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• W2026534287 abstract "The phagocyte oxidase (Phox) protein p40phox contains a Phox homology (PX) domain which, when expressed alone, interacts with phosphatidylinositol 3-phosphate (PtdIns (3)P). The functions of the PX domain in p40phox localization, association with the cytoskeleton, and superoxide production were examined in transgenic COS-7 cells expressing gp91phox, p22phox, p67phox, and p47phox (COSphox cells). Full-length p40phox exhibited a cytoplasmic localization pattern in resting cells. Upon stimulation with phorbol 12-myristate 13-acetate or fMet-Leu-Phe, p40phox translocated to plasma membrane in a p67phox- and p47phox-dependent manner. Heterologous expression of p40phox markedly enhanced superoxide production in phorbol 12-myristate 13-acetate - and fMet-Leu-Phe-stimulated COSphox cells. Unexpectedly, mutation of Arg-57 in the PX domain to Gln, which abrogated PtdIns (3)P binding, produced a dominant inhibitory effect on agonist-induced superoxide production and membrane translocation of p47phox and p67phox. The mutant p40phox (p40R57Q) displayed increased association with actin and moesin and was found enriched in the Triton X-100-insoluble fraction along with p67phox and p47phox. The enhanced cytoskeleton association of p67phox and p47phox and the dominant inhibitory effect produced by the p40R57Q were alleviated when a second mutation at Asp-289, which eliminated p40phox interaction with p67phox, was introduced. Likewise, cytochalasin B treatment abolished the dominant inhibitory effect of p40R57Q on superoxide production. These findings suggest a dual regulatory mechanism through the PX domain of p40phox; its interaction with the actin cytoskeleton may stabilize NADPH oxidase in resting cells, and its binding of PtdIns (3)P potentiates superoxide production upon agonist stimulation. Both functions require the association of p40phox with p67phox. The phagocyte oxidase (Phox) protein p40phox contains a Phox homology (PX) domain which, when expressed alone, interacts with phosphatidylinositol 3-phosphate (PtdIns (3)P). The functions of the PX domain in p40phox localization, association with the cytoskeleton, and superoxide production were examined in transgenic COS-7 cells expressing gp91phox, p22phox, p67phox, and p47phox (COSphox cells). Full-length p40phox exhibited a cytoplasmic localization pattern in resting cells. Upon stimulation with phorbol 12-myristate 13-acetate or fMet-Leu-Phe, p40phox translocated to plasma membrane in a p67phox- and p47phox-dependent manner. Heterologous expression of p40phox markedly enhanced superoxide production in phorbol 12-myristate 13-acetate - and fMet-Leu-Phe-stimulated COSphox cells. Unexpectedly, mutation of Arg-57 in the PX domain to Gln, which abrogated PtdIns (3)P binding, produced a dominant inhibitory effect on agonist-induced superoxide production and membrane translocation of p47phox and p67phox. The mutant p40phox (p40R57Q) displayed increased association with actin and moesin and was found enriched in the Triton X-100-insoluble fraction along with p67phox and p47phox. The enhanced cytoskeleton association of p67phox and p47phox and the dominant inhibitory effect produced by the p40R57Q were alleviated when a second mutation at Asp-289, which eliminated p40phox interaction with p67phox, was introduced. Likewise, cytochalasin B treatment abolished the dominant inhibitory effect of p40R57Q on superoxide production. These findings suggest a dual regulatory mechanism through the PX domain of p40phox; its interaction with the actin cytoskeleton may stabilize NADPH oxidase in resting cells, and its binding of PtdIns (3)P potentiates superoxide production upon agonist