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 CAMKIIinduced increases in Nox5 activity. The ability of CAMKII␣ 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.

Nebivolol prevents desensitization of β-adrenoceptor signaling and induction of cardiac hypertrophy in response to isoprenaline beyond β1-adrenoceptor blockage

“…It is important to note that CaMKII activation also plays a pivotal role in certain pathological conditions like chronic ␤-adrenoceptor stimulation-induced cardiac hypertrophy (22,31) and heart failure (40). The results of two recent studies have pointed out a possible aspect about the synergistic effects of CaMKII and NADPH oxidase activation and reactive oxygen species production (11,26) in diseased heart. It could be possible to point out a relationship between increased cardiac oxidative stress by stimulation of NADPH oxidase activity (36,37) and enhanced CaMKII activity (22,31), both induced by chronic isoprenaline infusion.…”

Molecular Biology of Atherosclerosis

“…Activated ERK1/2 further promotes ROS production by phosphorylating NOX5 and p47 phox (1375). CaMKII also activates NOX5 (1376). Nevertheless, endothelial ERK1/2 activated by PYK2 partially overcomes ANG II-induced endothelial eNOS dysfunction by enhancing eNOS activity through phosphorylation of Ser633 (Ser635 in bovine endothelial cells) (1736,1737,1994).…”

“…A likely mechanism is through CaMKII activation of PYK2 which can serve as a scaffold to bind and activate the Grb2-Sos pathway for ERK1/2 activation (894,1736). CaMKII also directly phosphorylates endothelial NOX5 to promote superoxide production (1376). There may also be a mechanical effect of shear stress on glypican and CD44 to assist in caveolin-1 phosphorylation and removal of its suppressive effect on eNOS (1760).…”

NADPH Oxidase 5 Is a Pro‐Contractile Nox Isoform and a Point of Cross‐Talk for Calcium and Redox Signaling‐Implications in Vascular Function

“…Unique to Nox5 is its long N‐terminal extension that contains 4 calcium (Ca 2+ )‐binding EF hands, which undergoes conformational change upon Ca 2+ binding 17, 18. Increased intracellular free Ca 2+ concentration ([Ca 2+ ] i ) is essential for the activity of Nox5,18, 19, 20 as evidenced in cell‐culture studies where ROS are not synthesized in Ca 2+ ‐free buffer or from cells with truncation of the N‐terminal region of Nox5 21. This is of particular significance in vascular smooth muscle cells (VSMCs), given that increased [Ca 2+ ] i is the major trigger for vascular contraction 22, 23.…”