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Endothelial nitric oxide synthase is regulated by ERK phosphorylation at Ser602.

ABSTRACT: eNOS (endothelial nitric oxide synthase) contains a MAPK (mitogen-activated protein kinase)-binding site associated with a major eNOS control element. Purified ERK (extracellular-signal-regulated kinase) phosphorylates eNOS with a stoichiometry of 2-3 phosphates per eNOS monomer. Phosphorylation decreases NO synthesis and cytochrome c reductase activity. Three sites of phosphorylation were detected by MS. All sites matched the SP and TP MAPK (mitogen-activated protein kinase) phosphorylation motif. Ser602 lies at the N-terminal edge of the 42-residue eNOS AI (autoinhibitory) element. The pentabasic MAPK-binding site lies at the opposite end of the AI, and other critical regulatory features are between them. Thr46 and Ser58 are located in a flexible region associated with the N terminus of the oxygenase domain. In contrast with PKC (protein kinase C), phosphorylation by ERK did not significantly interfere with CaM (calmodulin) binding as analysed by optical biosensing. Instead, ERK phosphorylation favours a state in which FMN and FAD are in close association and prevents conformational changes that expose reduced FMN to acceptors. The close associations between control sites in a few regions of the molecule suggest that control of signal generation is modulated by multiple inputs interacting directly on the surface of eNOS via overlapping binding domains and tightly grouped targets.

SUBMITTER: Salerno JC

PROVIDER: S-EPMC4166121 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Endothelial nitric oxide synthase is regulated by ERK phosphorylation at Ser602.

Salerno John C JC Ghosh Dipak K DK Razdan Raj R Helms Katy A KA Brown Christopher C CC McMurry Jonathan L JL Rye Emily A EA Chrestensen Carol A CA

Bioscience reports 20140917 5

eNOS (endothelial nitric oxide synthase) contains a MAPK (mitogen-activated protein kinase)-binding site associated with a major eNOS control element. Purified ERK (extracellular-signal-regulated kinase) phosphorylates eNOS with a stoichiometry of 2-3 phosphates per eNOS monomer. Phosphorylation decreases NO synthesis and cytochrome c reductase activity. Three sites of phosphorylation were detected by MS. All sites matched the SP and TP MAPK (mitogen-activated protein kinase) phosphorylation mot ...[more]

PMID: 25000310

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Project description:Icariside II, as a favonoid compound derived from epimedium, has been proved to involed in a variety of biological and pharmacological effects such as anti-inflammatory, anti-osteoporosis, anti-oxidation, anti-aging, and anti-cancer but its mechanism is unclear, especially in terms of its effect on post-transcriptional modification of endothelial nitric oxide synthase (eNOS). Phosphorylation of eNOS plays an important role in the synthesis of nitric oxide in endothelial cells, which is closely related to erectile dysfunction, atherosclerosis, Alzheimer's disease, and other diseases. Our study aims to investigate the effect and mechanism of Icariside II on the rapid phosphorylation of eNOS. In this study, human umbilical vein endothelial cells (HUVECs) were stimulated with Icariside II in the presence or absence of multiple inhibitors (1 µM), including LY294002 (PI3K-inhibitor), MK-2206 (AKT-inhibitor), Bisindolylmaleimide X (AMPK-inhibitor), H-89 (CaMKII-inhibitor), KN-62 (PKA-inhibitor), Dorsomorphin (PKC-inhibitor). The proliferation of HUVECs was assessed using cell counting kit-8 (CCK-8). The release of nitric oxide (NO) within HUVECs was detected via fluorescence probe (DAF-FM). Western blot was used to examine the effect of Icariside II on the expression of eNOS, phosphorylation of eNOS, and common signaling pathways proteins. In this study, Icariside II was found to promote the cell proliferation and rapid NO release in HUVECs. The phosphorylation of eNOS-Ser1177 was significantly increased after Icariside II stimulation and reached a peak at 10 min (p < 0.05). Meanwhile, the phosphorylation of eNOS-Thr495 was significantly decreased after 45 min of stimulation (p < 0.05). Following the intervention with multiple inhibitors, it was found that MK-2206 (AKT inhibitor), LY294002 (PI3K inhibitor), KN-62 (AMPK inhibitor), and Bisindolylmaleimide X (PKC inhibitor) could significantly inhibit the phosphorylation of eNOS-Ser1177 caused by Icariside II (p < 0.05), while MK-2206, LY294002, and Bisindolylmaleimide X reversed the alleviated phosphorylation of eNOS-Thr495. We concluded that Icariside can regulate rapid phosphorylation of eNOS- Ser1177 and eNOS-Thr495 via multiple signaling pathways, resulting in the up-regulation of eNOS and the increased release of NO.

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Endothelial nitric oxide synthase is regulated by ERK phosphorylation at Ser602.

Publications

Endothelial nitric oxide synthase is regulated by ERK phosphorylation at Ser602.

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