stimulation. Both functions require the association of p40phox with p67phox. Phox homology (PX) 3The abbreviations used are: PX domain, Phox homology domain; Phox, phagocyte oxidase; PtsIns (3)P, phosphatidylinositol 3-phosphate; CGD, chronic granulomatous disease; fMLF, fMet-Leu-Phe; FPR, formyl peptide receptor; CL, chemiluminescence; GFP, green fluorescence protein; PMA, phorbol 12-myristate 13-acetate; RT, room temperature; CPS, counts/s; PBS, phosphate-buffered saline. domains are evolutionarily conserved protein modules of 120-140 amino acids that bind phosphoinositides. Initially named for their presence in the two cytosolic factors of NADPH oxidase, p47phox and p40phox (1Ponting C.P. Protein Sci. 1996; 5: 2353-2357Crossref PubMed Scopus (266) Google Scholar), PX domains have been identified in more than 150 eukaryotic proteins including the sorting nexins (SNX1-15), vacuolar sorting and morphogenesis proteins (Vam7p, Vps5p, and Vps17p), yeast bud-emergence proteins (Bem1p and Bem3p), and phospholipase D2 (2Sato T.K. Overduin M. Emr S.D. Science. 2001; 294: 1881-1885Crossref PubMed Scopus (205) Google Scholar, 3Xu Y. Seet L.F. Hanson B. Hong W. Biochem. J. 2001; 360: 513-530Crossref PubMed Scopus (122) Google Scholar). The PX domains from these proteins interact with a variety of phosphoinositides. Published studies have shown that the PX domain in p40phox binds phosphatidylinositol 3-phosphate (PtdIns (3)P), and the PX domain in p47phox preferentially interacts with phosphatidylinositol 3,4-phosphates (4Kanai F. Liu H. Field S.J. Akbary H. Matsuo T. Brown G.E. Cantley L.C. Yaffe M.B. Nat. Cell Biol. 2001; 3: 675-678Crossref PubMed Scopus (501) Google Scholar, 5Ellson C.D. Gobert-Gosse S. Anderson K.E. Davidson K. Erdjument-Bromage H. Tempst P. Thuring J.W. Cooper M.A. Lim Z.Y. Holmes A.B. Gaffney P.R. Coadwell J. Chilvers E.R. Hawkins P.T. Stephens L.R. Nat. Cell Biol. 2001; 3: 679-682Crossref PubMed Scopus (362) Google Scholar, 6Ago T. Takeya R. Hiroaki H. Kuribayashi F. Ito T. Kohda D. Sumimoto H. Biochem. Biophys. Res. Commun. 2001; 287: 733-738Crossref PubMed Scopus (92) Google Scholar). A proposed function of the PX domain is membrane targeting of proteins containing this structural module. In studies using a green fluorescence protein (GFP)-fused PX domain of p40phox, membrane localization was observed in a phosphatidylinositol 3-kinase-dependent manner (4Kanai F. Liu H. Field S.J. Akbary H. Matsuo T. Brown G.E. Cantley L.C. Yaffe M.B. Nat. Cell Biol. 2001; 3: 675-678Crossref PubMed Scopus (501) Google Scholar, 5Ellson C.D. Gobert-Gosse S. Anderson K.E. Davidson K. Erdjument-Bromage H. Tempst P. Thuring J.W. Cooper M.A. Lim Z.Y. Holmes A.B. Gaffney P.R. Coadwell J. Chilvers E.R. Hawkins P.T. Stephens L.R. Nat. Cell Biol. 2001; 3: 679-682Crossref PubMed Scopus (362) Google Scholar). Membrane binding of the PX domains involves electrostatic interaction as well as membrane penetration by hydrophobic residues in the PX domain-containing proteins (7Stahelin R.V. Burian A. Bruzik K.S. Murray D. Cho W. J. Biol. Chem. 2003; 278: 14469-14479Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Structural analysis of the PX domain of p40phox reveals a positively charged binding pocket for the negatively charged PtdIns (3)P. Binding of p40phox to the phosphoinositide requires three conserved arginine residues (Arg-57, Arg-58, and Arg-105) that stabilize a critical lipid binding loop within the PX domain (8Bravo J. Karathanassis D. Pacold C.M. Pacold M.E. Ellson C.D. Anderson K.E. Butler P.J. Lavenir I. Perisic O. Hawkins P.T. Stephens L. Williams R.L. Mol. Cell. 2001; 8: 829-839Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Mutation of any one of the three arginines can cause a