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

描述(由申请人提供)： Na/K-ATPase是一种能量转导离子泵。这种酶是洋地黄的受体。 心脏里有毒品。我们最近的工作表明，该酶也是一种信号转导。 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，但哇巴因也需要 调节心脏钙离子和收缩能力。这一应用程序建立在这些新发现的基础上， 并提出了Na/K-ATPase转导的分子机制。 哇巴因信号。具体地说，我们提出了三个具体的目标来检验这一假设，即当Na/KATPase与哇巴因结合或被哇巴因激活时，它能够招募和 将一组蛋白质组装成不同的信号平台，传递细胞外信号 哇巴因信号进入不同的细胞隔间。在具体目标1中，我们计划使用 蛋白质组学方法破译具有潜在作用的蛋白质的组成 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与确定的候选者之间相互作用的实验 蛋白质。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。在特定目标2中，我们将使用体外分析来定义 调节Na/K-ATPase与其信号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 将测试已识别的结合结构域(S)在哇巴因诱导的 蛋白质的相互作用。在具体目标3中，我们计划首先确定蛋白质/蛋白质的作用 哇巴因激活Src过程中的相互作用。第二，我们将检验这一假设 小窝蛋白参与哇巴因激活的信号分支的组装，包括 导致MAPKs激活和L型钙通道的磷酸化。 我们认为，我们的建议是高度集中的，我们的研究结果将 有助于我们理解Na/K-ATPase的生物学以及 洋地黄药物的药理学，这最终将有助于新药的开发 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.