Serine 1179 Phosphorylation of Endothelial Nitric Oxide Synthase Increases Superoxide Generation and Alters Cofactor Regulation.

Hu Peng,Yugang Zhuang,Donghong He,Lishi Xie,Mark C Harbeck,Weiguo Chen

doi:10.1371/journal.pone.0142854

Hu Peng, Yugang Zhuang + Show 4 more

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https://doi.org/10.1371/journal.pone.0142854

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Endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. In addition to producing NO, eNOS can also generate superoxide (O2 -.) in the absence of the cofactor tetrahydrobiopterin (BH4). Previous studies have shown that bovine eNOS serine 1179 (Serine 1177/human) phosphorylation critically modulates NO synthesis. However, the effect of serine 1179 phosphorylation on eNOS superoxide generation is unknown. Here, we used the phosphomimetic form of eNOS (S1179D) to determine the effect of S1179 phosphorylation on superoxide generating activity, and its sensitivity to regulation by BH4, Ca2+, and calmodulin (CAM). S1179D eNOS exhibited significantly increased superoxide generating activity and NADPH consumption compared to wild-type eNOS (WT eNOS). The superoxide generating activities of S1179D eNOS and WT eNOS did not differ significantly in their sensitivity to regulation by either Ca2+ or CaM. The sensitivity of the superoxide generating activity of S1179D eNOS to inhibition by BH4 was significantly reduced compared to WT eNOS. In eNOS-overexpressing 293 cells, BH4 depletion with 10mM DAHP for 48 hours followed by 50ng/ml VEGF for 30 min to phosphorylate eNOS S1179 increased ROS accumulation compared to DAHP-only treated cells. Meanwhile, MTT assay indicated that overexpression of eNOS in HEK293 cells decreased cellular viability compared to control cells at BH4 depletion condition (P<0.01). VEGF-mediated Serine 1179 phosphorylation further decreased the cellular viability in eNOS-overexpressing 293 cells (P<0.01). Our data demonstrate that eNOS serine 1179 phosphorylation, in addition to enhancing NO production, also profoundly affects superoxide generation: S1179 phosphorylation increases superoxide production while decreasing sensitivity to the inhibitory effect of BH4 on this activity.

Highlights

Nitric oxide (NO) synthesized by the endothelial NO synthase plays important and diverse roles in numerous biological processes, including vascular tone regulation, vascular remodeling, and angiogenesis [1,2,3]
We compared the activities of wild type endothelial NO synthase (eNOS) (WT eNOS) and S1179D eNOS by measuring the L-[C14]arginine to L[C14]citrulline conversion rate
These results confirmed that L[C14]citrulline production was generated from eNOS and eNOS activity increased by phosphorylation

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Introduction

Nitric oxide (NO) synthesized by the endothelial NO synthase (eNOS) plays important and diverse roles in numerous biological processes, including vascular tone regulation, vascular remodeling, and angiogenesis [1,2,3]. Recent studies have shown that phosphorylation of eNOS enhances eNOS activity and alters eNOS enzyme characteristics. Studies of VEGF and shear-stress-mediated phosphorylation of eNOS indicated that the serine/threonine protein kinase Akt/PKB mediates the activation of eNOS, leading to increase NO production [9]. In the absence of Larginine and BH4, by the same mechanism used to generate NO [4, 11]. It is unclear if Serine 1179 phosphorylation regulates O2-. It is not known if O2-. generation by eNOS is regulated by Ca2+, CaM, and BH4 in the same manner as NO generation

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