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

描述（由申请人提供）： Na/K-ATP酶是一种能量转换的离子泵。这种酶是洋地黄的受体 心脏里的药物我们最近的工作表明，酶也是一个信号转导器。 值得注意的是，我们已经表明，该酶的信号转导功能不仅是 参与控制细胞生长和基因表达，但也需要哇巴因， 调节心脏钙和收缩力。这个应用程序是建立在这些新的发现， 并建议定义Na/K-ATP酶转导的分子机制， 哇巴因信号。具体来说，我们提出了三个具体的目的来检验Na/KATP酶的假设，当它与哇巴因结合或被哇巴因激活时，能够招募和激活细胞。 将一组蛋白质组装成不同的信号平台， 哇巴因信号进入不同的细胞区室。在具体目标1中，我们计划使用 蛋白质组学方法来破译蛋白质的组成， 与Na/K-ATP酶相互作用。然后，我们将使用免疫沉淀和体外结合 用于确认Na/K-ATP酶与所鉴定的候选物之间的相互作用的测定 proteins.最后，我们将研究哇巴因如何调节这些蛋白的磷酸化， 酶相关蛋白。在具体目标2中，我们将使用体外试验来定义 介导Na/K-ATP酶及其信号传导伙伴之间相互作用的结构域。 根据这些研究的结果，共定位成像和BRET分析 以测试所鉴定的结合结构域在哇巴因诱导的细胞凋亡中的作用。 蛋白质相互作用在具体目标3中，我们计划首先确定蛋白质/蛋白质的作用 哇巴因诱导Src激活中的相互作用。第二，我们将检验以下假设： 小窝蛋白参与哇巴因激活的信号分支的组装， 导致MAPK活化和L型钙通道磷酸化的那些。 我们认为，我们的建议重点突出，我们的研究成果将 对我们理解Na/K-ATP酶的生物学以及 洋地黄药物的药理学，这将最终有助于开发新的 治疗方法。

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.