significant reduction in binding of PtdIns (3)P (4Kanai F. Liu H. Field S.J. Akbary H. Matsuo T. Brown G.E. Cantley L.C. Yaffe M.B. Nat. Cell Biol. 2001; 3: 675-678Crossref PubMed Scopus (501) Google Scholar, 5Ellson C.D. Gobert-Gosse S. Anderson K.E. Davidson K. Erdjument-Bromage H. Tempst P. Thuring J.W. Cooper M.A. Lim Z.Y. Holmes A.B. Gaffney P.R. Coadwell J. Chilvers E.R. Hawkins P.T. Stephens L.R. Nat. Cell Biol. 2001; 3: 679-682Crossref PubMed Scopus (362) Google Scholar). Studies have been conducted for the function of p40phox in NADPH oxidase activation since its initial discovery as a p67phox-associated protein (9Wientjes F.B. Hsuan J.J. Totty N.F. Segal A.W. Biochem. J. 1993; 296: 557-561Crossref PubMed Scopus (262) Google Scholar, 10Someya A. Nagaoka I. Yamashita T. FEBS Lett. 1993; 330: 215-218Crossref PubMed Scopus (72) Google Scholar, 11Tsunawaki S. Mizunari H. Nagata M. Tatsuzawa O. Kuratsuji T. Biochem. Biophys. Res. Commun. 1994; 199: 1378-1387Crossref PubMed Scopus (97) Google Scholar). These studies have resulted in different and sometimes conflicting observations. Evidence supporting a positive regulatory role of p40phox came from studies using both cell-free reconstitution and whole-cell assays. The possible mechanisms for p40phox-mediated potentiation of NADPH oxidase include increasing the affinity of p47phox for flavocytochrome b558 (12Cross A.R. Biochem. J. 2000; 349: 113-117Crossref PubMed Google Scholar), binding to membrane-associated PtdIns (3)P through its PX domain (5Ellson C.D. Gobert-Gosse S. Anderson K.E. Davidson K. Erdjument-Bromage H. Tempst P. Thuring J.W. Cooper M.A. Lim Z.Y. Holmes A.B. Gaffney P.R. Coadwell J. Chilvers E.R. Hawkins P.T. Stephens L.R. Nat. Cell Biol. 2001; 3: 679-682Crossref PubMed Scopus (362) Google Scholar) and cooperation with p67phox for membrane translocation of the cytosolic complex (13Kuribayashi F. Nunoi H. Wakamatsu K. Tsunawaki S. Sato K. Ito T. Sumimoto H. EMBO J. 2002; 21: 6312-6320Crossref PubMed Scopus (125) Google Scholar). Other investigators, using essentially the same cells and cell-free reconstitution assays, found p40phox to be a negative regulator for NADPH oxidase. The negative regulatory mechanisms include SH3 domain-mediated interference of p40phox association with other cytosolic factors (14Sathyamoorthy 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) and inhibition of p67phox membrane translocation (15Vergnaud S. Paclet M.H. El Benna J. Pocidalo M.A. Morel F. Eur. J. Biochem. 2000; 267: 1059-1067Crossref PubMed Scopus (59) Google Scholar). More recent studies have examined the roles of p40phox in NADPH oxidase activation using transfected cells and mouse models. Suh et al. (16Suh C.I. Stull N.D. Li X.J. Tian W. Price M.O. Grinstein S. Yaffe M.B. Atkinson S. Dinauer M.C. J. Exp. Med. 2006; 203: 1915-1925Crossref PubMed Scopus (122) Google Scholar) reported that p40phox is required for FcγR receptor-mediated superoxide generation after phagocytosis, a function that was lost when critical residues for PtdIns (3)P binding were mutated. Ellson et al. (17Ellson C.D. Davidson K. Ferguson G.J. O'Connor R. Stephens L.R. Hawkins P.T. J. Exp. Med. 2006; 203: 1927-1937Crossref PubMed Scopus (149) Google Scholar) found that neutrophils from p40phox knock-out mice displayed defective oxidant production in response to several types of stimuli. Moreover, replacement of the mouse p40phox gene with one that contains a Arg-58 to Ala mutation caused embryonic lethality in homozygous offspring, with the heterozygous mice displaying compromised ability to kill Staphylococcus aureus (18Ellson C. Davidson K. Anderson K. Stephens L.R. Hawkins P.T. EMBO J. 2006; 25: 4468-4478Crossref PubMed Scopus (108) Google Scholar). These