title: Endoplasmic reticulum Ca2+ release modulates endothelial nitric-oxide synthase via extracellular signal-regulated kinase (ERK) 1/2-mediated serine 635 phosphorylation.
abstract: Endothelial nitric-oxide synthase (eNOS) plays a central role in cardiovascular regulation. eNOS function is critically modulated by Ca(2+) and protein phosphorylation, but the interrelationship between intracellular Ca(2+) mobilization and eNOS phosphorylation is poorly understood. Here we show that endoplasmic reticulum (ER) Ca(2+) release activates eNOS by selectively promoting its Ser-635/633 (bovine/human) phosphorylation. With bovine endothelial cells, thapsigargin-induced ER Ca(2+) release caused a dose-dependent increase in eNOS Ser-635 phosphorylation, leading to elevated NO production. ER Ca(2+) release also promoted eNOS Ser-633 phosphorylation in mouse vessels in vivo. This effect was independent of extracellular Ca(2+) 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 Ca(2+) release-induced eNOS Ser-635 phosphorylation, whereas inhibiting protein kinase A or Ca(2+)/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 Ca(2+) 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 Ca(2+) release enhances eNOS Ser-635 phosphorylation and function via ERK1/2 activation. Because ER Ca(2+) is commonly mobilized by agonists or physicochemical stimuli, the identified ER Ca(2+)-ERK1/2-eNOS Ser-635 phosphorylation pathway may have a broad role in the regulation of endothelial function.
authors: Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Molecular and Cellular Biochemistry, The Ohio State University College of Medicine, Columbus, Ohio 43210, USA.
authors: Zhihong
authors: Z
authors: Xiao
authors: Tingting
authors: T
authors: Wang
authors: Honghua
authors: H
authors: Qin
authors: Chao
authors: C
authors: Huang
authors: Youmei
authors: Y
authors: Feng
authors: Yong
authors: Y
authors: Xia
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000235:genetics; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D012694:Serine / Q000235:genetics / Q000378:metabolism
chemical_list: D012694:Serine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D002118:Calcium