. Wilkenfeld C. Lundquist K.F. Weissmann G. J. Biol. Chem. 1988; 263: 11098-11105Abstract Full Text PDF PubMed Google Scholar, 29Bonser R.W. Thompson N.T. Randall R.W. Garland L.G. Biochem. J. 1989; 264: 617-620Crossref PubMed Scopus (166) Google Scholar, 30Rossi F. Grzeskowiak M. Della Bianca V. Calzetti F. Gandini G. Biochem. Biophys. Res. Commun. 1990; 168: 320-327Crossref PubMed Scopus (122) Google Scholar, 31Agwu D.E. McPhail L.C. Sozzani S. Bass D.A. McCall C.E. J. Clin. Invest. 1991; 88: 531-539Crossref PubMed Scopus (147) Google Scholar, 32Bauldry S.A. Elsey K.L. Bass D.A. J. Biol. Chem. 1992; 267: 25141-25152Abstract Full Text PDF PubMed Google Scholar, 33Bauldry S.A. Nasrallah V.N. Bass D.A. J. Biol. Chem. 1992; 267: 323-330Abstract Full Text PDF PubMed Google Scholar, 34Hsu M.F. Raung S.L. Tsao L.T. Kuo S.C. Wang J.P. Br. J. Pharmacol. 1997; 120: 917-925Crossref PubMed Scopus (8) Google Scholar). Thus, it has been hypothesized that phospholipase D and its product, PA, play important roles in the signal transduction mechanisms leading to superoxide anion production. Recently, our laboratory developed and characterized a cell-free system for NADPH oxidase activation, which was synergistically activated by PA + diacylglycerol (35Qualliotine-Mann D. Agwu D.E. Ellenburg M.D. McCall C.E. McPhail L.C. J. Biol. Chem. 1993; 268: 23843-23849Abstract Full Text PDF PubMed Google Scholar, 36McPhail L.C. Qualliotine-Mann D. Waite K.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7931-7935Crossref PubMed Scopus (89) Google Scholar). NADPH oxidase activation was enhanced by ATP and reduced by protein kinase inhibitors. Furthermore, PA induced the phosphorylation of several neutrophil proteins. These data suggest that phosphorylation-dependent mechanisms are involved in the activation of NADPH oxidase in this cell-free system. We have reported that p47 phox is phosphorylated in this system by a novel, cytosolic PA-activated protein kinase (36McPhail L.C. Qualliotine-Mann D. Waite K.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7931-7935Crossref PubMed Scopus (89) Google Scholar, 37Waite K.A. Wallin R. Qualliotine-Mann D. McPhail L.C. J. Biol. Chem. 1997; 272: 15569-15578Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Now we report that p22 phox also is phosphorylated in the system in a PA-dependent manner. Phosphorylation of p22 phox was first observed by Garcia and Segal (38Garcia R.C. Segal A.W. Biochem. J. 1988; 252: 901-904Crossref PubMed Scopus (35) Google Scholar) in intact cells. We have now characterized the phosphorylation of p22 phox in vitro and show that a potentially novel, PA-activated protein kinase and conventional but not other protein kinase C (PKC) isoforms are able to phosphorylate this protein. Escherichia coli, containing a plasmid encoding GST-p22 (amino acids 127–195) or GST-p47, p22 phox, and p47 phox antibodies were kindly provided by Dr. Tom Leto (National Institutes of Health). The fusion proteins were prepared as described previously (39Leto T.L. Adams A.G. de Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (245) Google Scholar). Purified, relipidated flavocytochrome b 558was a generous gift of Dr. Michael Kleinberg (University of Maryland) (3Rotrosen D. Yeung C.L. Leto T.L. Malech H.L. Kwong C.H. Science. 1992; 256: 1459-1462Crossref PubMed Scopus (315) Google Scholar, 40Harper A.M. Dunne M.J. Segal A.W. Biochem. J. 1984; 219: 519-527Crossref PubMed Scopus (50) Google Scholar). The mAb 44.1 to p22 phox (41Burritt J.B. Quinn M.T. Jutila M.A. Bond C.W. Jesaitis A.J. J. Biol. Chem. 1995; 270: 16974-16980Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) was the kind gift of Dr. Algirdas J. Jesaitis (Montana State University). Anti-mouse IgG and anti-rabbit IgG were from Transduction Labs (Lexington, KY). Phosphatidylinositol (pig liver), phosphatidylcholine (pig liver), phosphatidylethanolamine (pig liver), phosphatidylserine (PS, bovine brain), cardiolipin (heart), 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], egg lecithin-derived PA, 1,2-dicapryl-sn-glycero-3-phosphate (di10:0PA), 1,2-dioleoyl-sn-glycero-3-phosphate, and 1,2-distearoyl-sn-glycero-3-phosphate were obtained from Avanti Polar Lipids (Alabaster, AL). The 