title: Phosphorylation of p47phox is required for receptor-mediated NADPH oxidase/NOX2 activation in Epstein-Barr virus-transformed human B lymphocytes.
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.
authors.affiliation: INSERM, U773, Centre de Recherche Biomédicale Bichat Beaujon CRB3 Paris F-75018, France ; Université Paris 7 site Bichat UMRS 773, Paris F-75018, France.
authors.forename: Sahra Amel
authors.initials: SA
authors.lastname: Belambri
authors.forename: Margarita
authors.initials: M
authors.lastname: Hurtado-Nedelec
authors.forename: Abderrahmane
authors.initials: A
authors.lastname: Senator
authors.forename: Karama
authors.initials: K
authors.lastname: Makni-Maalej
authors.forename: Michèle
authors.initials: M
authors.lastname: Fay
authors.forename: Marie-Anne
authors.initials: MA
authors.lastname: Gougerot-Pocidalo
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Marie
authors.forename: Pham My-Chan
authors.initials: PM
authors.lastname: Dang
authors.forename: Jamel
authors.initials: J
authors.lastname: El-Benna
keywords: B lymphocytes; NADPH oxidase; NOX2; ROS; p47phox; pansorbin; phosphorylation