title: NOXO1 phosphorylation on serine 154 is critical for optimal NADPH oxidase 1 assembly and activation.
abstract: Reactive oxygen species (ROS) production by NADPH oxidase 1 (NOX1), which is mainly expressed in colon epithelial cells, requires the membrane-bound component p22(PHOX) and the cytosolic partners NOX organizer 1 (NOXO1), NOX activator 1 (NOXA1), and Rac1. Contrary to that of its phagocyte counterpart NOX2, the molecular basis of NOX1 regulation is not clear. Because NOXO1 lacks the phosphorylated region found in its homolog p47(PHOX), the current view is that NOX1 activation occurs without NOXO1 phosphorylation. Here, however, we demonstrate that phorbol myristate acetate (PMA) stimulates NOXO1 phosphorylation in a transfected human embryonic kidney (HEK) 293 epithelial cell model via protein kinase C and identify Ser-154 as the major phosphorylated site. Endogenous NOXO1 from T84 colon epithelial cells was also phosphorylated, suggesting that NOXO1 phosphorylation is physiologically relevant. In transfected HEK-293 cells, PMA-induced phosphorylation on Ser-154 enhanced NOXO1 binding to NOXA1 (+97%) and to the p22(PHOX) C-terminal region (+384%), increased NOXO1 colocalization with p22(PHOX), and allowed optimal ROS production by NOX1 as demonstrated by the use of S154A and S154D mutants compared with that by wild-type NOXO1 (P<0.05). Pulldown experiments revealed that phos-phorylation on Ser-154 was sufficient to markedly enhance NOXO1 binding to NOXA1, which in turn acts as a molecular switch, allowing optimal interaction of NOXO1 with p22(PHOX). This study unexpectedly revealed that full assembly and activation of NOX1 is a tightly regulated process in which NOXO1 phosphorylation on Ser-154 is the initial trigger.
authors: Institut National de Santé et de Recherche Médicale (INSERM) U773, Centre de Recherche Biomédicale Bichat Beaujon CRB3, Paris, France.
authors: Maya
authors: M
authors: Debbabi
authors: Yolande
authors: Y
authors: Kroviarski
authors: Odile
authors: O
authors: Bournier
authors: Marie-Anne
authors: MA
authors: Gougerot-Pocidalo
authors: Jamel
authors: J
authors: El-Benna
authors: Pham My-Chan
authors: PM
authors: Dang
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D057809:HEK293 Cells; D006801:Humans; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D010586:Phagocytes / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection / Q000379:methods
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C476419:NOXO1 protein, human; D017382:Reactive Oxygen Species; D013481:Superoxides; D012694:Serine; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate