Abstract 326: Regulation of Endothelial NO Synthase Serine 1179 Phosphorylation and Function by Protein Phosphatase 1
Endothelial NO synthase (eNOS) function is critically modulated by protein phosphorylation. In particular, phosphorylation of eNOS serine 1179/1177 (bovine/human) residue has emerged as a central mechanism of eNOS regulation in response to hormones, share stress, oxidant stress, etc. While the kinases that phosphorylate eNOS S1179 have been well established, the dephosphorylating pathways are not completely understood. It was reported that eNOS S1179 is dephosphorylated by protein phosphatase 2A (PP2A). Here we provide in vitro and cell culture evidence demonstrating that eNOS S1179 phosphorylation and function are also critically modulated by protein phosphatase 1 (PP1). Incubating isolated bovine eNOS with endothelial cell lysates resulted in marked S1179 dephosphorylation. This dephosphorylation was dose-dependently reversed by the endogenous PP1 specific protein phosphatase inhibitor-2, suggesting that eNOS S1179 was dephosphorylated by PP1. Indeed, purified PP1 dephospho-rylated eNOS S1179. Compared to the effect of purified PP2A, PP1 exhibited near two-fold higher potency in dephosphorylating eNOS S1179 in vitro (186±32.9% of PP2A, P<0.01, n=3). To determine the role of PP1 in modulating eNOS S1179 phosphorylation in cells, we treated bovine aortic endothelial cells with PP1 specific inhibitor tautomycetin (2.5 μM). Tautomycetin markedly enhanced eNOS S1179 phosphorylation in cells. To corroborate the results obtained by pharmacological inhibition, we knocked down intracellular PP1 with siRNA. PP1 knockdown markedly increased eNOS S1179 phosphorylation. Finally, blockade of both PP1 and PP2A resulted in more dramatic increases of eNOS S1179 phosphorylation than blocking PP2A alone. Consequently, PP1/PP2A-inhibited cells displayed increased eNOS activity than PP2A-inhibited cells (29.4±2.5 versus 20.9±3.8 pmol/mg/min, P<0.05, n=3). Taken together, both in vitro evidence with purified phosphatases and cell culture experiments with PP1 inhibitors or RNAi demonstrated that besides PP2A, PP1 also played a crucial role in regulating eNOS S1179 phosphorylation. Intervening PP1 may be another important approach to modulate eNOS S1179 phosphorylation and function in cardiovascular regulation and diseases.