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. 1996 Sep 1;184(3):893–902. doi: 10.1084/jem.184.3.893

Involvement of p40phox in activation of phagocyte NADPH oxidase through association of its carboxyl-terminal, but not its amino-terminal, with p67phox

PMCID: PMC2192801  PMID: 9064349

Abstract

Phagocyte NADPH oxidase, dormant in resting cells, is activated upon cell stimulation to produce superoxide anion, a precursor of microbicidal oxidants. Active NADPH oxidase is found on the membrane as an enzyme complex, composed of membrane-integrated cytochrome b558 (gp91phox and p22phox subunits) and two cytosolic factors (p47phox and p67phox), each of the latter containing two src homology 3 (SH3) domains. Recently, we radioactively identified a third cytosolic factor, p40phox, as a molecule that associates with p67phox in human neutrophils. Although it has been found that this p40phox protein is defective in patients with chronic granulomatous disease (CGD) who lack p67phox, evidence to functionally relate it to the NADPH oxidase system has hitherto been lacking. In this study, we raised separate antibodies against both the COOH- and NH2-terminal polypeptides of p40phox as well as against the COOH-terminal polypeptide of p67phox to examine the mode of interaction between p40phox and p67phox in a complex. The antibody against the COOH terminus of p67phox was able to communoprecipitate p40phox in conjunction with p67phox itself as was expected. Very interestingly, however, the antibody against the COOH terminus of p40phox completely dissociated the p67phox molecule from the p40phox- p67phox complex unit without any detectable coimmunoprecipitation of p67phox, despite their tight association, whereas that against the NH2 terminus of p40phox had absolutely no dissociation effect. Similar results were found regarding their effects on the O2-generating ability of cytosol in a cell-free activation system, i.e., inhibition was noted with the COOH terminus antibody but not with that for the NH2 terminus of p40phox. However, this dissociation did not affect the translocation of the cytosolic components including p47phox to the membrane. Once the NADPH oxidase was activated, the antibody for the COOH terminus did not show any inhibitory effect on catalysis by the activated enzyme. The stimulators of NADPH oxidase, MA and SDS, did not dissociate the p40phox-p67phox complex. These results provide the first demonstration that p40phox is practically involved in the activation of NADPH oxidase through the association of its COOH-terminal, but not its NH2-terminal, with p67phox.