1-oleoyl-sn-glycero-3-phosphate and 1-oleoyl-2-acetyl glycerol were from Serdary Research Laboratories (London, Canada). All lipids were prepared by sonication in water as described previously (35Qualliotine-Mann D. Agwu D.E. Ellenburg M.D. McCall C.E. McPhail L.C. J. Biol. Chem. 1993; 268: 23843-23849Abstract Full Text PDF PubMed Google Scholar). Reagents used for SDS-polyacrylamide gel electrophoresis (PAGE) were from Bio-Rad. Nitrocellulose was from Schleicher & Schuell. NEN Life Science Products provided [γ-32P]ATP (2 mCi/ml) and polyvinylidene fluoride membrane (PolyScreen). Isolymph was from Gallard-Schlesinger Industries (Carle Place, NY). Dextran T-500, CNBr-activated Sepharose 4B beads, and glutathione-Sepharose beads were obtained from Amersham Pharmacia Biotech. Life Technologies, Inc. provided phosphate-buffered saline and Hanks' balanced salt solution. The bicinchoninic acid assay kit, GelCodeTM Blue Stain and SuperSignalTM Enhanced Chemiluminescence reagent were provided by Pierce. Calbiochem (La Jolla, CA) supplied octyl-B-glucopyranoside and purified PKC (rat brain). Human recombinant PKC isoforms were purchased from PanVera (Madison, WI). Cellulose thin layer electrophoresis plates were supplied by Merck (Darmstadt, Germany). Staurosporine, microcystin, and GF109203X were from Alexis (San Diego, CA). The 1-(5-isoquinolinesulfonyl)piperazine was synthesized by Dr. Michael J. Thomas (Wake Forest University School of Medicine) (42Gerard C. McPhail L.C. Marfat A. Stimler-Gerard N.P. Bass D.A. McCall C.E. J. Clin. Invest. 1986; 77: 61-65Crossref PubMed Scopus (154) Google Scholar). All other reagents were from Sigma. Neutrophils were purified, as described previously (43McPhail L.C. Snyderman R. J. Clin. Invest. 1983; 72: 192-200Crossref PubMed Scopus (156) Google Scholar). The cells were then treated with diisopropyl fluorophosphate (1.71 mm), resuspended (2 × 108 cells/ml) in sonication buffer (11% sucrose, 50 mmNa x PO4, pH 7.0, 130 mm NaCl, 5 mm EGTA, 10 μm benzamidine, 10 μg/ml leupeptin, 10 μm pepstatin, 1 μg/ml aprotinin, and 1 mm phenylmethylsulfonyl fluoride) and sonicated to ∼90% breakage as determined by phase contrast microscopy. Membrane and cytosolic fractions were separated using a 15%/40% discontinuous sucrose gradient, as described previously (44Sergeant S. McPhail L.C. J. Immunol. 1997; 159: 2877-2885PubMed Google Scholar, 45Caldwell S.E. McCall C.E. Hendricks C.L. Leone P.A. Bass D.A. McPhail L.C. J. Clin. Invest. 1988; 81: 1485-1496Crossref PubMed Scopus (61) Google Scholar). Protein concentrations were determined using the bicinchoninic acid protocol with bovine serum albumin as a standard. Neutrophils from patients with chronic granulomatous disease (CGD) were prepared in a similar fashion; however, the sonication buffer contained 11% sucrose, 130 mm NaCl, 5 mm EGTA, and 1 mm phenylmethylsulfonyl fluoride. Individuals from two families (four patients total) were obtained. Two males (CBI and CBII) from one family have ∼10% of normal cytochromeb 558 levels (46Woodman R.C. Newburger P.E. Anklesaria P. Erickson R.W. Rae J. Cohen M.S. Curnutte J.T. Blood. 1995; 85: 231-241Crossref PubMed Google Scholar, 47Newburger P.E. Skalnik D.G. Hopkins P.J. Eklund E.A. Curnutte J.T. J. Clin. Invest. 1994; 94: 1205-1211Crossref PubMed Google Scholar); whereas, one male and one female from a second family (JB and EB) have no detectable cytochromeb 558. 2S. Strum and L. C. McPhail, unpublished observation. Reaction mixtures (150 μl) contained 50 mm Na x PO4, pH 7.0, 1 mmEGTA, 5 mm MgCl2, either neutrophil membrane fractions (3.1–6.25 μg of protein) or another substrate, and a lipid activator (usually 10 μm di10:0PA) (36McPhail L.C. Qualliotine-Mann D. Waite K.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7931-7935Crossref PubMed Scopus (89) Google Scholar, 37Waite K.A. Wallin R. Qualliotine-Mann D. McPhail L.C. J. Biol. Chem. 1997; 272: 15569-15578Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). 10 μm [γ-32P]ATP (∼10 μCi) and neutrophil cytosol (12.5–25 μg of protein) or another source of protein kinase were added, and the reaction mixture was allowed to incubate for the times indicated in figure legends. The reaction was stopped by the addition of Laemmli sample buffer (48Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar) for analysis by SDS-PAGE, autoradiography, and densitometry. 