findings demonstrate a physiological function of p40phox in regulating NADPH oxidase activation that involves its PX domain. Despite recent progress in p40phox research, the function of p40phox in resting cells remains undefined. p40phox was originally discovered as a p67phox-associated protein (9Wientjes F.B. Hsuan J.J. Totty N.F. Segal A.W. Biochem. J. 1993; 296: 557-561Crossref PubMed Scopus (262) Google Scholar, 10Someya A. Nagaoka I. Yamashita T. FEBS Lett. 1993; 330: 215-218Crossref PubMed Scopus (72) Google Scholar, 11Tsunawaki S. Mizunari H. Nagata M. Tatsuzawa O. Kuratsuji T. Biochem. Biophys. Res. Commun. 1994; 199: 1378-1387Crossref PubMed Scopus (97) Google Scholar). In unprimed neutrophils, p40phox forms a complex with p67phox, whereas p47phox was not a part of the complex (19Brown G.E. Stewart M.Q. Liu H. Ha V.L. Yaffe M.B. Mol. Cell. 2003; 11: 35-47Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Neutrophils from chronic granulomatous disease (CGD) patients who lack p67phox contain very little p40phox (11Tsunawaki S. Mizunari H. Nagata M. Tatsuzawa O. Kuratsuji T. Biochem. Biophys. Res. Commun. 1994; 199: 1378-1387Crossref PubMed Scopus (97) Google Scholar, 15Vergnaud S. Paclet M.H. El Benna J. Pocidalo M.A. Morel F. Eur. J. Biochem. 2000; 267: 1059-1067Crossref PubMed Scopus (59) Google Scholar), suggesting that interaction between the two cytosolic factors helps to stabilize their structures. Moreover, p40phox, like the other cytosolic factors, associates with the actin cytoskeleton in resting neutrophils and with membrane skeleton in activated neutrophils (20El Benna J. Dang P.M. Andrieu V. Vergnaud S. Dewas C. Cachia O. Fay M. Morel F. Chollet-Martin S. Hakim J. Gougerot-Pocidalo M.A. J. Leukocyte Biol. 1999; 66: 1014-1020Crossref PubMed Scopus (44) Google Scholar). One of the proteins that helps to mediate protein association with the actin cytoskeleton is moesin, which interacts with the PX domain of p40phox (21Wientjes F.B. Reeves E.P. Soskic V. Furthmayr H. Segal A.W. Biochem. Biophys. Res. Commun. 2001; 289: 382-388Crossref PubMed Scopus (69) Google Scholar). Based on these findings, we speculate that the PX domain in p40phox may have dual regulatory functions through its interaction with the actin cytoskeleton and with PtdIns (3)P. In the current study, we employed a COS-7-based whole-cell reconstitution system (22Price M.O. McPhail L.C. Lambeth J.D. Han C.H. Knaus U.G. Dinauer M.C. Blood. 2002; 99: 2653-2661Crossref PubMed Scopus (101) Google Scholar) to examine the effects of a full-length p40phox and a PX domain mutant on NADPH oxidase activity. We observed that expression of the wild type p40phox could enhance superoxide generation in response to both PMA and fMet-Leu-Phe (fMLF), a finding consistent with recent publications suggesting that p40phox enhances NADPH oxidase activation (16Suh C.I. Stull N.D. Li X.J. Tian W. Price M.O. Grinstein S. Yaffe M.B. Atkinson S. Dinauer M.C. J. Exp. Med. 2006; 203: 1915-1925Crossref PubMed Scopus (122) Google Scholar, 17Ellson C.D. Davidson K. Ferguson G.J. O'Connor R. Stephens L.R. Hawkins P.T. J. Exp. Med. 2006; 203: 1927-1937Crossref PubMed Scopus (149) Google Scholar, 18Ellson C. Davidson K. Anderson K. Stephens L.R. Hawkins P.T. EMBO J. 2006; 25: 4468-4478Crossref PubMed Scopus (108) Google Scholar). Surprisingly, an Arg to Gln mutation at position 57 (R57Q), which abolishes p40phox interaction with PtdIns (3)P through its PX domain (4Kanai F. Liu H. Field S.J. Akbary H. Matsuo T. Brown G.E. Cantley L.C. Yaffe M.B. Nat. Cell Biol. 2001; 3: 675-678Crossref PubMed Scopus (501) Google Scholar), switched p40phox to a different mode of action. It not only abrogated the potentiation effect but also produced