Nitric oxide (NO) produced in the endothelium by the enzyme endothelial nitric oxide synthase (eNOS) is an important vasoactive compound. Impaired regulation of eNOS has been proposed to account for the vascular defects underlying cardiovascular diseases such as hypertension, diabetes and atherosclerosis. NO generation by eNOS is tightly controlled by the availability of cofactors and substrates, modulation of phosphorylation state, protein-protein interactions, and intracellular localization. However, evaluation of these mechanisms has not fully accounted for the reduced synthesis of vascular NO observed in disease states. In this proposal we have identified a novel regulatory influence on eNOS activity, tyrosine phosphorylation. In preliminary data, we demonstrate that eNOS is phosphorylated on tyrosine 83. The phosphorylation of this residue by the protein tyrosine kinase Src, increases eNOS activity 3-5 fold. Mutation of this residue to a non-phosphorylatable analogue or blockade of Src activity reduces both basal and stimulated NO release. These observations support the central hypothesis of this application that phosphorylation of Tyr-83 is key regulator of eNOS activity and that impaired phosphorylation of Tyr-83 may contribute to endothelial dysfunction. To test this hypothesis, 3 specific aims are proposed:
Aim 1 will determine the molecular mechanisms by which Tyr-83 phosphorylation enhances eNOS activity. Our goal is to identify changes in eNOS cofactor or substrate affinity and new eNOS protein binding partners that are regulated by Tyr-83 phosphorylation.
Aim 2 will determine the mechanisms leading to eNOS Tyr-83 phosphorylation. We will investigate the role of hsp90 and subcellular location as key mechanisms controlling the where and when of Tyr-83 phosphorylation.
Aim 3 will determine the physiological role of Tyr-83 phosphorylation. We have recently developed a phospho specific antibody for Tyr-83 which will enable us to identify the contribution of Tyr-83 phosphorylation to eNOS function in endothelial cells, intact blood vessel and in animal models of human disease. These studies will identify a new mechanism controlling eNOS activity and will contribute to our understanding of the dysregulation of eNOS in cardiovascular disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL085827-04
Application #
7786993
Study Section
Atherosclerosis and Inflammation of the Cardiovascular System Study Section (AICS)
Program Officer
Wood, Katherine
Project Start
2007-04-01
Project End
2012-03-31
Budget Start
2010-04-01
Budget End
2011-03-31
Support Year
4
Fiscal Year
2010
Total Cost
$367,500
Indirect Cost
Name
Georgia Regents University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
966668691
City
Augusta
State
GA
Country
United States
Zip Code
30912
Chen, Feng; Kumar, Sanjiv; Yu, Yanfang et al. (2014) PKC-dependent phosphorylation of eNOS at T495 regulates eNOS coupling and endothelial barrier function in response to G+ -toxins. PLoS One 9:e99823
Chen, Feng; Yu, Yanfang; Haigh, Steven et al. (2014) Regulation of NADPH oxidase 5 by protein kinase C isoforms. PLoS One 9:e88405
Ruan, Ling; Torres, Christina M; Buffett, Ryan J et al. (2013) Calcineurin-mediated dephosphorylation of eNOS at serine 116 affects eNOS enzymatic activity indirectly by facilitating c-Src binding and tyrosine 83 phosphorylation. Vascul Pharmacol 59:27-35
Elms, Shawn; Chen, Feng; Wang, Yusi et al. (2013) Insights into the arginine paradox: evidence against the importance of subcellular location of arginase and eNOS. Am J Physiol Heart Circ Physiol 305:H651-66
Pandey, Deepesh; Patel, Anand; Patel, Vijay et al. (2012) Expression and functional significance of NADPH oxidase 5 (Nox5) and its splice variants in human blood vessels. Am J Physiol Heart Circ Physiol 302:H1919-28
Qian, Jin; Chen, Feng; Kovalenkov, Yevgeniy et al. (2012) Nitric oxide reduces NADPH oxidase 5 (Nox5) activity by reversible S-nitrosylation. Free Radic Biol Med 52:1806-19
Chen, Feng; Yu, Yanfang; Qian, Jin et al. (2012) Opposing actions of heat shock protein 90 and 70 regulate nicotinamide adenine dinucleotide phosphate oxidase stability and reactive oxygen species production. Arterioscler Thromb Vasc Biol 32:2989-99
Qian, Jin; Fulton, David J R (2012) Exogenous, but not Endogenous Nitric Oxide Inhibits Adhesion Molecule Expression in Human Endothelial Cells. Front Physiol 3:3
Rao, Xiaoquan; Zhong, Jixin; Zhang, Shu et al. (2011) Loss of methyl-CpG-binding domain protein 2 enhances endothelial angiogenesis and protects mice against hind-limb ischemic injury. Circulation 123:2964-74
Pandey, D; Fulton, D J R (2011) Molecular regulation of NADPH oxidase 5 via the MAPK pathway. Am J Physiol Heart Circ Physiol 300:H1336-44

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