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Selected References

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  1. Abo A., Boyhan A., West I., Thrasher A. J., Segal A. W. Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245. J Biol Chem. 1992 Aug 25;267(24):16767–16770. [PubMed] [Google Scholar]
  2. Abo A., Webb M. R., Grogan A., Segal A. W. Activation of NADPH oxidase involves the dissociation of p21rac from its inhibitory GDP/GTP exchange protein (rhoGDI) followed by its translocation to the plasma membrane. Biochem J. 1994 Mar 15;298(Pt 3):585–591. doi: 10.1042/bj2980585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Chanock S. J., el Benna J., Smith R. M., Babior B. M. The respiratory burst oxidase. J Biol Chem. 1994 Oct 7;269(40):24519–24522. [PubMed] [Google Scholar]
  5. Chuang T. H., Bohl B. P., Bokoch G. M. Biologically active lipids are regulators of Rac.GDI complexation. J Biol Chem. 1993 Dec 15;268(35):26206–26211. [PubMed] [Google Scholar]
  6. Clark R. A., Volpp B. D., Leidal K. G., Nauseef W. M. Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation. J Clin Invest. 1990 Mar;85(3):714–721. doi: 10.1172/JCI114496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Curnutte J. T., Kuver R., Scott P. J. Activation of neutrophil NADPH oxidase in a cell-free system. Partial purification of components and characterization of the activation process. J Biol Chem. 1987 Apr 25;262(12):5563–5569. [PubMed] [Google Scholar]
  8. Dinauer M. C., Pierce E. A., Erickson R. W., Muhlebach T. J., Messner H., Orkin S. H., Seger R. A., Curnutte J. T. Point mutation in the cytoplasmic domain of the neutrophil p22-phox cytochrome b subunit is associated with a nonfunctional NADPH oxidase and chronic granulomatous disease. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11231–11235. doi: 10.1073/pnas.88.24.11231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dusi S., Donini M., Rossi F. Mechanisms of NADPH oxidase activation: translocation of p40phox, Rac1 and Rac2 from the cytosol to the membranes in human neutrophils lacking p47phox or p67phox. Biochem J. 1996 Mar 1;314(Pt 2):409–412. doi: 10.1042/bj3140409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Finan P., Shimizu Y., Gout I., Hsuan J., Truong O., Butcher C., Bennett P., Waterfield M. D., Kellie S. An SH3 domain and proline-rich sequence mediate an interaction between two components of the phagocyte NADPH oxidase complex. J Biol Chem. 1994 May 13;269(19):13752–13755. [PubMed] [Google Scholar]
  11. Fuchs A., Dagher M. C., Vignais P. V. Mapping the domains of interaction of p40phox with both p47phox and p67phox of the neutrophil oxidase complex using the two-hybrid system. J Biol Chem. 1995 Mar 17;270(11):5695–5697. doi: 10.1074/jbc.270.11.5695. [DOI] [PubMed] [Google Scholar]
  12. Heyworth P. G., Curnutte J. T., Nauseef W. M., Volpp B. D., Pearson D. W., Rosen H., Clark R. A. Neutrophil nicotinamide adenine dinucleotide phosphate oxidase assembly. Translocation of p47-phox and p67-phox requires interaction between p47-phox and cytochrome b558. J Clin Invest. 1991 Jan;87(1):352–356. doi: 10.1172/JCI114993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Iyer S. S., Pearson D. W., Nauseef W. M., Clark R. A. Evidence for a readily dissociable complex of p47phox and p67phox in cytosol of unstimulated human neutrophils. J Biol Chem. 1994 Sep 2;269(35):22405–22411. [PubMed] [Google Scholar]
  14. Jouan A., Dagher M. C., Fuchs A., Foucaud-Gamen J., Vignais P. V. Immunoaffinity purification of an oxidase-activating cytosolic complex from bovine neutrophils. Biochem Biophys Res Commun. 1993 Dec 30;197(3):1296–1302. doi: 10.1006/bbrc.1993.2618. [DOI] [PubMed] [Google Scholar]