a dominant inhibitory effect on superoxide generation. We found an increased association of p40R57Q with actin and moesin compared with the wild type p40phox. In cells expressing p40R57Q, more cytosolic factors were targeted to the Triton X-100-insoluble fraction than in cells expressing the wild type p40phox. The dominant inhibitory effect of p40R57Q was eliminated when the cells were treated with cytochalasin B, which prevents actin polymerization, or when the association of p40phox with p67phox was eliminated. These intriguing findings suggest that p40phox can positively and negatively regulate NADPH oxidase through its PX domain interaction with PtdIns (3)P and with the actin cytoskeleton. Materials—PMA, fMLF, isoluminol, cytochalasin B, anti-moesin, and anti-FLAG monoclonal antibodies were purchased from Sigma-Aldrich. Horseradish peroxidase was obtained from Roche Applied Science. The anti-p67phox (against amino acids 317-469) and early endosome antigen 1 monoclonal antibodies were purchased from BD Transduction Laboratories (Lexington, KY). Anti-actin and anti-p22phox polyclonal antibodies (against amino acids 1-195) were acquired from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-p40phox (against a C-terminal 6-histidine-tagged full-length human p40phox) and anti-p47phox (against a glutathione S-transferasefused full-length human p47phox) polyclonal antibodies were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Plasmid Constructs—Preparation and characterization of the expression constructs of formyl peptide receptor (FPR), protein kinase Cδ and p40phox were described in a previous publication (23He R. Nanamori M. Sang H. Yin H. Dinauer M.C. Ye R.D. J. Immunol. 2004; 173: 7462-7470Crossref PubMed Scopus (38) Google Scholar). The full-length cDNA encoding the human p40phox was subcloned in-frame with GFP in pEGFP-N1 vector (Clontech, Palo Alto, CA) to produce a p40phox protein fused to the N terminus of GFP. Point mutations of p40phox were generated with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The oligonucleotide primers used for the PCR-based mutagenesis were 5′-GTACCTCATCTACCAACGCTACCGCCAGTTC-3′ and its reverse and complementary sequence for the R57Q mutant and 5′-CTGAATTACCGGGCCGCTGAGGGGGATC-3′ and its reverse and complement primer for the D289A mutant. Both primer pairs were used for construction of the double mutant p40R57Q/D289A. All DNA constructs were verified by automated sequencing. Cell Culture and Transient Transfection—The transgenic COSphox and COS91/22 cells were generated as described previously (22Price M.O. McPhail L.C. Lambeth J.D. Han C.H. Knaus U.G. Dinauer M.C. Blood. 2002; 99: 2653-2661Crossref PubMed Scopus (101) Google Scholar). COS91/22 expresses gp91phox and p22phox. Subsequent transfection resulted in COSphox, which expresses p67phox and p47phox in addition to gp91phox and p22phox (22Price M.O. McPhail L.C. Lambeth J.D. Han C.H. Knaus U.G. Dinauer M.C. Blood. 2002; 99: 2653-2661Crossref PubMed Scopus (101) Google Scholar). The stable cell lines were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum and antibiotics for proper selection (22Price M.O. McPhail L.C. Lambeth J.D. Han C.H. Knaus U.G. Dinauer M.C. Blood. 