Phosphorylation of serine282 in NADPH oxidase activator 1 by Erk desensitizes EGF-induced ROS generation.
Accumulating evidence indicates that protein phosphorylation regulates Nox activity. In this report, we show that serine282 residue of Nox activator 1 (NoxA1) is phosphorylated by Erk in response to EGF resulting in desensitization of Nox1 activity. Specifically, murine NoxA1 is detected as two independent protein bands in SDS PAGE, and the form of protein with higher mobility shifted to and merged with the one with lower mobility in response to EGF treatment. Pretreatment with PD98059 resulted in inhibition of NoxA1 migration in response to EGF indicating that Erk was involved in the process. Site-directed mutagenesis showed that S282A mutant but not S239A mutant failed to respond to EGF, demonstrating that serine282 is the target amino acid of Erk. Expression of S282A mutant of NoxA1 in these cells led to increased superoxide anion production in response to EGF compared to expression of the wild type, whereas the expression of S282E, a phosphomimetic mutant, resulted in significantly decreased superoxide anion generation. We also tested whether the phosphorylation of serine282 of NoxA1 affects Rac activation. Expression of S282A mutant NoxA1 up-regulated the Rac activity, whereas expression of S282E mutant led to the abrogation of Rac activation. Taken together, these results demonstrate that phosphorylation of NoxA1 is a part of the feedback mechanism that functions through activation of Rac with a net outcome of negative modulation of Nox1 activity.
Oh
Hyunjin
H
Department of Life Sciences, Ewha Womans University, 11-1 Daehyun-Dong, Seodaemoon-Gu, Seoul 120-750, South Korea.
Jung
Hye Young
HY
Kim
Jaesang
J
Bae
Yun Soo
YS
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D025461:Feedback, Physiological; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D005419:Flavonoids; C476420:NOXA1 protein, human; D017382:Reactive Oxygen Species; D012694:Serine; D004815:Epidermal Growth Factor; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one