DESCRIPTION (provided by applicant): 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.
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