Tyrosine phosphorylation of endothelial nitric oxide synthase

描述（由申请人提供）：由内皮一氧化氮合酶（eNOS）在内皮中产生的一氧化氮（NO）是一种重要的血管活性化合物。eNOS的调节受损已被认为是导致心血管疾病如高血压、糖尿病和动脉粥样硬化的血管缺陷的原因。eNOS的NO生成受到辅因子和底物的可用性、磷酸化状态的调节、蛋白质-蛋白质相互作用和细胞内定位的严格控制。然而，这些机制的评价还没有完全解释在疾病状态下观察到的血管NO合成减少。在这个提议中，我们已经确定了一种新的调控eNOS活性的影响，酪氨酸磷酸化。在初步数据中，我们证明eNOS是磷酸化酪氨酸83。蛋白酪氨酸激酶Src对该残基的磷酸化使eNOS活性增加3-5倍。将该残基突变为不可磷酸化的类似物或阻断Src活性可降低基础和刺激的NO释放。这些观察结果支持本申请的中心假设，即Tyr-83的磷酸化是eNOS活性的关键调节剂，并且Tyr-83的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辅因子或底物亲和力的变化以及由Tyr-83磷酸化调控的新eNOS蛋白结合伴侣。目的2将确定导致eNOS Tyr-83磷酸化的机制。我们将研究热休克蛋白90和亚细胞定位的作用，作为控制酪氨酸-83磷酸化的位置和时间的关键机制。目的3将确定Tyr-83磷酸化的生理作用。我们最近开发了Tyr-83的磷酸化特异性抗体，这将使我们能够鉴定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。这些研究将确定一个新的机制控制eNOS活性，并将有助于我们了解eNOS在心血管疾病中的失调。

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.