Phosphatases, local structures and signaling in heart

心脏中的磷酸酶、局部结构和信号传导

• 批准号: 6662936
• 负责人: TERRY B. ROGERS
• 金额: $ 22.65万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6662936
• 关键词: beta adrenergic receptor; biological signal transduction; calcium channel; cardiac myocytes; confocal scanning microscopy; cytoskeletal proteins; enzyme mechanism; green fluorescent proteins; laboratory rat; newborn animals; phosphoprotein phosphatase; protein kinase; protein localization; protein sequence; protein structure function; receptor expression; tissue /cell culture; transfection; voltage /patch clamp

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.

描述（由申请人提供）： 心脏中的局部信号传导包括激酶-磷酸酶平衡， 功能蛋白的磷酸化状态。由于对激酶的研究只能揭示部分 信号级联，磷酸酶的重要性是低估。新的结果由 项目负责人表明，抑制丝氨酸/苏氨酸磷酸酶PP 1和PP 2A会导致 L-型钙离子浓度增加和t-小管细胞骨架蛋白的错误定位。此外，PI 发现PP 2A的B靶向亚基被分离到心室内的战略位点， 肌细胞，包括质膜的条纹状结构和局灶性区域。因此，这 该项目将定义磷酸酶亚细胞靶向、细胞骨架 结构和心肌细胞中的Ca 2+信号。目标是一系列相互关联的 这些实验与同事们在这个PPG上的努力混合在一起。目标1将定义 磷酸酶PP 2A在心肌细胞中靶向的功能位点。这个系列将 使用完整细胞研究与分子方法相结合。目标2将建立在 （1）的结果，并将鉴定PP 2A B靶向中的特定氨基酸序列基序 这些亚基决定其分离成离散的心脏细胞结构。病毒定向基因 GFP融合蛋白的转移将用于分析。目标3将确定以下方面的作用： 磷酸酶和t-小管细胞骨架蛋白对L-型钙通道的调节 活动本系列将检查正常人和心脏病患者的局部激酶/磷酸酶平衡， 在项目2和项目3中开发的失败动物模型。T-小管细胞骨架结构的作用 将使用项目2中开发的新型显性阴性探针检查控制伊卡的能力。 目标4将提供与上述目标（1）和（2）的功能联系，并将研究如何改变 PP 2A靶向改变信号转导级联。这个系列将利用有趣的 一项新的研究结果表明，PP 2A B亚单位过表达导致β-肾上腺素能反应减弱， 培养的心肌细胞。精确的分子分析将得到以下研究的补充： 来自项目3中开发的心力衰竭应激激活模型的细胞， 也可以看到这些B亚基的增加。 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，以关键 研究局部磷酸化，细胞骨架结构和Ca 2+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 PPG中的所有项目负责人。