@article {Pandeymol.110.070193, author = {Deepesh Pandey and Jean-Philippe Gratton and Ruslan Rafikov and Stephen M Black and David Fulton}, title = {Calcium/Calmodulin- dependent Kinase II mediates the Phosphorylation and Activation of NADPH Oxidase 5}, elocation-id = {mol.110.070193}, year = {2011}, doi = {10.1124/mol.110.070193}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {Excessive synthesis of reactive oxygen species (ROS) contributes to the pathology of many human diseases and originates from changes in the expression and posttranslational regulation of the transmembrane NADPH oxidases (Nox). Nox5 is a novel Nox isoform whose activity is regulated by intracellular calcium levels. We recently 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 if calcium activated kinases such as CAMKII are involved. We found that Nox5 activity in BAEC was suppressed by 2 doses of the CAMKII inhibitor, KN-93. In co-transfected COS-7 cells, WT 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 onT494 and S498 as detected by phosphorylation state-specific antibodies. MS analysis revealed the phosphorylation of additional, novel sites at S475, S502 and S675. Of these phosphorylation sites, mutation of only S475 to alanine prevented CAMKII-induced increases in Nox5 activity. The ability of CAMKIIα to phosphorylate S475 in intact cells was supported by the binding of Nox5 to phosphoprotein-affinity columns and via MS/MS analysis. Collectively these results suggest that CAMKII can positively regulate Nox5 activity via the phosphorylation of S475.}, issn = {0026-895X}, URL = {https://molpharm.aspetjournals.org/content/early/2011/06/03/mol.110.070193}, eprint = {https://molpharm.aspetjournals.org/content/early/2011/06/03/mol.110.070193.full.pdf}, journal = {Molecular Pharmacology} }