Phosphorylation of NADPH oxidase activator 1 (NOXA1) on serine 282 by MAP kinases and on serine 172 by protein kinase C and protein kinase A prevents NOX1 hyperactivation.
NADPH oxidase activator 1 (NOXA1) together with NADPH oxidase organizer 1 (NOXO1) are key regulatory subunits of the NADPH oxidase NOX1. NOX1 is expressed mainly in colon epithelial cells and could be involved in mucosal innate immunity by producing reactive oxygen species (ROS). Contrary to its phagocyte counterpart NOX2, the mechanisms involved in NOX1 activation and regulation remain unclear. Here we report that NOX1 activity is regulated through MAP kinase (MAPK), protein kinase C (PKC), and protein kinase A (PKA)-dependent phosphorylation of NOXA1. We identified Ser-282 as target of MAPK and Ser-172 as target of PKC and PKA in vitro and in a transfected human embryonic kidney 293 (HEK293) cell model using site directed mutagenesis and phosphopeptide mapping analysis. In HEK293 cells, phosphorylation of these sites occurred at a basal level and down-regulated constitutive NOX1 activity. Indeed, S172A and S282A single mutants of NOXA1 significantly up-regulated constitutive NOX1-derived ROS production, and S172A/S282A double mutant further increased it, as compared to wild-type NOXA1. Furthermore, phosphorylation of NOXA1 on Ser-282 and Ser-172 decreased its binding to NOX1 and Rac1. These results demonstrated a critical role of NOXA1 phosphorylation on Ser-282 and Ser-172 in preventing NOX1 hyperactivation through the decrease of NOXA1 interaction to NOX1 and Rac1.
Kroviarski
Yolande
Y
INSERM U773, Centre de Recherche Biomédicale Bichat Beaujon, Paris, France.
Debbabi
Maya
M
Bachoual
Rafik
R
Périanin
Axel
A
Gougerot-Pocidalo
Marie-Anne
MA
El-Benna
Jamel
J
Dang
Pham My-Chan
PM
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D047468:Immunoprecipitation; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C476420:NOXA1 protein, human; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D012694:Serine; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C402799:NOX1 protein, human; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D020928:Mitogen-Activated Protein Kinases; D020744:rac GTP-Binding Proteins