Am J Blood Res 2012;2(3):187-193
Original Article
Phosphorylation of p47phox is required for receptor-mediated NADPH
oxidase/NOX2 activation in Epstein-Barr virus-transformed human B
lymphocytes
Sahra Amel Belambri, Margarita Hurtado-Nedelec, Abderrahmane Senator, Karama Makni-Maalej, Michèle Fay, Marie-Anne
Gougerot-Pocidalo, Jean-Claude Marie, Pham My-Chan Dang, Jamel El-Benna
INSERM, U773, Centre de Recherche Biomédicale Bichat Beaujon CRB3, Paris F-75018, France; Université Paris 7 site
Bichat, UMRS 773, Paris F-75018, France; AP-HP, Centre Hospitalier Universitaire Xavier Bichat, UF Dysfonctionnements
Immunitaires, Paris, F-75018; Laboratoire de Biochimie Appliquée, Equipe de recherche: Stress oxydatif et inflammation,
Département de Biologie, Faculté des Sciences, Université Ferhat Abbas, Sétif, Algéria
Received September 17, 2012; accepted October 10, 2012; Epub October 20, 2012; Published October 30, 2012
Abstract: The phagocyte NADPH oxidase (NOX2) is known to be expressed in Epstein-Barr virus (EBV)-transformed human B
lymphocytes. Phosphorylation of the NOX2 cytosolic subunit p47phox is required for phorbol myristate acetate (PMA)-induced
NOX2 activation in EBV-transformed B lymphocytes, however the role of this process in receptor-mediated NOX2 activation is
not known. Here, we used pansorbin which acts by cross linking cell surface IgG and transfected cells with mutated p47phox
to address if the phosphorylation of this subunit is required for receptor-mediated NOX2 activation. We show that pansorbin
induced NOX2 activation in a time and concentration-dependent manner, albeit at levels only of 20% of those induced by PMA.
GF109203X, a PKC selective inhibitor, inhibited pansorbin as well as PMA-induced NOX2 activation. Using specific
anti-phospho serine antibodies we showed that pansorbin induced p47phox phosphorylation on Ser304, 315, 320, 328, and
345 and kinetics of these phosphorylations preceed NOX2 activation. To determine whether the phosphorylation of p47phox is
required for pansorbin-induced NOX2 activation, we transfected EBV-transformed lymphocytes deficent in p47phox with a
plasmid expressing wild type p47phox or p47phox with all the phosphorylated serines mutated to alanines,
p47phoxS(303-379)A. Results show that pansorbin-induced NOX2 activation was greatly decreased in lymphocytes
expressing the mutant as compared to the wild-type p47phox. These results show that pansorbin induced p47phox
phosphorylation on multiple sites in EBV-transformed B lymphocytes and this process is required for pansorbin-induced
NADPH oxidase activation in these cells. (AJBR1209003).
Keywords: NADPH oxidase, NOX2, p47phox, B lymphocytes, pansorbin, ROS, phosphorylation
Address all correspondence to:
Dr. Jamel El-Benna, INSERM, U773, CRB3, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, Paris F-75018, France.
Tél: 33157277723; E-mail: jamel.elbenna@inserm.fr
Original Article
Phosphorylation of p47phox is required for receptor-mediated NADPH
oxidase/NOX2 activation in Epstein-Barr virus-transformed human B
lymphocytes
Sahra Amel Belambri, Margarita Hurtado-Nedelec, Abderrahmane Senator, Karama Makni-Maalej, Michèle Fay, Marie-Anne
Gougerot-Pocidalo, Jean-Claude Marie, Pham My-Chan Dang, Jamel El-Benna
INSERM, U773, Centre de Recherche Biomédicale Bichat Beaujon CRB3, Paris F-75018, France; Université Paris 7 site
Bichat, UMRS 773, Paris F-75018, France; AP-HP, Centre Hospitalier Universitaire Xavier Bichat, UF Dysfonctionnements
Immunitaires, Paris, F-75018; Laboratoire de Biochimie Appliquée, Equipe de recherche: Stress oxydatif et inflammation,
Département de Biologie, Faculté des Sciences, Université Ferhat Abbas, Sétif, Algéria
Received September 17, 2012; accepted October 10, 2012; Epub October 20, 2012; Published October 30, 2012
Abstract: The phagocyte NADPH oxidase (NOX2) is known to be expressed in Epstein-Barr virus (EBV)-transformed human B
lymphocytes. Phosphorylation of the NOX2 cytosolic subunit p47phox is required for phorbol myristate acetate (PMA)-induced
NOX2 activation in EBV-transformed B lymphocytes, however the role of this process in receptor-mediated NOX2 activation is
not known. Here, we used pansorbin which acts by cross linking cell surface IgG and transfected cells with mutated p47phox
to address if the phosphorylation of this subunit is required for receptor-mediated NOX2 activation. We show that pansorbin
induced NOX2 activation in a time and concentration-dependent manner, albeit at levels only of 20% of those induced by PMA.
GF109203X, a PKC selective inhibitor, inhibited pansorbin as well as PMA-induced NOX2 activation. Using specific
anti-phospho serine antibodies we showed that pansorbin induced p47phox phosphorylation on Ser304, 315, 320, 328, and
345 and kinetics of these phosphorylations preceed NOX2 activation. To determine whether the phosphorylation of p47phox is
required for pansorbin-induced NOX2 activation, we transfected EBV-transformed lymphocytes deficent in p47phox with a
plasmid expressing wild type p47phox or p47phox with all the phosphorylated serines mutated to alanines,
p47phoxS(303-379)A. Results show that pansorbin-induced NOX2 activation was greatly decreased in lymphocytes
expressing the mutant as compared to the wild-type p47phox. These results show that pansorbin induced p47phox
phosphorylation on multiple sites in EBV-transformed B lymphocytes and this process is required for pansorbin-induced
NADPH oxidase activation in these cells. (AJBR1209003).
Keywords: NADPH oxidase, NOX2, p47phox, B lymphocytes, pansorbin, ROS, phosphorylation
Address all correspondence to:
Dr. Jamel El-Benna, INSERM, U773, CRB3, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, Paris F-75018, France.
Tél: 33157277723; E-mail: jamel.elbenna@inserm.fr
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