5× NaCl Solution (5m NaCl, 25 mm EDTA, 25 mm EGTA, 5 μm staurosporine, 25 mm sodium orthovanadate, 5 μm microcystin, 125 mm NaF, 5 mm p-nitrophenylphosphate, 50 μmbenzamidine, 50 μg/ml leupeptin, 50 μm pepstatin, 5 μg/ml aprotinin, and 5 mm phenylmethylsulfonyl fluoride) was used to quench reactions that were used for solubilization and subsequent immunoprecipitation (see below). For phosphorylation by conventional and novel PKC isoforms, previously described conditions were used (42Gerard C. McPhail L.C. Marfat A. Stimler-Gerard N.P. Bass D.A. McCall C.E. J. Clin. Invest. 1986; 77: 61-65Crossref PubMed Scopus (154) Google Scholar, 44Sergeant S. McPhail L.C. J. Immunol. 1997; 159: 2877-2885PubMed Google Scholar, 49Park J.-W. Hoyal C.R. El Benna J. Babior B.M. J. Biol. Chem. 1997; 272: 11035-11043Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). For PKCζ experiments, 100-μl reaction mixtures contained 25 mm Tris, pH 7.5, 5 mm MgCl2, 0.5 mm EGTA, and 1 mm dithiothreitol with or without the addition of 100 μg/ml PS. For PKC reaction mixtures containing GST-p47, 25 μg/reaction whale myoglobin was added. 3Whale myoglobin was added to reaction mixtures containing low total protein to prevent loss of GST-p47 phox during processing (unpublished observation). Proteins were analyzed by separation on SDS-PAGE, transferred to nitrocellulose, and analyzed by autoradiography/densitometry or PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA). Western blotting was performed to confirm p22 phox and GST-p47 protein levels (described below). Membrane fractions were solubilized as described previously (50Quinn M.T. Parkos C.A. Jesaitis A.J. Methods Enzymol. 1995; 255: 476-487Crossref PubMed Scopus (18) Google Scholar) except that additional protease and phosphatase inhibitors (25 mm NaF, 1 mm p-nitrophenylphosphate, 5 mmNa3VO4, 1 μm microcystin, 10 μm benzamidine, 10 μg/ml leupeptin, 10 μmpepstatin, 1 μg/ml aprotinin, and 1 mmphenylmethylsulfonyl fluoride) were included in the buffers used. Two methods for immunoprecipitation were used. In the first method, CNBr-activated Sepharose beads were conjugated to either mAb 44.1 or the isotype control, according to manufacturer's directions (Amersham Pharmacia Biotech). Solubilized membrane was precleared with isotype control antibodies-conjugated beads and then incubated with the mAb 44.1-conjugated (∼150 μg of solubilized membrane protein/15 μl antibody-conjugated beads) at 4 °C for 16 h. Alternatively, protein A-Sepharose and either the isotype control antibody or mAb 44.1 were incubated with the solubilized membrane (500 μg solubilized membrane/20 μg antibody) at 4 °C for 16 h. The beads were washed, and the immunoprecipitated proteins were analyzed by 14 or 8–15% SDS-PAGE. Protein samples were prepared for analysis by SDS-PAGE using Laemmli sample buffer (48Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar) and separated on 8–15, 7, or 14% SDS-PAGE. Gels were stained with either Coomassie Brilliant Blue R-250 or GelCode Blue Stain (Pierce), destained, dried, and analyzed by autoradiography and densitometry. In some experiments, proteins were transferred electrophoretically (51Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44893) Google Scholar) to nitrocellulose for autoradiography and Western blot analysis. Autoradiographs were analyzed by scanning densitometry (PDI, Huntington Station, NY). Western blot analysis for p47 phox was performed as described previously (37Waite K.A. Wallin R. Qualliotine-Mann D. McPhail L.C. J. Biol. Chem. 1997; 272: 15569-15578Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). For p22 phox Western blotting, a transfer buffer optimized for flavocytochrome b 558 (192 mm glycine, 25 mm Tris, 20% methanol, 0.1% SDS) was used (51Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44893) Google Scholar, 52Verhoeven A.J. Bolscher B.G.J.M. Meerhof L.J. Van Zwieten R. Keijer J. Weening R.S. Roos D. Blood. 