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

描述（由申请人提供）： Na/K-ATP酶是一种能量转换离子泵。该酶充当洋地黄的受体 心中有药。我们最近的工作表明酶也是一种信号转导器。 值得注意的是，我们已经证明酶的信号转导功能不仅是 参与细胞生长和基因表达的控制，也是哇巴因 调节心脏钙和收缩力。该应用程序是建立在这些新颖的发现之上的， 并建议定义 Na/K-ATP 酶转导的分子机制 乌本信号。具体来说，我们提出了三个具体目标来检验这样的假设：Na/KATPase 在与哇巴因结合或被哇巴因激活时，能够招募和 将一组蛋白质组装到不同的信号平台中，以传递细胞外信号 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 破译蛋白质组成的蛋白质组学方法有可能 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 确认 Na/K-ATP 酶与已识别候选物之间相互作用的测定 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。在具体目标 2 中，我们将使用体外测定来定义 介导 Na/K-ATP 酶及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 将进行测试已识别的结合域在哇巴因诱导的中的作用 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 哇巴因诱导的 Src 激活中的相互作用。其次，我们将检验以下假设： Caveolins 参与哇巴因激活的信号分支的组装，包括 那些导致 MAPK 激活和 L 型钙通道磷酸化的物质。 我们相信我们的建议是高度集中的，我们的研究结果将 极大地有助于我们了解 Na/K-ATP 酶的生物学以及 洋地黄药物的药理学，最终将有助于新型药物的开发 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.