Activation of p47 , a Cytosolic Subunit of the Leukocyte NADPH Oxidase

The leukocyte NADPH oxidase catalyzes the reduction of oxygen to superoxide (O⨪2) at the expense of NADPH in phagocytes and B lymphocytes. The enzyme is dormant in resting cells but becomes active when the cells are exposed to appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47PHOX becomes phosphorylated on several serines and migrates to the plasma membrane. We report here that p47PHOX-deficient B lymphoblasts expressing the p47PHOX S359A/S370A or p47PHOX S359K/S370K double mutation show dramatically reduced levels of enzyme activity and phosphorylation of p47PHOX as compared with the same cells expressing wild type p47PHOX. In addition, these mutant p47PHOX proteins fails to translocate to the plasma membrane when the cells are stimulated. In contrast, normal phosphorylation and translocation are seen in mutants containing aspartate or glutamate at positions 359 and 370, but oxidase activity is still greatly reduced. These results imply that a negative charge at position 359 and/or 370 is sufficient to allow the phosphorylation and translocation of p47PHOX to take place but that features unique to a phosphorylated hydroxyamino acid are required to support O⨪2 production. These findings, plus those from an earlier study (Inanami, O., Johnson, J. L., McAdara, J. K., El Benna, J., Faust, L. P., Newburger, P. E., and Babior, B. M. (1998) J. Biol. Chem. 273, 9539–9543), suggest that oxidase activation requires 1) the sequential phosphorylation of at least two serines on p47PHOX: Ser-359 or Ser-370, followed by Ser-303 or Ser-304; and 2) the translocation of p47PHOX to the membrane at some point after the first phosphorylation takes place. The leukocyte NADPH oxidase catalyzes the reduction of oxygen to superoxide (O⨪2) at the expense of NADPH in phagocytes and B lymphocytes. The enzyme is dormant in resting cells but becomes active when the cells are exposed to appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47PHOX becomes phosphorylated on several serines and migrates to the plasma membrane. We report here that p47PHOX-deficient B lymphoblasts expressing the p47PHOX S359A/S370A or p47PHOX S359K/S370K double mutation show dramatically reduced levels of enzyme activity and phosphorylation of p47PHOX as compared with the same cells expressing wild type p47PHOX. In addition, these mutant p47PHOX proteins fails to translocate to the plasma membrane when the cells are stimulated. In contrast, normal phosphorylation and translocation are seen in mutants containing aspartate or glutamate at positions 359 and 370, but oxidase activity is still greatly reduced. These results imply that a negative charge at position 359 and/or 370 is sufficient to allow the phosphorylation and translocation of p47PHOX to take place but that features unique to a phosphorylated hydroxyamino acid are required to support O⨪2 production. These findings, plus those from an earlier study (Inanami, O., Johnson, J. L., McAdara, J. K., El Benna, J., Faust, L. P., Newburger, P. E., and Babior, B. M. (1998) J. Biol. Chem. 273, 9539–9543), suggest that oxidase activation requires 1) the sequential phosphorylation of at least two serines on p47PHOX: Ser-359 or Ser-370, followed by Ser-303 or Ser-304; and 2) the translocation of p47PHOX to the membrane at some point after the first phosphorylation takes place. The leukocyte NADPH oxidase catalyzes the reduction of oxygen to superoxide (O⨪2) at the expense of NADPH in phagocytes and B lymphocytes as shown in Equation 1 (1Chanock S.J. El Benna J. Smith R.M. Babior B.M. J. Biol. Chem. 1994; 269: 24519-24522Abstract Full Text PDF PubMed Google Scholar). 2O2+NADPH→2O⨪2+NADP++H+Equation 1 It is a multicomponent enzyme comprising a membrane-associated flavocytochrome known as cytochrome b 558 and at least three cytosolic subunits as follows: p47PHOX, p67PHOX, and the small G protein Rac2 (2Heyworth P.G. Curnutte J.T. Nauseef W.M. Volpp B.D. Pearson D.W. Rosen H. Clark R.A. J. Clin. Invest. 1991; 87: 352-356Crossref PubMed Scopus (305) Google Scholar). 