Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase

Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phospho rylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylatio n coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphory lating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated wi th eNOS, PP1 is responsible for dephosphorylation of Thr-495 based on its s pecificity for this site in both eNOS and the corresponding synthetic phosp hopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Tr eatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-m ediated dephosphorylation of Ser-1177, These results show that regulation o f eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases.