1. Endoplasmic Reticulum Ca2+ Release Modulates Endothelial Nitric-oxide Synthase via Extracellular Signal-regulated Kinase (ERK) 1/2-mediated Serine 635 Phosphorylation.
- Author
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Zhihong Xiao, Tingting Wang, Honghua Qin, Chao Huang, Youmei Feng, and Yong Xia
- Subjects
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NITRIC-oxide synthases , *PHOSPHORYLATION , *ENDOPLASMIC reticulum , *PROTEIN kinases , *BRADYKININ , *ALANINE - Abstract
Endothelial nitric-oxide synthase (eNOS) plays a central role in cardiovascular regulation. eNOS function is critically modulated by Ca2+ and protein phosphorylation, but the interrelationship between intracellular Ca2~ mobilization and eNOS phosphorylation is poorly understood. Here we show that endoplasmic reticulum (ER) Ca2+ release activates eNOS by selectively promoting its Ser-635/633 (bovine/human) phosphorylation. With bovine endothelial cells, thapsigargin-induced ER Ca2+ release caused a dose-dependent increase in eNOS Ser-635 phosphorylation, leading to elevated NO production. ER Ca2~ release also promoted eNOS Ser-633 phosphorylation in mouse vessels in vivo. This effect was independent of extracellular Ca~+ and selective to Ser-635 because the phosphorylation status of other eNOS sites, including Ser-1179 or Thr-497, was unaffected in thapsigargin-treated cells. Blocking ERK1/2 abolished ER Ca2~ release-induced eNOS Ser-635 phosphorylation, whereas inhibiting protein kinase A or Ca2~/calmodulin-dependent protein kinase II had no effect. Protein phosphorylation assay confirmed that ERK1/2 directly phosphorylated the eNOS Ser-635 residue in vitro. Further studies demonstrated that ER Ca2~ release-induced ERK1/2 activation mediated the enhancing action of purine or bradykinin receptor stimulation on eNOS Ser-635/633 phosphorylation in bovine/human endothelial cells. Mutating the Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. Taken together, these studies reveal that ER Ca2+ release enhances eNOS Ser-635 phosphorylation and function via ERK1/2 activation. Because ER Ca2+ is commonly mobilized by agonists or physicochemical stimuli, the identified ER Ca2+-ERK1/2-eNO5 Ser-635 phosphorylation pathway may have a broad role in the regulation of endothelial function. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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