1A fourth cytosolic subunit, p40PHOX, is associated with p47PHOX and p67PHOX in a cytosolic complex, but its function is unknown (34Someya A. Nagaoka I. Yamashita T. FEBS Lett. 1993; 330: 215-218Crossref PubMed Scopus (72) Google Scholar). 1A fourth cytosolic subunit, p40PHOX, is associated with p47PHOX and p67PHOX in a cytosolic complex, but its function is unknown (34Someya A. Nagaoka I. Yamashita T. FEBS Lett. 1993; 330: 215-218Crossref PubMed Scopus (72) Google Scholar). The enzyme is dormant in resting cells but becomes active when the cells are exposed to appropriate stimuli. During oxidase activation, the highly basic p47PHOX component becomes phosphorylated on up to nine serines near the C terminus and migrates to the plasma membrane, where it associates with cytochrome b 558, carrying with it to the membrane the subunit p67PHOX and possibly Rac2 (3Hayakawa T. Suzuki K. Suzuki S. Andrews P.C. Babior B.M. J. Biol. Chem. 1986; 261: 9109-9115Abstract Full Text PDF PubMed Google Scholar, 4Ohtsuka T. Okamura N. Ishibashi S. Biochim. Biophys. Acta. 1986; 888: 332-337Crossref PubMed Scopus (40) Google Scholar, 5Caldwell 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, 6Rotrosen D. Leto T.L. J. Biol. Chem. 1990; 265: 19910-19915Abstract Full Text PDF PubMed Google Scholar, 7El Benna J. Faust L.P. Babior B.M. J. Biol. Chem. 1994; 269: 23431-23436Abstract Full Text PDF PubMed Google Scholar, 8El Benna J. Ruedi J.M. Babior B.M. J. Biol. Chem. 1994; 269: 6729-6734Abstract Full Text PDF PubMed Google Scholar, 9Heyworth P.G. Bohl B.P. Bokoch G.M. Curnutte J.T. J. Biol. Chem. 1994; 269: 30749-30752Abstract Full Text PDF PubMed Google Scholar, 10Dusi S. Donini M. Rossi F. Biochem. J. 1996; 314: 409-412Crossref PubMed Scopus (110) Google Scholar, 11Faust L.P. El Benna J. Babior B.M. Chanock S.J. J. Clin. Invest. 1995; 96: 1499-1505Crossref PubMed Scopus (155) Google Scholar). Previously we have shown that a mutant p47PHOX containing a single alanine mutation at Ser-379 is unable to translocate to the membrane, resulting in a loss of enzyme activity. This serine has been shown to be phosphorylated in wild type p47PHOX, albeit to a very low level (11Faust L.P. El Benna J. Babior B.M. Chanock S.J. J. Clin. Invest. 1995; 96: 1499-1505Crossref PubMed Scopus (155) Google Scholar). Its phosphorylation, however, is not necessary for the remaining serines to be phosphorylated either in vivo orin vitro (12El Benna J. Faust L.P. Johnson J.L. Babior B.M. J. Biol. Chem. 1996; 271: 6374-6378Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). During earlier studies using several p47PHOX mutants containing an alanine instead of a serine at a single potential phosphorylation site, we showed that no individual serine phosphate was required for the phosphorylation of the remaining serines to take place, although, as discussed above, Ser-379 is necessary for enzyme activity (11Faust L.P. El Benna J. Babior B.M. Chanock S.J. J. Clin. Invest. 1995; 96: 1499-1505Crossref PubMed Scopus (155) Google Scholar). In this study we investigate the effect of double mutations involving serines 359 and 370 on the phosphorylation of p47PHOX and the activity of the leukocyte NADPH oxidase. Cloning of the plasmid constructs used in the transfection experiments was performed by standard techniques (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The mutant constructs S359A/S370A, S359D/S370D, and S359E/S370E were generated by a two-step polymerase chain reaction method using an appropriate mutant oligonucleotide to make the desired mutation (Table I). The polymerase chain reaction-generated fragments were subsequently substituted into the EBOpLPP-based expression vector containing the wild type p47PHOX cDNA. The mutant construct S310–348A was created by single strand mutagenesis of the EBOpLPP-based expression vector as described previously using primers listed in Table I (11Faust L.P. El Benna J. Babior B.M. Chanock S.J. J. Clin. Invest. 