Interactions of Na+/K+ ATPase with its signaling partners

Na /K ATP 酶与其信号伙伴的相互作用

• 批准号: 7010371
• 负责人: Zijian Xie
• 金额: $ 22.72万
• 依托单位: UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7010371
• 关键词: biological signal transduction; calcium channel; caveolins; cell growth regulation; enzyme mechanism; immunoprecipitation; laboratory rat; mitogen activated protein kinase; myocardium; ouabain; phosphorylation; protein kinase; protein localization; protein protein interaction; proteomics; protooncogene; sodium potassium exchanging ATPase; tissue /cell culture

DESCRIPTION (provided by applicant): Na/K-ATPase is an energy transducing ion pump. The enzyme serves as a receptor for digitalis drugs in the heart. Our recent work demonstrates that the enzyme is also a signal transducer. Significantly, we have shown that the signal transduction function of the enzyme is not only involved in control of cell growth and gene expression, but also required for ouabain to regulate cardiac calcium and contractility. This application is built upon these novel findings, and is proposed to define the molecular mechanisms by which Na/K-ATPase transduces the ouabain signals. Specifically,we propose three Specific Aims to test the hypothesis that Na/KATPase, when it binds to ouabain or is activated by ouabain, is capable of recruiting and assembling a group of proteins into different signaling platforms that relays the extracellular ouabain signal into different cellular compartments. In Specific Aim 1, we plan to use proteomic approaches to decipher the composition of the proteins that have the potential to interact with the Na/K-ATPase. We shall then use immunoprecipitation and in vitro binding assays to confirm the interactions between the Na/K-ATPase and the identified candidate proteins. Finally, we shall examine how ouabain regulates the phosphorylation of these enzyme-associated proteins. In Specific Aim 2, we shall use in vitro assays to define the domains that mediate the interactions between the Na/K-ATPase and its signaling partners. Depending on the outcomes of these studies, both co-localization imaging and BRET analysis will be performed to test the role of the identified binding domain(s) in ouabain-induced protein interactions. In Specific Aim 3, we plan first to determine the role of protein/protein interaction in ouabain-induced activation of Src. Second, we shall test the hypothesis that caveolins are involved in assembly of the ouabain-activated signaling branches including those leading to the activationof MAPKs and phosphorylation of the L-type calcium channel. We believe that our proposal is highly focused and that the outcomes of our study shall contribute significantly to our understandingof the biology of the Na/K-ATPase as well as the pharmacology of digitalis drugs, which will ultimately aid in the development of novel therapeutic approaches.

描述（由申请人提供）： Na/k-ATPase是一种能量转导的离子泵。该酶充当digitalis的受体 心脏中的毒品。我们最近的工作表明该酶也是信号传感器。 值得注意的是，我们已经表明，酶的信号转导函数不仅是 参与控制细胞生长和基因表达，但也需要 调节心脏钙和收缩力。该应用是基于这些新颖的发现， 并建议定义Na/k-atpase传递的分子机制 Ouabain信号。具体而言，我们提出了三个特定的目的，以检验Na/Katpase与ouabain结合或被Ouabain激活的假设，能够招募和 将一组蛋白质组装到不同的信号平台中，这些信号平台传达细胞外 Ouabain信号进入不同的细胞室。在特定目标1中，我们计划使用 蛋白质组学方法是破译具有潜力的蛋白质的组成 与Na/K-ATPase相互作用。然后，我们将使用免疫沉淀和体外结合 确认Na/K-ATPase与已确定候选者之间的相互作用的测定法 蛋白质。最后，我们将研究瓦巴因如何调节这些磷酸化 酶相关蛋白。在特定目标2中，我们将使用体外测定法来定义 介导NA/K-ATPase及其信号伴侣之间相互作用的域。 根据这些研究的结果，共定位成像和BRET分析 将执行以测试已识别结合域在ouaabain诱导的 蛋白质相互作用。在特定目标3中，我们计划首先确定蛋白质/蛋白质的作用 Ouabain诱导的SRC激活中的相互作用。其次，我们将检验以下假设 小窝蛋白参与了Ouabain激活的信号分支的组装 那些导致MAPK激活的人和L型钙通道的磷酸化。 我们认为，我们的建议非常集中，我们的研究结果应 有助于我们理解Na/K-ATPase的生物学以及 Digitalis药物的药理学，最终将有助于开发新颖 治疗方法。