Phosphatases, local structures and signaling in heart

描述(由申请人提供)： 心脏中的局部信号包括一种激酶-磷酸酶平衡，控制着 功能蛋白的磷酸化状态。因为对激酶的研究只能揭示 在信号级联中，磷酸酶的重要性被低估。的最新结果 项目负责人表明，抑制丝氨酸/苏氨酸磷酸酶PP1和PP2A会导致 L型LCA增加和一个T管细胞骨架蛋白的错误定位。此外，私家侦探还有 发现PP2A的B靶向亚基被分离到脑室内的战略位置 肌细胞，包括质膜的纹状结构和灶状区。因此，这一点 该项目将确定磷酸酶亚细胞靶向、细胞骨架 结构和心肌细胞中的钙信号转导。这些目标是一系列相互关联的 与同事们在这个PPG上的努力相结合的实验。目标1将定义 磷酸酶PP2A在心肌细胞中的靶向功能部位。本系列将 使用完整细胞研究和分子方法相结合的方法。AIM 2将在此基础上发展 结果(1)和将确定特定的氨基酸序列基序在PP2A B-靶向 决定其分离到离散的心脏细胞结构的亚基。病毒导向基因 分析中将使用GFP融合蛋白的转移。目标3将定义 磷酸酶和T管细胞骨架蛋白在L钙通道调节中的作用 活动。本系列将研究正常和心脏局部的激酶/磷酸酶平衡。 项目2和项目3中开发的失败动物模型：T管细胞骨架结构的作用 将用在项目2中开发的新的显性负性探针来检查ICA的控制。 目标4将提供与上述目标(1)和(2)的功能链接，并将检查如何进行更改 在PP2A靶向改变信号转导通路中。这一系列将充分利用耐人寻味的 新结果表明，PP2A B亚单位过度表达会导致迟钝的β-肾上腺素能反应 培养的心肌细胞。精确的分子分析将得到以下研究的补充 在项目3中，来自心力衰竭应激激活模型的细胞被开发出来，其中戏剧性的 这些B亚基的增加也可以看到。 该项目将使用一系列互补的方法，包括电压钳位，高 分辨率共聚焦成像、腺病毒基因转移和生化分析 研究局部磷酸化、细胞骨架结构和钙信号之间的联系。 心。该项目的另一个优点是灵活的计划将利用以下结果 PPG中的所有项目负责人。

DESCRIPTION (provided by applicant): Local signaling in heart includes a kinase-phosphatase balance that controls the phosphorylation state of functional proteins. As studies on kinases can only reveal part of the signaling cascade, the importance of phosphatases is underappreciated. New results by the Project Leader show that inhibition of serine/threonine phosphatases, PP1 and PP2A, lead to increases in L-type lCa and mislocalization of a t-tubule cytoskeletal protein. Also, the PI has found that B-targeting subunits of PP2A are segregated to strategic sites within ventricular myocytes, including striated structures and focal regions of the plasma membrane. Thus, this project will define the links between phosphatase subcellular targeting, cytoskeletal architecture, and Ca2+ signaling in cardiac myocytes. The aims are a series of interrelated experiments that are co-mingled with the efforts of colleagues on this PPG. Aim 1 will define the functional sites to which the phosphatase PP2A is targeted in cardiac myocytes. This series will use a combination of intact cell studies combined with molecular methods. Aim 2 will build on the results of (1) and will identify the specific amino acid sequence motifs in PP2A B-targeting subunits that determine its segregation to discrete cardiac cell structures. Virus directed gene transfer of GFP fusion proteins will be used in the analysis. Aim 3 will define the role of phosphatases and t-tubule cytoskeleton proteins in the regulation of L-type Ca2+ channel activity. This series will examine the local kinase/ phosphatase balance in normal and heart failure animal models developed in Projects 2 and 3. The role of t-tubule cytoskeletal structure in control of ICa will be examined with novel dominant negative probes developed in Project 2. Aim 4 will provide a functional link to Aims (1) and (2) above, and will examine how alterations in PP2A targeting change signal transduction cascades. This series will capitalize on intriguing new results that PP2A B subunit overexpression leads to a blunted beta-adrenergic response in cultured cardiac myocytes. The precise molecular analysis will be complemented by studies in cells from stress-activated models of heart failure developed in Project 3, where dramatic increases in these B subunits are also seen. This project will use a series of complementary approaches including voltage clamp, high resolution confocal imaging, adenoviral gene transfer, and biochemical analyses to critically examine the links between local phosphorylation, cytoskeletal structures, and Ca2+ signaling in heart. Another strength of this project is that the flexible plans will capitalize on the results from all of the Project Leaders in the PPG.