1995; 96: 1499-1505Crossref PubMed Scopus (155) Google Scholar). The mutant construct S359K/S370K was generated from the EBOpLPP wild type vector using the Chameleon mutagenesis kit (Stratagene) following the manufacturer's instructions and using the mutant oligonucleotides listed in Table I. The sequence of all mutant constructs was confirmed by dideoxynucleotide-based sequencing (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Epstein-Barr virus-transformed p47PHOX-deficient B cells were co-transfected with SV40 and EBOpLPP-derived p47PHOX expression vectors and expanded under hygromycin selection as described elsewhere (14Chanock S.J. Faust L.P. Barrett D. Bizal C. Maly F.E. Newburger P.E. Ruedi J.M. Smith R.M. Babior B.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10174-10177Crossref PubMed Scopus (42) Google Scholar). Transfected cells were cultured as described elsewhere (14Chanock S.J. Faust L.P. Barrett D. Bizal C. Maly F.E. Newburger P.E. Ruedi J.M. Smith R.M. Babior B.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10174-10177Crossref PubMed Scopus (42) Google Scholar), except they were maintained at a higher cell density (1 × 106/ml), which was found to significantly increase their oxidase activity.Table IOligonucleotides used in the mutagenesis of p47PHOXMutationOligonucleotideSourceS303A/S304ACG GAT GGC CGC GCG CCG GGG CGG CGC21S303D/S304DCG GAT TTC TTC GCG CCG GG21S303E/S304ECG GAT GTC GTC GCG CCG GG21S310ATG GAT GGC GTG CGC ATT CCG GAT GGA11S315ACCG CGC GCG CTG ATG GAT11S320ATC CTG GGC GAG CCG CTTCCG CGA11S328ACG TAC GGC GTT GCG GCG ATA GGC11S345A/S348AACC GGG GGC GTT GCG GCG ATA GGC11S359AG TTT AGC GCG CTG CGT CTG CC11S359DG TTT GTC GCG CTG CGTNewS359EG TTT CTC GCG CTG CGTNewS370AGTC GGC GGC AGG CCT CGG GGG CAC11S370DTC GGC GTC CGG TCG CGG GGG CNewS370ETC GGC CTC CGG TCG CGG GGG CNewS370KGGTGCCCCCGCGGCCGAAGGCCGACCTCATCCTGNewS359KGGGCACCGCCGCCTGCGGTTTCTTGCGCTGCGTCTGCCGNewEboMluIGTGGAGTGGGGGCGCGCGTCAGCCCCCACNew Open table in a new tab Recombinant fusion proteins composed of an upstream glutathione S-transferase (GST) linked to a downstream p47PHOX, either wild type or mutant, were isolated from Escherichia coli that had been transformed with pGEX-1λT plasmids containing cDNA inserts encoding the downstream proteins, as previously reported (15Park J.-W. El Benna J. Scott K.E. Christensen B.L. Chanock S.J. Babior B.M. Biochemistry. 1994; 33: 2907-2911Crossref PubMed Scopus (85) Google Scholar). The fusion proteins were purified by affinity chromatography on glutathione-agarose as described elsewhere (15Park J.-W. El Benna J. Scott K.E. Christensen B.L. Chanock S.J. Babior B.M. Biochemistry. 1994; 33: 2907-2911Crossref PubMed Scopus (85) Google Scholar). Excess glutathione was removed from the solution of purified recombinant protein by dialysis against relaxation buffer (100 mm KCl, 3 mm NaCl, 3.5 mmMgCl2, and 10 mm Pipes, pH 8.0), and the protein concentration was determined using the Bio-Rad assay kit with bovine serum albumin as the standard. Leukocyte NADPH oxidase activity was measured by chemiluminescence. Assays using whole cells were carried out as described elsewhere (16Thrasher A. Chetty M. Casimir C. Segal A.W. Blood. 1992; 80: 1125-1129Crossref PubMed Google Scholar), except that 3 × 106 cells and 5 IU horseradish peroxidase were used in a final volume of 0.35 ml. The cell suspensions were placed in a 96-well microtiter plate, warmed to 37 °C, then activated at the same temperature with phorbol myristate acetate (PMA, 1 μg/ml). Chemiluminescence was then measured at 45-s intervals using a Luminoskan luminometer (Labsystems Research, Finland) at 37 °C. For measuring leukocyte NADPH oxidase activity in a cell-free system, reaction mixtures contained 1.6 × 106 cell eq of neutrophil membranes, 106 cell eq of lymphoblast cytosol (prepared by sonicating a suspension of lymphoblasts in Dulbecco's phosphate-buffered saline for three 10-s intervals at 4° C and then removing particles by centrifugation for 15 min at the same temperature in an Eppendorf microcentrifuge), 160 μm NADPH, 1 mm luminol, 5 IU horseradish and relaxation buffer in a final volume of 0.35 ml. The enzyme was activated by the of μm and was measured at 10-s intervals using a Luminoskan luminometer at Recombinant p47PHOX was and as described previously J.