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Evaluation of p47phox Phosphorylation in Human Neutrophils Using Phospho-Specific Antibodies

  • Sahra Amel Belambri
  • Pham My-Chan Dang
  • Jamel El-Benna
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1124)

Abstract

Superoxide anions production by neutrophils plays a key role in host defense against pathogens and in inflammation. The enzyme responsible for this process is called the NADPH oxidase. It is a multicomponent enzyme comprised of a membrane-bound flavocytochrome b558 and several cytosolic proteins (p47phox, p67phox, p40phox, and p21rac1/2). The phosphorylation of p47phox is essential for the activation of the complex in intact cells. Until recently, analysis of the phosphorylation of p47phox in neutrophils required radioactive labeling, which implied the use of high amount of radioactive (32P)-orthophosphoric acid, high number of cells, and protein recovery by immunoprecipitation. In this study, we describe a radioactive-free technique to analyze the phosphorylation of p47phox in cell lysates, based on the use of phospho-specific antibodies, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting. This technique could be used to quickly and easily study the phosphorylation of p47phox under different conditions, such as testing the effects of pharmacological agents in this process or assessing the activation status of neutrophils in situ.

Keywords

Neutrophils p47phox-phosphorylation NADPH oxidase NOX2 Phospho-specific antibodies 

Notes

Acknowledgments

This work was supported by grants from INSERM, CNRS, University Paris7, the Labex Inflammex, and the association Vaincre la mucoviscidose (VLM).