2002; 99: 2653-2661Crossref PubMed Scopus (101) Google Scholar). Cells plated in 90-mm (diameter) tissue culture dishes (0.5-1 × 106 cells per dish) were transfected using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. A total of 6.5 μg of DNA was used in each transfection. Transient transfection efficiency of 45-50% was routinely obtained based the expression of a co-transfected GFP construct using flow cytometry. The human myelomonoblastic cell line PLB-985 was maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/ml penicillin, 50 μg/ml streptomycin, and 2 mml-glutamine. The cells were grown in suspension at a density between 2 × 105/ml and 1 × 106/ml. Cell line Nucleofector Kit V (Amaxa Biosystems, Cologne, Germany) was used for transient transfection of 3 × 106 PLB-985 cells with 5 μg of DNA using Program C-023. Transfection efficiency was ∼30% as determined by flow cytometry based on the fluorescence of a co-expressed green fluorescent protein. Measurement of NADPH Oxidase Activity—Superoxide produced by COSphox and PBL-985 cells was determined using an isoluminol-enhanced chemiluminescence assay, as previously described (23He R. Nanamori M. Sang H. Yin H. Dinauer M.C. Ye R.D. J. Immunol. 2004; 173: 7462-7470Crossref PubMed Scopus (38) Google Scholar, 24Dahlgren C. Karlsson A. J. Immunol. Methods. 1999; 232: 3-14Crossref PubMed Scopus (656) Google Scholar). Oxidant production was inhibited by superoxide dismutase (250 units) as reported previously (23He R. Nanamori M. Sang H. Yin H. Dinauer M.C. Ye R.D. J. Immunol. 2004; 173: 7462-7470Crossref PubMed Scopus (38) Google Scholar). The assay buffer contained horseradish peroxidase (see below) to offset the possible effect of myeloperoxidase. Briefly, COSphox cells were harvested with enzyme-free cell dissociation buffer (Invitrogen). Both COSphox and PLB-985 cells were collected by centrifugation and resuspended in RPMI 1640 containing 0.5% bovine serum albumin at 1-3 × 106 cells/ml. Cells were incubated in the dark with 100 μm isoluminol and 40 units/ml horseradish peroxidase at room temperature for 10 min, and 200-μl aliquots were transferred into 6-mm diameter wells of a 96-well, flat-bottom, white tissue culture plate (E&K Scientific, Campbell, CA). Chemiluminescence (CL) was measured at 37 °C in a Wallac 1420 Multilabel Counter (PerkinElmer Life Sciences). The CL CPS were continually recorded at 1-min intervals for 5-15 min before and 20-40 min after stimulation with PMA (200 ng/ml) or fMLF (1 μm). The relative amount of superoxide produced was calculated based on the integrated CL during the first 20 min after agonist stimulation. Western Blotting—Protein samples were loaded on a 12% SDS-polyacrylamide gel. After electrophoresis, proteins were transferred to nitrocellulose membranes (Schleicher & Schuell). The blots were blocked with 5% nonfat dry milk in TBS/T buffer (20 mm Tris-HCl, pH 7.6, 137 mm NaCl, 0.1% Tween 20) for 2 h at RT.The blots were washed with TBS/T and incubated with primary antibodies (0.2-1 μg/ml) overnight at 4 °C. Anti-rabbit (Bio-Rad) or anti-mouse (Calbiochem) peroxidase-conjugated secondary antibodies were added to the membranes at a dilution ratio of 1:3000, and incubation was continued to for 1 h at RT.The protein bands on the membrane were visualized by chemiluminescence (Pierce). Immunoprecipitation—Twenty-four hours after transfection the cells were lysed in a buffer containing 20 mm Tris-HCl, pH 7.4, 1 mm dithiothreitol, 100 mm NaCl, 1 mm EDTA, 5 mm MgCl2, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and 1× protease inhibitor mixture set I (Calbiochem). For immunoprecipitation with moesin and actin, a buffer containing 1% sodium deoxycholate, 10 mm Tris, pH 7.4, 0.1% SDS, 150 mm NaCl, 1% Nonidet P-40, 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 1× protease inhibitor mixture set I was used instead. The cell lysates were cleared of debris by centrifugation at 14,000 × g for 10 min at 4 °C. Protein content in the cell lysate was measured using a DC Protein Assay (Bio-Rad) and standardized before immunoprecipitation with the anti-FLAG monoclonal antibody (5 μg/ml) at 4 °C overnight. Protein A/G PLUS-agarose was added to the samples for 1.5 h at 4 °C. The beads were washed twice in washing buffer (20 mm Tris-HCl, pH 7.4, 1 mm dithiothreitol, 100 mm NaCl, 1 mm EDTA, 5 mm MgCl2, 0.1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride) and then once in PBS. The beads were resuspended in 50 μl of 2 × SDS-PAGE loading buffer and boiled for 5 min. The samples were analyzed by Western blotting. Cell Fractionation—Cell fractionation was performed as described (25Zhan Y. He D. Newburger P.E. Zhou G.W. J. Cell. Biochem. 