-W. Babior B.M. Biochemistry. PubMed Scopus (61) Google Scholar). In or mutant recombinant p47PHOX was at temperature for min with of in the or of μm The were then placed on for 5 min the protein by the of acid to a final concentration of was as a The were on for 10 min and then at for 10 min at °C. The proteins were in of and were in a after the of Transfected lymphoblasts were for 15 min at with as described elsewhere Curnutte J.T. J. M. Babior B.M. N. J. PubMed Scopus Google Scholar). The cells were then in phosphate-buffered saline and at 37 for 10 (1 was then to the and the was for a The cells were then in of relaxation pH 1 mm mm containing 1 and were carried out at °C. The cells were by for 5 and 10 to and The was × to from The membrane was in the relaxation buffer and then in buffer and for 3 Transfected lymphoblasts and cells were at × in phosphate-buffered for three 10-s and at for 15 min in an Eppendorf volume of buffer was then to the and the proteins were by on a The proteins were then to and the p47PHOX were using a of a purified against a from p47PHOX as described elsewhere J.-W. M. Ruedi J.M. Smith R.M. Babior B.M. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). was measured by with a of p47PHOX from Transfected B lymphoblasts expressing wild type or mutant p47PHOX were at 37 in Dulbecco's 10 mm pH mm The cells were then to containing and for an at 37 °C. The cells were activated for min with (1 after which their p47PHOX was isolated and purified by chromatography as described previously Benna J. Faust L.P. Babior B.M. J. Biol. Chem. 1994; 269: 23431-23436Abstract Full Text PDF PubMed Google Scholar). of p47PHOX with protein C was performed by a reaction containing 1 of recombinant p47PHOX, of protein 10 mm 1 5 of and 5 in a volume of for min at 37 °C. The were by the of 10 of and the proteins were then by on a to the method of D.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The proteins were by and was by the from the and measuring their using A of of was from of the and This was from the Previously it has been shown that a p47PHOX double of serine with alanine at Ser-303 and of p47PHOX the activation of the leukocyte NADPH but this activity was by the with glutamate or Johnson J.L. El Benna J. Faust L.P. Newburger P.E. Babior B.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The glutamate are to the effect of serine phosphorylation J.L. R.M. 1990; PubMed Scopus Google Scholar). the that the double mutant was active either that the effect of phosphorylation is to the increase in serines and or that activation the of a the phosphorylated serines and of the we report a loss in oxidase activation in p47PHOX-deficient B cells expressing the double mutant p47PHOX S359A/S370A In this however, oxidase activity was not by the with glutamate or aspartate or the p47PHOX p47PHOX S359D/S370D, p47PHOX reported here is the activity of the mutant p47PHOX in which serines and were to with p47PHOX-deficient B lymphocytes expressing this mutant of the oxidase activity of cells expressing p47PHOX, that these serines are not for oxidase activity. This with the previously reported that the double mutants p47PHOX (11Faust L.P. El Benna J. Babior B.M. Chanock S.J. J. Clin. Invest. 1995; 96: 1499-1505Crossref PubMed Scopus (155) Google and p47PHOX Johnson J.L. El Benna J. Faust L.P. Newburger P.E. Babior B.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google have no effect on oxidase The of oxidase activation in p47PHOX-deficient B cells expressing the double mutants was not to a in the expression of these as their levels of expression were found to be to that of the wild type protein and Table this loss of activity not to be to an of the mutant proteins to into a from lymphocytes expressing the double mutants were in a cell-free oxidase activated with and were found to support oxidase activity to the same as wild type p47PHOX a method to in of activation J.