title: Phosphorylation of Noxo1 at threonine 341 regulates its interaction with Noxa1 and the superoxide-producing activity of Nox1.
abstract: Superoxide production by Nox1, a member of the Nox family NAPDH oxidases, requires expression of its regulatory soluble proteins Noxo1 (Nox organizer 1) and Noxa1 (Nox activator 1) and is markedly enhanced upon cell stimulation with phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC). The mechanism underlying PMA-induced enhancement of Nox1 activity, however, remains to be elucidated. Here we show that, in response to PMA, Noxo1 undergoes phosphorylation at multiple sites, which is inhibited by the PKC inhibitor GF109203X. Among them, Thr341 in Noxo1 is directly phosphorylated by PKC in vitro, and alanine substitution for this residue reduces not only PMA-induced Noxo1 phosphorylation but also PMA-dependent enhancement of Nox1-catalyzed superoxide production. Phosphorylation of Thr341 allows Noxo1 to sufficiently interact with Noxa1, an interaction that participates in Nox1 activation. Thus phosphorylation of Noxo1 at Thr341 appears to play a crucial role in PMA-elicited activation of Nox1, providing a molecular link between PKC-mediated signal transduction and Nox1-catalyzed superoxide production. Furthermore, Ser154 in Noxo1 is phosphorylated in both resting and PMA-stimulated cells, and the phosphorylation probably participates in a PMA-independent constitutive activity of Nox1. Ser154 may also be involved in protein kinase A (PKA) mediated regulation of Nox1; this serine is the major residue that is phosphorylated by PKA in vitro. Thus phosphorylation of Noxo1 at Thr341 and at Ser154 appears to regulate Nox1 activity in different manners.
  Noxo1 binds to p22phox by pull down (1, 2, 3) Noxo1 binds to Noxo1 by pull down (View interaction) Noxa1 binds to Noxo1 by pull down (1, 2, 3, 4, 5).
authors: Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.
authors: Asataro
authors: A
authors: Yamamoto
authors: Ryu
authors: R
authors: Takeya
authors: Masaki
authors: M
authors: Matsumoto
authors: Keiichi I
authors: KI
authors: Nakayama
authors: Hideki
authors: H
authors: Sumimoto
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D002853:Chromatography, Liquid; D006224:Cricetinae; D003412:Cricetulus; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006801:Humans; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013481:Superoxides / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C476420:NOXA1 protein, human; C476419:NOXO1 protein, human; D013481:Superoxides; D013912:Threonine; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C402799:NOX1 protein, human; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate
keywords: NADPH oxidase; Nox1; Noxa1; Noxo1; protein kinase A; protein kinase C