2004; 92: 795-809Crossref PubMed Scopus (33) Google Scholar), with a modification in buffer composition. Briefly, 24 h after transfection, the cells were lysed in a buffer containing 20 mm Tris-HCl, pH 7.4, 100 mm NaCl, 1 mm EDTA, 5mm MgCl2, and 1% Triton X-100 at 4 °C for 20 min. Cell lysates were then centrifuged at 14,000 × g for 15 min to separate Triton X-100-soluble and -insoluble fractions. The insoluble fraction was dissolved in 500 μl of 1 × SDS-PAGE loading buffer and boiled for 5 min. The samples were analyzed by Western blotting. Membrane Translocation Assay—Twenty-four hours after transfection, COSphox or COS91/22 cells were stimulated with or without agonists. Cells (1 × 107/sample) were lysed with ice-cold hypotonic buffer (20 mm Tris-HCl, pH 7.4, 2 mm EDTA, 2 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and 1:50 dilution of Protease inhibitor mixture set I). The lysate was then subjected to 3 cycles of freeze/thaw in liquid nitrogen and at 37 °C. Samples were then centrifuged, and the pellets were washed twice in the hypotonic buffer and resuspended in the same buffer containing 1% Triton X-100. The samples were mixed for 30 min at 4 °C to dissociate membrane-bound proteins and then spun down at 14,000 rpm for 10 min at 4 °C. The supernatant were collected as the Triton-soluble membrane fraction. The proteins in the sample were detected by Western blotting. Immunofluorescence Microscopy—Confocal microscopy was performed using indirect immunofluorescence. Six hours after transfection, cells were seeded on glass coverslips pre-coated with 50 μg/ml poly-l-lysine (Sigma) and grown for 18 h in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum. Cells were stimulated with or without PMA (200 ng/ml) for 5 min, washed 3 times in PBS, and fixed with 3% paraformaldehyde in PBS for 15 min at RT. Cells were washed 3 more times in PBS at RT and permeabilized with 0.2% Triton X-100 in PBS for 15 min at RT. Coverslips were blocked with 5% bovine serum albumin in PBS for 1 h at RT. Cells were washed 3 times in PBS and incubated with the primary antibodies in PBS containing 5% bovine serum albumin overnight at 4 °C. Anti-p47phox and anti-p67phox were used at 2 μg/ml each. After washing 5 times with PBST (0.2% Tween 20 in PBS) at RT, cells were incubated with rhodamine red-X-conjugated goat anti-rabbit IgG (secondary antibody; Jackson ImmunoResearch Laboratories, West Grove, PA) at 1.5 μg/ml for 1 h at RT. After additional washes with PBST and H2O, coverslips were mounted on glass slides using the ProLong Gold antifade reagent with 4′, 6-diamidino-2-phenylindole (Molecular Probes). Fluorescence images were captured with a Zeiss LSM 510 confocal microscope equipped with heliumneon, argon, and krypton laser sources. Statistic Analysis—Data were analyzed by paired Student's t test using the PRISM software (Version 4.0, GraphPad, San Diego, CA). Localization of the Full-length p40phox in Transfected Cells—The PX domain of p40phox is thought to preferentially bind PtdIns (3)P for its membrane targeting (4Kanai F. Liu H. Field S.J. Akbary H. Matsuo T. Brown G.E. Cantley L.C. Yaffe M.B. Nat. Cell Biol. 2001; 3: 675-678Crossref PubMed Scopus (501) Google Scholar, 6Ago T. Takeya R. Hiroaki H. Kuribayashi F. Ito T. Kohda D. Sumimoto H. Biochem. Biophys. Res. Commun. 