Tyrosine phosphorylation of endothelial nitric oxide synthase

描述（由申请人提供）：一氧化氮（NO）由内皮一氧化氮合酶（eNOS）在内皮细胞中产生，是一种重要的血管活性化合物。eNOS的调节受损被认为是高血压、糖尿病和动脉粥样硬化等心血管疾病的血管缺陷的原因。eNOS产生NO受到辅助因子和底物的可用性、磷酸化状态的调节、蛋白-蛋白相互作用和细胞内定位的严格控制。然而，对这些机制的评估并没有完全解释在疾病状态下观察到的血管NO合成减少的原因。在这项提议中，我们已经确定了对eNOS活性的一种新的调控影响，酪氨酸磷酸化。在初步数据中，我们证明eNOS在酪氨酸83上被磷酸化。酪氨酸激酶Src磷酸化该残基，使eNOS活性增加3-5倍。该残基突变为非磷酸化类似物或阻断Src活性可减少基础和刺激的NO释放。这些观察结果支持了本研究的中心假设，即Tyr-83的磷酸化是eNOS活性的关键调节因子，而Tyr-83磷酸化受损可能导致内皮功能障碍。为了验证这一假设，提出了3个具体目标：目标1将确定Tyr-83磷酸化增强eNOS活性的分子机制。我们的目标是确定eNOS辅助因子或底物亲和力的变化以及受tyr83磷酸化调节的新的eNOS蛋白结合伙伴。目的2将确定导致eNOS Tyr-83磷酸化的机制。我们将研究hsp90和亚细胞定位作为控制Tyr-83磷酸化的地点和时间的关键机制的作用。目的3将确定Tyr-83磷酸化的生理作用。我们最近开发了一种针对Tyr-83的磷酸化特异性抗体，这将使我们能够确定Tyr-83磷酸化对内皮细胞、完整血管和人类疾病动物模型中eNOS功能的贡献。这些研究将确定控制eNOS活性的新机制，并有助于我们理解心血管疾病中eNOS的失调。

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.