References

  1. 1.
    Nauseef WM (2007) How human neutrophils kill and degrade microbes: an integrated view. Immunol Rev 219:88–102PubMedCrossRefGoogle Scholar
  2. 2.
    El-Benna J, Dang PM, Gougerot-Pocidalo MA, Elbim C (2005) Phagocyte NADPH oxidase: a multicomponent enzyme essential for host defenses. Arch Immunol Ther Exp (Warsz) 3:199–206Google Scholar
  3. 3.
    Babior BM (1999) NADPH oxidase: an update. Blood 93:1464–1476PubMedGoogle Scholar
  4. 4.
    Chanock SJ, El Benna J, Smith RM, Babior BM (1994) The respiratory burst oxidase. J Biol Chem 269:24519–24522PubMedGoogle Scholar
  5. 5.
    Groemping Y, Rittinger K (2005) Activation and assembly of the NADPH oxidase: a structural perspective. Biochem J 386:401–416PubMedCrossRefGoogle Scholar
  6. 6.
    DeLeo FR, Quinn MT (1996) Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins. J Leukoc Biol 60:677–691PubMedGoogle Scholar
  7. 7.
    Quinn MT, Gauss KA (2004) Structure and regulation of the neutrophil respiratory burst oxidase: comparison with nonphagocyte oxidases. J Leukoc Biol 76:760–781PubMedCrossRefGoogle Scholar
  8. 8.
    El-Benna J, Dang PMC, Gougerot-Pocidalo MA, Marie JC, Braut-Boucher F (2009) p47phox, the phagocyte NADPH oxidase/NOX2 organizer: structure, phosphorylation and implication in diseases. Exp Mol Med 41:217–225PubMedCrossRefGoogle Scholar
  9. 9.
    El-Benna J, Faust LP, Babior BM (1994) 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 269:23431–23436PubMedGoogle Scholar
  10. 10.
    El Benna J, Faust LP, Johnson JL, Babior BM (1996) Phosphorylation of the respiratory burst oxidase subunit p47phox as determined by two-dimensional phosphopeptide mapping. Phosphorylation by protein kinase C, protein kinase A, and a mitogen-activated protein kinase. J Biol Chem 271:6374–6378PubMedCrossRefGoogle Scholar
  11. 11.
    Faust LR, el Benna J, Babior BM, Chanock SJ (1995) The phosphorylation targets of p47phox, a subunit of the respiratory burst oxidase. Functions of the individual target serines as evaluated by site-directed mutagenesis. J Clin Invest 96:1499–1505PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Belambri SA, Hurtado-Nedelec M, Senator A, Makni-Maalej K, Fay M, Gougerot-Pocidalo MA, Marie JC, Dang PM, El-Benna J (2012) Phosphorylation of p47phox is required for receptor-mediated NADPH oxidase/NOX2 activation in Epstein-Barr virus-transformed human B lymphocytes. Am J Blood Res 2:187–193PubMedCentralPubMedGoogle Scholar
  13. 13.
    El-Benna J, Dang PMC, Gougerot-Pocidalo MA (2008) Priming of the neutrophil NADPH oxidase activation: role of p47phox phosphorylation and NOX2 mobilization to the plasma membrane. Semin Immunopathol 30:279–289PubMedCrossRefGoogle Scholar
  14. 14.
    Sheppard FR, Kelher MR, Moore EE, McLaughlin NJ, Banerjee A, Silliman CC (2005) Structural organization of the neutrophil NADPH oxidase: phosphorylation and translocation during priming and activation. J Leukoc Biol 78:1025–1042PubMedCrossRefGoogle Scholar
  15. 15.
    Dewas C, Dang PM, Gougerot-Pocidalo MA, El-Benna J (2003) TNF-alpha induces phosphorylation of p47(phox) in human neutrophils: partial phosphorylation of p47phox is a common event of priming of human neutrophils by TNF-alpha and granulocyte-macrophage colony-stimulating factor. J Immunol 171:4392–4398PubMedGoogle Scholar
  16. 16.
    Dang PMC, Stensballe A, Boussetta T, Raad H, Dewas C, Kroviarski Y, Hayem G, Jensen ON, Gougerot-Pocidalo MA, El-Benna J (2006) A specific p47phox-serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites. J Clin Invest 116:2033–2043PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    DeLeo FR, Renee J, McCormick S, Nakamura M, Apicella M, Weiss JP, Nauseef WM (1998) Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly. J Clin Invest 101:455–463PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Boussetta T, Gougerot-Pocidalo MA, Hayem G, Ciappelloni S, Raad H, Arabi Derkawi R, Bournier O, Kroviarski Y, Zhou XZ, Malter JS, Lu PK, Bartegi A, Dang PM, El-Benna J (2010) The prolyl isomerase Pin1 acts as a novel molecular switch for TNF-alpha-induced priming of the NADPH oxidase in human neutrophils. Blood 116:5795–5802PubMedCrossRefGoogle Scholar
  19. 19.
    Makni-Maalej K, Boussetta T, Hurtado-Nedelec M, Belambri SA, Gougerot-Pocidalo MA, El-Benna J (2012) The TLR7/8 agonist CL097 primes N-formyl-methionyl-leucyl-phenylalanine-stimulated NADPH oxidase activation in human neutrophils: critical role of p47phox phosphorylation and the proline isomerase Pin1. J Immunol 189:4657–4665PubMedCrossRefGoogle Scholar
  20. 20.
    El-Benna J, Dang PM (2007) Analysis of protein phosphorylation in human neutrophils. Methods Mol Biol 412:85–96PubMedCrossRefGoogle Scholar
  21. 21.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  22. 22.
    Amrein PC, Stossel TP (1980) Prevention of degradation of human polymorphonuclear leukocyte proteins by diisopropylfluorophosphate. Blood 56:442–447PubMedGoogle Scholar
  23. 23.
    Towbin H, Staehlin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Sahra Amel Belambri
    • 1
  • Pham My-Chan Dang
    • 2
  • Jamel El-Benna
    • 2
  1. 1.Département de Biologie, Faculté des SciencesUniversité Ferhat AbbasSétifAlgeria
  2. 2.Faculté de MedecineCentre de Recherche Biomédicale Bichat Beaujon, INSERM U773ParisFrance

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