2001; 287: 733-738Crossref PubMed Scopus (92) Google Scholar, 26Ellson C.D. Andrews S. Stephens L.R. Hawkins P.T. J. Cell Sci. 2002; 115: 1099-1105Crossref PubMed Google Scholar). Previous studies have shown that an isolated PX domain fused to a GFP was localized primarily in early endosome (4Kanai F. Liu H. Field S.J. Akbary H. Matsuo T. Brown G.E. Cantley L.C. Yaffe M.B. Nat. Cell Biol. 2001; 3: 675-678Crossref PubMed Scopus (501) Google Scholar, 5Ellson C.D. Gobert-Gosse S. Anderson K.E. Davidson K. Erdjument-Bromage H. Tempst P. Thuring J.W. Cooper M.A. Lim Z.Y. Holmes A.B. Gaffney P.R. Coadwell J. Chilvers E.R. Hawkins P.T. Stephens L.R. Nat. Cell Biol. 2001; 3: 679-682Crossref PubMed Scopus (362) Google Scholar), an intracellular organelle enriched with PtdIns (3)P (27Ellson C.D. Anderson K.E. Morgan G. Chilvers E.R. Lipp P. Stephens L.R. Hawkins P.T. Curr. Biol. 2001; 11: 1631-1635Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). In this study we examined the full-length p40phox for its intracellular localization and redistribution before and after agonist stimulation. A full-length p40phox fused to GFP (p40phox-GFP) was transfected into COSphox cells, a stable cell line of COS-7 that expresses gp91phox, p22phox, p47phox, and p67phox but lacks p40phox (22Price M.O. McPhail L.C. Lambeth J.D. Han C.H. Knaus U.G. Dinauer M.C. Blood. 2002; 99: 2653-2661Crossref PubMed Scopus (101) Google Scholar). Imaging analysis of the transfected COSphox cells (Fig. 1, A-F) revealed cytoplasmic localization of the GFP fluorescence in resting state (Fig. 1, A and C). Slightly more intense fluorescence was observed in the perinuclear region and in membrane ruffles. In comparison, an antibody against early endosome antigen 1 (EEA-1) stained punctate structures in unstimulated cells (Fig. 1B). There were very few punctate structures with both green (p40phox-GFP) and red (anti-early endosome antigen 1) fluorescence (Fig. 1C). Upon stimulation with PMA (Fig. 1, D-F) or fMLF (data not shown), there was a marked increase in plasma membrane-associated green fluorescence (Fig. 1, D and F). PMA stimulation did not increase or decrease double-stained fluorescence in the periphery of the cells (Fig. 1F), indicating the absence of fusion between early endosome and the plasma membrane. To determine whether agonist-induced membrane translocation of p40phox requires p67phox and p47phox, p40phox-GFP was expressed in COS91/22 cells, a stable cell line of COS-7 expressing gp91phox and p22phox but not p67phox and p47phox (22Price M.O. McPhail L.C. Lambeth J.D. Han C.H. Knaus U.G. Dinauer M.C. Blood. 2002; 99: 2653-2661Crossref PubMed Scopus (101) Google Scholar). As shown in Fig. 1, G-L, the GFP fluorescence remained cytoplasmic in the resting state (G and I) as well as following PMA stimulation (J and L). This result suggests that p40phox membrane translocation requires the presence of p67phox and p47phox, which is consistent with the notion that p40phox translocates to plasma membrane in a complex with p67phox and p47phox. A recent study conducted by Ueyama et al. (28Ueyama T. Tatsuno T. Kawasaki T. Tsujibe S. Shirai Y. Sumimoto H. Leto T.L. Saito N. Mol. Biol. Cell. 2007; 18: 441-454Crossref PubMed Scopus (74) Google Scholar) showed that in the RAW267.4 macrophage cell line, which contains very low level of endogenous p67phox, PMA and fMLF was unable to induce membrane transloc" @default.
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• W2026534287 title "Characterization of a Mutation in the Phox Homology Domain of the NADPH Oxidase Component p40phox Identifies A Mechanism for Negative Regulation of Superoxide Production" @default.
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