  15. Leto T. L., Adams A. G., de Mendez I. Assembly of the phagocyte NADPH oxidase: binding of Src homology 3 domains to proline-rich targets. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10650–10654. doi: 10.1073/pnas.91.22.10650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Leto T. L., Lomax K. J., Volpp B. D., Nunoi H., Sechler J. M., Nauseef W. M., Clark R. A., Gallin J. I., Malech H. L. Cloning of a 67-kD neutrophil oxidase factor with similarity to a noncatalytic region of p60c-src. Science. 1990 May 11;248(4956):727–730. doi: 10.1126/science.1692159. [DOI] [PubMed] [Google Scholar]
  17. Leusen J. H., Bolscher B. G., Hilarius P. M., Weening R. S., Kaulfersch W., Seger R. A., Roos D., Verhoeven A. J. 156Pro-->Gln substitution in the light chain of cytochrome b558 of the human NADPH oxidase (p22-phox) leads to defective translocation of the cytosolic proteins p47-phox and p67-phox. J Exp Med. 1994 Dec 1;180(6):2329–2334. doi: 10.1084/jem.180.6.2329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lomax K. J., Leto T. L., Nunoi H., Gallin J. I., Malech H. L. Recombinant 47-kilodalton cytosol factor restores NADPH oxidase in chronic granulomatous disease. Science. 1989 Jul 28;245(4916):409–412. doi: 10.1126/science.2547247. [DOI] [PubMed] [Google Scholar]
  19. Mayer B. J., Baltimore D. Signalling through SH2 and SH3 domains. Trends Cell Biol. 1993 Jan;3(1):8–13. doi: 10.1016/0962-8924(93)90194-6. [DOI] [PubMed] [Google Scholar]
  20. McPhail L. C. SH3-dependent assembly of the phagocyte NADPH oxidase. J Exp Med. 1994 Dec 1;180(6):2011–2015. doi: 10.1084/jem.180.6.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mizunari H., Kakinuma K., Suzuki K., Namiki H., Kuratsuji T., Tsunawaki S. Nucleoside-triphosphate binding of the two cytosolic components of the respiratory burst oxidase system: evidence for its inhibition by the 2',3'-dialdehyde derivative of NADPH and desensitization in their translocated states. Biochim Biophys Acta. 1993 Dec 16;1220(1):21–30. doi: 10.1016/0167-4889(93)90092-4. [DOI] [PubMed] [Google Scholar]
  22. Morel F., Doussiere J., Vignais P. V. The superoxide-generating oxidase of phagocytic cells. Physiological, molecular and pathological aspects. Eur J Biochem. 1991 Nov 1;201(3):523–546. doi: 10.1111/j.1432-1033.1991.tb16312.x. [DOI] [PubMed] [Google Scholar]
  23. Nauseef W. M., Volpp B. D., McCormick S., Leidal K. G., Clark R. A. Assembly of the neutrophil respiratory burst oxidase. Protein kinase C promotes cytoskeletal and membrane association of cytosolic oxidase components. J Biol Chem. 1991 Mar 25;266(9):5911–5917. [PubMed] [Google Scholar]
  24. Park J. W., Babior B. M. The translocation of respiratory burst oxidase components from cytosol to plasma membrane is regulated by guanine nucleotides and diacylglycerol. J Biol Chem. 1992 Oct 5;267(28):19901–19906. [PubMed] [Google Scholar]
  25. Park J. W., Benna J. E., Scott K. E., Christensen B. L., Chanock S. J., Babior B. M. Isolation of a complex of respiratory burst oxidase components from resting neutrophil cytosol. Biochemistry. 1994 Mar 15;33(10):2907–2911. doi: 10.1021/bi00176a021. [DOI] [PubMed] [Google Scholar]
  26. Park J. W., Ma M., Ruedi J. M., Smith R. M., Babior B. M. The cytosolic components of the respiratory burst oxidase exist as a M(r) approximately 240,000 complex that acquires a membrane-binding site during activation of the oxidase in a cell-free system. J Biol Chem. 1992 Aug 25;267(24):17327–17332. [PubMed] [Google Scholar]
  27. Parkos C. A., Dinauer M. C., Jesaitis A. J., Orkin S. H., Curnutte J. T. Absence of both the 91kD and 22kD subunits of human neutrophil cytochrome b in two genetic forms of chronic granulomatous disease. Blood. 1989 May 1;73(6):1416–1420. [PubMed] [Google Scholar]
  28. Parkos C. A., Dinauer M. C., Walker L. E., Allen R. A., Jesaitis A. J., Orkin S. H. Primary structure and unique expression of the 22-kilodalton light chain of human neutrophil cytochrome b. Proc Natl Acad Sci U S A. 1988 May;85(10):3319–3323. doi: 10.1073/pnas.85.10.3319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Quinn M. T., Evans T., Loetterle L. R., Jesaitis A. J., Bokoch G. M. Translocation of Rac correlates with NADPH oxidase activation. Evidence for equimolar translocation of oxidase components. J Biol Chem. 1993 Oct 5;268(28):20983–20987. [PubMed] [Google Scholar]