-W. Babior B.M. Biochemistry. PubMed Scopus (61) Google confirmed that the mutant protein S359A/S370A normal (Table This method the that a that oxidase activation the of to type and mutant S359A/S370A recombinant protein were found to to the same with and activation with in reduced levels of We suggest that the mutant proteins active the in their acid of p47PHOX in p47PHOX-deficient B cells with plasmids expressing and mutant recombinant the of three Open table in a new tab activity in a cell-free containing and mutant of recombinant p47PHOX. The assay was performed as described under and These results the for p47PHOX-deficient B lymphoblasts expressing of p47PHOX. The results are as a of the wild type and the of three of of with on the of and mutant S359A/S370A recombinant p47PHOX by and mutant recombinant p47PHOX were with for min in the or of μm as described under and were for by The results shown are from three in The contained of Open table in a new tab These the of three and mutant recombinant p47PHOX were with for min in the or of μm as described under and were for by The results shown are from three in The contained of It is that the activation of the leukocyte NADPH oxidase is by the of p47PHOX to the plasma membrane, where it associates with cytochrome b 558 (2Heyworth P.G. Curnutte J.T. Nauseef W.M. Volpp B.D. Pearson D.W. Rosen H. Clark R.A. J. Clin. Invest. 1991; 87: 352-356Crossref PubMed Scopus (305) Google Scholar, D. H. Leto T. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar). investigate serine 359 and 370 are for this we plasma from p47PHOX-deficient B cells expressing wild type or double mutant of and after activation with of these showed that mutant p47PHOX S359A/S370A and p47PHOX S359K/S370K were unable to translocate to the membrane, the of p47PHOX and p47PHOX S359E/S370E to be normal and Table These results suggest that the of a negative charge here by the of the with aspartate or glutamate be required for the translocation of p47PHOX. It has previously been that the of p47PHOX be in resting at least in by an involving the highly basic with a concentration of negative charge elsewhere on the of the (11Faust L.P. El Benna J. Babior B.M. Chanock S.J. J. Clin. Invest. 1995; 96: 1499-1505Crossref PubMed Scopus (155) Google Scholar). of a negative by either serine phosphorylation or the of serine by a as the glutamate or aspartate described the the and the to allow of the oxidase. The translocation results described here to with this as the allow translocation the not the of p47PHOX to the of p47PHOX to the membrane in p47PHOX-deficient B cells expressing and mutant recombinant the of three Open table in a new tab These the of three the of serines 359 and 370 for the phosphorylation of p47PHOX, we phosphorylated fusion proteins of wild type and mutant p47PHOX. The results and Table showed that was a reduction in the level of phosphorylation of as compared with the wild type and the mutant of p47PHOX. It that the of the at positions 359 and 370 phosphorylation of p47PHOX, in to the greatly reduced level of phosphorylation in the alanine mutant with the the effect of the wild type with to vitro of and mutant of recombinant were from the and the was measured by The the of two Open table in a new tab The were from the and the was measured by The the of two The reduction in phosphorylation with in vitro was with and p47PHOX S359K/S370K in and Table In contrast, the levels of in vivo phosphorylation of p47PHOX S359D/S370D, p47PHOX and double mutant Johnson J.L. El Benna J. Faust L.P. Newburger P.E. Babior B.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), were to that seen in the wild type vivo phosphorylation of and mutant of recombinant of was determined by and the results are the of three or Open table in a new tab The of was determined by and the results are the of three or It has been known for some that p47PHOX becomes phosphorylated when the oxidase is activated (3Hayakawa T. Suzuki K. Suzuki S. Andrews P.C. Babior B.M. J. Biol. Chem. 1986; 261: 9109-9115Abstract Full Text PDF PubMed Google Scholar, B.M. T. Suzuki K. Suzuki S. Clin. Scholar, A.W. P.G. S. PubMed Scopus Google Scholar, N. Curnutte J.T. Babior B.M. J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar, P.G. Biochim. Biophys. Acta. 1990; PubMed Scopus Google Scholar, D. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1991; Full Text PDF PubMed Google and that the serines for this phosphorylation Ser-303 and Ser-379 in the C terminus of the protein (3Hayakawa T. Suzuki K. Suzuki S. Andrews P.C. Babior B.M. J. Biol. Chem. 1986; 261: 9109-9115Abstract Full Text PDF PubMed Google Scholar, 4Ohtsuka T. Okamura N. Ishibashi S. Biochim. Biophys. Acta. 1986; 888: 332-337Crossref PubMed Scopus (40) Google Scholar, 5Caldwell 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, 6Rotrosen D. Leto T.L. J. Biol. Chem. 1990; 265: 19910-19915Abstract Full Text PDF PubMed Google Scholar, 7El Benna J. Faust L.P. Babior B.M. J. Biol. Chem. 1994; 269: 23431-23436Abstract Full Text PDF PubMed Google Scholar). that the phosphorylation of p47PHOX is required for oxidase activity has been using cell-free the activation with phosphorylated J. Clin. Invest. PubMed Scopus Google Scholar, Benna J. J.-W. Ruedi J.M. Babior B.M. 1995; PubMed Scopus Google Scholar, J.-W. El Benna J. Babior B.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). discussed Ser-379 is known to be for oxidase the level of phosphorylation of this serine is very low (11Faust L.P. El Benna J. Babior B.M. Chanock S.J. J. Clin. Invest. 1995; 96: 1499-1505Crossref PubMed Scopus (155) Google Scholar), the by its phosphorylation in oxidase activation is The study has a the phosphorylation of p47PHOX and oxidase The and mutations of 359 and 370 in p47PHOX S359A/S370A and p47PHOX the phosphorylation of p47PHOX and its translocation to the membrane, oxidase activity after translocation and phosphorylation of p47PHOX were by the alanine with the glutamate or but oxidase activation in the and S359E/S370E mutants still It that are two for the serine at 359 or a serine phosphate at of these positions is required for oxidase activation to take place. A negative charge not this as and mutations not activity. a serine phosphate at of these positions is required for translocation and for phosphorylation of the remaining This the for a negative as the of the serine with glutamate or aspartate was to phosphorylation and translocation to wild type discussed the mutation of the p47PHOX serines and to had no effect on the phosphorylation of the remaining serines but oxidase activity. The activity was however, with glutamate or in positions and Johnson J.L. El Benna J. Faust L.P. Newburger P.E. Babior B.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). glutamate or aspartate in positions 359 and 370 of p47PHOX, phosphorylation and translocation of the mutants p47PHOX and S359E/S370E were it is not to the of the or mutations at positions 359 or 370 to support oxidase The results for the double mutant p47PHOX however, suggest that the function of mutants and S359E/S370E is not to a in or the of a as in the of in is to of the charge of the highly basic C terminus as previously In either we that a is exposed of the active oxidase. In suggest that p47PHOX requires at least three serines for oxidase activation as follows: a serine at position that is for oxidase activity and translocation but not for phosphorylation of the remaining serines (11Faust L.P. El Benna J. Babior B.M. Chanock S.J. J. Clin. Invest. 1995; 96: 1499-1505Crossref PubMed Scopus (155) Google a phosphorylated serine at position 359 or 370 that is required for oxidase activity and be phosphorylated to allow translocation as as phosphorylation of the remaining serines and a phosphorylated serine at position or that is for oxidase activity but is not necessary for the phosphorylation of the remaining serines or for the translocation of p47PHOX to the membrane Johnson J.L. El Benna J. Faust L.P. Newburger P.E. Babior B.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We that with to p47PHOX, the activation of the leukocyte NADPH oxidase the following 1) phosphorylation of Ser-359 and/or translocation of the phosphorylated subunit to the phosphorylation of Ser-303 and/or the activation of p47PHOX. The of and be but the phosphorylation of Ser-359 or is an first for the of p47PHOX in the activation of the oxidase. phosphorylation of Ser-379 is required for oxidase activity under