1989; 73: 1686-1694Crossref PubMed Google Scholar). Blots were blocked for 1 h with 5% milk in TBS-T (10 mm Tris, 100 mm NaCl, and 0.1% Tween-20), incubated with mAb 44.1 (1:1000 dilution) for 4 h at room temperature, and washed for 2 h. The blot was incubated with anti-mouse horseradish peroxidase-conjugated secondary antibody for 1 h followed by six 10-min washes. Proteins were visualized using SuperSignalTM Enhanced Chemiluminescence (Pierce). Lipid phosphorous assays were performed as described previously (53Rouser G. Siakotos A.N. Fleischer S. Lipids. 1966; 1: 85-86Crossref PubMed Scopus (1317) Google Scholar), using NaH2PO4 (5–50 nmol) for the standard curve. Phosphorylated p22 phox was excised from polyvinylidene difluoride membranes and subjected to hydrolysis with 6 nHCl for 1.5 h at 110 °C (54Boyle W.J. Van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1275) Google Scholar). Phosphoamino acids were separated by thin layer electrophoresis at 1100 V for 45 min in water:acetic acid:pyridine (189:10:1, pH 3.5) (11El Benna J. Dang P.M.-C. Gaudry M. Fay M. Morel F. Hakim J. Gougerot-Pocidalo M.-A. J. Biol. Chem. 1997; 272: 17204-17208Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 54Boyle W.J. Van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1275) Google Scholar). H3[32P]O4 was used as a standard for the migration of inorganic phosphate. As shown in Fig. 1 A (left two lanes), when neutrophil membrane and cytosolic fractions were incubated with di10:0PA, a 22-kDa protein was phosphorylated. Because this phosphoprotein was the appropriate size for the light chain of the flavocytochrome, we hypothesized it could be p22 phox . Neutrophil membrane fractions from patients with flavocytochrome b 558-deficient CGD were substituted for normal membrane fractions in the reaction mixture. Membrane fractions from a total of four patients, two with the X-linked form of CGD and two with the autosomal recessive form, were tested. The two patients with X-linked CGD were previously characterized as having ∼10% of the normal levels of flavocytochromeb 558 (46Woodman R.C. Newburger P.E. Anklesaria P. Erickson R.W. Rae J. Cohen M.S. Curnutte J.T. Blood. 1995; 85: 231-241Crossref PubMed Google Scholar, 47Newburger P.E. Skalnik D.G. Hopkins P.J. Eklund E.A. Curnutte J.T. J. Clin. Invest. 1994; 94: 1205-1211Crossref PubMed Google Scholar), whereas the sibling pair with the autosomal recessive form had no detectable flavocytochromeb 558.2 As shown in Fig. 1 A (right two lanes), the substitution of neutrophil membrane fractions from a patient having ∼10% normal levels of flavocytochrome b 558 resulted in markedly reduced levels of the phosphorylated 22-kDa protein. Similar results were obtained with membrane fractions from the other X-linked patient, and no PA-dependent phosphorylation of a 22-kDa band was observed using membrane fractions from the two patients totally deficient in flavocytochrome b 558 (data not shown). To further confirm that p22 phox was phosphorylated, we performed immunoprecipitation experiments using a monoclonal antibody to the protein (41Burritt J.B. Quinn M.T. Jutila M.A. Bond C.W. Jesaitis A.J. J. Biol. Chem. 1995; 270: 16974-16980Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Following phosphorylation in the presence of cytosol, membrane fractions were reisolated, and solubilized proteins were subjected to immunoprecipitation with the p22 phox -specific antibody. As shown in Fig. 1 B (left two lanes), a phosphorylated 22-kDa protein was immunoprecipitated from membrane fractions incubated with neutrophil cytosol in the presence of di10:0PA. Very little phosphorylation was observed in immunoprecipitates from membrane fractions incubated in the presence of neutrophil cytosol without the addition of di10:0PA. In contrast, no phosphorylated proteins were immunoprecipitated with the isotype control antibody (Fig. 1 B, right two lanes). We performed two additional experiments to verify that p22 phox is phosphorylated by a PA-dependent protein kinase present in neutrophil cytosol. First, purified, relipidated flavocytochromeb 558 was mixed with neutrophil cytosolic fractions in the absence or presence of di10:0PA. In the presence of purified flavocytochrome b 558, di10:0PA clearly induces the phosphorylation of a protein migrating at 22 kDa (Fig. 1 C, right three lanes). Because only a faint band at 22 kDa is observed in the absence of the purified