  30. Ren R., Mayer B. J., Cicchetti P., Baltimore D. Identification of a ten-amino acid proline-rich SH3 binding site. Science. 1993 Feb 19;259(5098):1157–1161. doi: 10.1126/science.8438166. [DOI] [PubMed] [Google Scholar]
  31. Rotrosen D., Yeung C. L., Katkin J. P. Production of recombinant cytochrome b558 allows reconstitution of the phagocyte NADPH oxidase solely from recombinant proteins. J Biol Chem. 1993 Jul 5;268(19):14256–14260. [PubMed] [Google Scholar]
  32. Rotrosen D., Yeung C. L., Leto T. L., Malech H. L., Kwong C. H. Cytochrome b558: the flavin-binding component of the phagocyte NADPH oxidase. Science. 1992 Jun 5;256(5062):1459–1462. doi: 10.1126/science.1318579. [DOI] [PubMed] [Google Scholar]
  33. Someya A., Nagaoka I., Yamashita T. Purification of the 260 kDa cytosolic complex involved in the superoxide production of guinea pig neutrophils. FEBS Lett. 1993 Sep 13;330(2):215–218. doi: 10.1016/0014-5793(93)80276-z. [DOI] [PubMed] [Google Scholar]
  34. Sumimoto H., Kage Y., Nunoi H., Sasaki H., Nose T., Fukumaki Y., Ohno M., Minakami S., Takeshige K. Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5345–5349. doi: 10.1073/pnas.91.12.5345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thrasher A. J., Keep N. H., Wientjes F., Segal A. W. Chronic granulomatous disease. Biochim Biophys Acta. 1994 Oct 21;1227(1-2):1–24. doi: 10.1016/0925-4439(94)90100-7. [DOI] [PubMed] [Google Scholar]
  36. Tsunawaki S., Mizunari H., Nagata M., Tatsuzawa O., Kuratsuji T. A novel cytosolic component, p40phox, of respiratory burst oxidase associates with p67phox and is absent in patients with chronic granulomatous disease who lack p67phox. Biochem Biophys Res Commun. 1994 Mar 30;199(3):1378–1387. doi: 10.1006/bbrc.1994.1383. [DOI] [PubMed] [Google Scholar]
  37. Tsunawaki S., Mizunari H., Namiki H., Kuratsuji T. NADPH-binding component of the respiratory burst oxidase system: studies using neutrophil membranes from patients with chronic granulomatous disease lacking the beta-subunit of cytochrome b558. J Exp Med. 1994 Jan 1;179(1):291–297. doi: 10.1084/jem.179.1.291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Volpp B. D., Nauseef W. M., Donelson J. E., Moser D. R., Clark R. A. Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7195–7199. doi: 10.1073/pnas.86.18.7195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wientjes F. B., Hsuan J. J., Totty N. F., Segal A. W. p40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains. Biochem J. 1993 Dec 15;296(Pt 3):557–561. doi: 10.1042/bj2960557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Woodman R. C., Ruedi J. M., Jesaitis A. J., Okamura N., Quinn M. T., Smith R. M., Curnutte J. T., Babior B. M. Respiratory burst oxidase and three of four oxidase-related polypeptides are associated with the cytoskeleton of human neutrophils. J Clin Invest. 1991 Apr;87(4):1345–1351. doi: 10.1172/JCI115138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. de Mendez I., Garrett M. C., Adams A. G., Leto T. L. Role of p67-phox SH3 domains in assembly of the NADPH oxidase system. J Biol Chem. 1994 Jun 10;269(23):16326–16332. [PubMed] [Google Scholar]
  42. el Benna J., Faust L. P., Babior B. M. The phosphorylation of the respiratory burst oxidase component p47phox during neutrophil activation. Phosphorylation of sites recognized by protein kinase C and by proline-directed kinases. J Biol Chem. 1994 Sep 23;269(38):23431–23436. [PubMed] [Google Scholar]
  43. el Benna J., Ruedi J. M., Babior B. M. Cytosolic guanine nucleotide-binding protein Rac2 operates in vivo as a component of the neutrophil respiratory burst oxidase. Transfer of Rac2 and the cytosolic oxidase components p47phox and p67phox to the submembranous actin cytoskeleton during oxidase activation. J Biol Chem. 1994 Mar 4;269(9):6729–6734. [PubMed] [Google Scholar]

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