Insulin-stimulated phosphorylation of endothelial nitric oxide synthase at serine-615 contributes to nitric oxide synthesis

Ritchie, S. A., Kohlhaas, C. F., Boyd, A. R., Yalla, K. C., Walsh, K., Connell, J. M.C. and Salt, I. P. (2010) Insulin-stimulated phosphorylation of endothelial nitric oxide synthase at serine-615 contributes to nitric oxide synthesis. Biochemical Journal, 426(1), pp. 85-90. (doi:10.1042/BJ20091580)

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Publisher's URL: http://dx.doi.org/10.1042/BJ20091580

Abstract

Insulin stimulates endothelial NO (nitric oxide) synthesis via PKB (protein kinase B)/Akt-mediated phosphorylation and activation of eNOS (endothelial NO synthase) at Ser-1177. In previous studies, we have demonstrated that stimulation of eNOS phosphorylation at Ser-1177 may be required, yet is not sufficient for insulin-stimulated NO synthesis. We therefore investigated the role of phosphorylation of eNOS at alternative sites to Ser-1177 as candidate parallel mechanisms contributing to insulin-stimulated NO synthesis. Stimulation of human aortic endothelial cells with insulin rapidly stimulated phosphorylation of both Ser-615 and Ser-1177 on eNOS, whereas phosphorylation of Ser-114, Thr-495 and Ser-633 was unaffected. Insulin-stimulated Ser-615 phosphorylation was abrogated by incubation with the PI3K (phosphoinositide 3-kinase) inhibitor wortmannin, infection with adenoviruses expressing a dominant-negative mutant PKB/Akt or pre-incubation with TNFα (tumour necrosis factor α), but was unaffected by high culture glucose concentrations. Mutation of Ser-615 to alanine reduced insulin-stimulated NO synthesis, whereas mutation of Ser-615 to aspartic acid increased NO production by NOS in which Ser-1177 had been mutated to an aspartic acid residue. We propose that the rapid PKB-mediated stimulation of phosphorylation of Ser-615 contributes to insulin-stimulated NO synthesis.

Item Type:Articles
Keywords:Endothelial nitric oxide synthase (eNOS), endothelium, insulin, nitric oxide, phosphorylation, protein kinase B (PKB), signalling
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Salt, Dr Ian and Yalla, Dr Krishna and Connell, Professor John and Boyd, Mr Alasdair and Akehurst, Dr Christine
Authors: Ritchie, S. A., Kohlhaas, C. F., Boyd, A. R., Yalla, K. C., Walsh, K., Connell, J. M.C., and Salt, I. P.
Subjects:Q Science > QH Natural history > QH345 Biochemistry
College/School:College of Medical Veterinary and Life Sciences
College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
Journal Name:Biochemical Journal
Journal Abbr.:Biochem. j.
Publisher:Portland Press Ltd.
ISSN:0264-6021
ISSN (Online):1470-8728
Published Online:20 November 2009
Copyright Holders:Copyright © 2010 Biochemical Society
First Published:First published in Biochemical Journal 426:85-90
Publisher Policy:Reproduced in accordance with the copyright policy of the publisher.
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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
379881The role of Tumour Necrosis Factor-alpha and Adiponectin in insulin-stimulated endothelial nitric oxide productionStuart RitchieMedical Research Council (MRC)G84/6589Cardiovascular Science