flavocytochromeb 558 (Fig. 1 C, left two lanes), these results indicate that the 22-kDa band is p22 phox . In the second experiment, we tested whether a recombinant form of p22 phox could undergo PA-dependent phosphorylation. We used a GST-p22 phox fusion protein containing amino acids 127–195 of p22 phox, comprising the putative cytosolic tail of the protein (55Imajoh-Ohmi S. Tokita K. Ochiai H. Nakamura M. Kanegasaki S. J. Biol. Chem. 1992; 267: 180-184Abstract Full Text PDF PubMed Google Scholar). This region of the protein also contains three protein kinase phosphorylation motifs (Wisconsin Package version 9.0, Genetics Computing Group, Madison, WI). Therefore, it might be expected that this portion of the protein would contain a PA-dependent phosphorylation site(s). As shown in Fig. 1 D, in the presence of di10:0PA and neutrophil cytosolic fractions, the p22 phox peptide was phosphorylated. Thus, the cytosolic portion of p22 phox contains one or more PA-dependent phosphorylation site(s). Therefore, p22 phox can be phosphorylated by a PA-dependent protein kinase, presumably present in neutrophil cytosol. In none of these experiments did we observe evidence of phosphorylation of the flavocytochrome b heavy chain, gp91 phox (data not shown). To characterize the phosphorylation of p22 phox by a PA-activated protein kinase, we varied the concentration of di10:0PA from 0 to 300 μm, and the time of incubation from 0 to 120 min. Optimal phosphorylation of p22 phox was obtained with incubation of neutrophil cytosol and membrane fractions in the presence of 10 μm di10:0PA for 60 min (data not shown). This concentration of PA is within the range of that measured in stimulated neutrophils (56Agwu D.E. McPhail L.C. Wykle R.L. McCall C.E. Biochem. Biophys. Res. Commun. 1989; 159: 79-86Crossref PubMed Scopus (35) Google Scholar), thus indicating that phosphorylation is mediated by a physiological concentration of PA. We next determined the lipid specificity for the induction of p22 phox phosphorylation. At 10 μm, only di10:0 PA (PA) clearly induced p22 phox phosphorylation. However, the addition of 10 μm PS or phosphatidylinositol (PI) caused a faint darkening over background in the 22 kDa range (Fig. 2 A,top panel). A concentration curve with PS (0–300 μm) revealed a low level of p22 phox phosphorylation with a maximum at 100 μm (data not shown). We then screened various lipids at 100 μm for their ability to induce p22 phox phosphorylation. At this concentration, PS, phosphatidylinositol (PI), phosphatidylglycerol (PG), and 1-oleoyl-sn-glycero-3-phosphate (LPA) induced low levels of p22 phox phosphorylation (Fig. 2 A). However, di10:0PA clearly induced the highest response. We examined the ability of PA species with different acyl chain compositions to induce p22 phox phosphorylation. As shown in Fig. 2 B, di10:0PA (10:0), at either 10 or 100 μm, induced the highest level of p22 phox phosphorylation; however, both 1,2-dioleoyl-sn-glycero-3-phosphate (18:1) and the physiological egg lecithin-derived PA (egg) also had activity. In contrast, 1,2-distearoyl-sn-glycero-3-phosphate (18:0) was unable to induce the phosphorylation of p22 phox . Thus, PA-induced phosphorylation of p22 phox shows selectivity for short or unsaturated acyl chains over long, saturated acyl chains. To identify the type of protein kinase that mediates p22 phox phosphorylation, we tested the effect of several protein kinase inhibitors. 1-(5-isoquinolinesulfonyl)piperazine, an H-7 analog, selectively blocks protein Ser/Thr kinases (42Gerard C. McPhail L.C. Marfat A. Stimler-Gerard N.P. Bass D.A. McCall C.E. J. Clin. Invest. 1986; 77: 61-65Crossref PubMed Scopus (154) Google Scholar). Staurosporine inhibits both protein Ser/Thr and tyrosine kinases (57Meyer T. Regenass U. Fabbro D. Alteri E. Rosel J. Muller M. Caravatti G. Matter A. Int. J. Cancer. 1989; 43: 851-856Crossref PubMed Scopus (435) Google Scholar, 58Ruegg U.T. Burgess G.M. Trends Pharmaco" @default.
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• W2054526825 title "A Phosphatidic Acid-activated Protein Kinase and Conventional Protein Kinase C Isoforms Phosphorylate p22 , an NADPH Oxidase Component" @default.
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