title: Calcium/calmodulin-dependent kinase II mediates the phosphorylation and activation of NADPH oxidase 5.
abstract: Excessive synthesis of reactive oxygen species contributes to the pathology of many human diseases and originates from changes in the expression and posttranslational regulation of the transmembrane NADPH oxidases (Noxes). Nox5 is a novel Nox isoform whose activity is regulated by intracellular calcium levels. We have reported that the activity and calcium-sensitivity of Nox5 can also be modulated by direct phosphorylation. However, the kinases that phosphorylate Nox5 have not been identified, and thus, the goal of this study was to determine whether calcium-activated kinases such as calcium/calmodulin-dependent kinase II (CAMKII) are involved. We found that Nox5 activity in bovine aortic endothelial cells was suppressed by two doses of the CAMKII inhibitor 2-(N-[2-hydroxyethyl])-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylamine (KN-93). In cotransfected COS-7 cells, wild-type and constitutively active CAMKII, but not a dominant-negative, robustly increased basal Nox5 activity. The ability of CAMKII to increase Nox5 activity was also observed with fixed calcium concentrations in an isolated enzyme activity assay. CAMKII did not elevate intracellular calcium or activate other Nox enzymes. In vitro phosphorylation assays revealed that CAMKII can directly phosphorylate Nox5 on Thr494 and Ser498 as detected by phosphorylation state-specific antibodies. Mass spectrometry (MS) analysis revealed the phosphorylation of additional, novel sites at Ser475, Ser502, and Ser675. Of these phosphorylation sites, mutation of only Ser475 to alanine prevented CAMKII-induced increases in Nox5 activity. The ability of CAMKIIα to phosphorylate Ser475 in intact cells was supported by the binding of Nox5 to phosphoprotein-affinity columns and via MS/MS analysis. Together, these results suggest that CAMKII can positively regulate Nox5 activity via the phosphorylation of Ser475.
authors.affiliation: Vascular Biology Center, Medical College of Georgia, Augusta, GA, USA.
authors.forename: Deepesh
authors.initials: D
authors.lastname: Pandey
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Gratton
authors.forename: Ruslan
authors.initials: R
authors.lastname: Rafikov
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Black
authors.forename: David J R
authors.initials: DJ
authors.lastname: Fulton
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019255:NADPH Oxidases / Q000378:metabolism*; D010766:Phosphorylation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D019255:NADPH Oxidases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002118:Calcium