Phosphatases and Local signaling in Heart

描述（由申请人提供）：许多调查有助于定义 心脏中主要Ca 2+信号循环的分子元素， 兴奋-收缩（EC）耦合级联。由于磷酸化 反应一直是这一调节机制的突出焦点， 蛋白磷酸酶是心肌细胞中被低估的酶， 发信号。PI有令人惊讶的新的初步结果， 丝氨酸/苏氨酸磷酸酶，PP 1和PP 2A，导致蛋白质的快速离域， 一种与横小管相关的细胞骨架蛋白， EC偶联、离子通道活性和收缩事件的变化。这些 结果与新出现的主题一致，即细胞内信号传导 特异性包括激酶和磷酸酶与 控制蛋白质局部动态平衡的特定亚细胞位点 磷酸化这一假设将从一个新的角度进行探讨， 确定蛋白磷酸酶2A（PP 2A）的空间分离在 心肌细胞中的信号传导。 该项目围绕三个相互关联的目标组织。(1)有哪些 兴奋-收缩偶联中局部事件的生理特性 由磷酸酶调控的级联反应心脏的基本性质 将表征由蛋白磷酸酶调节的肌细胞。(2)如何 亚细胞靶向PP 2A调节Ca ~（2+）信号和兴奋 心脏细胞的收缩耦合新的病毒基因转移方法将是 用于过表达PP 2A的靶向亚基。功能影响将是 评估。(3)PP 2A的亚细胞靶向如何调节信号 心肌细胞中的信号转导途径这些实验将利用 PI的一项新发现，PP 2A靶向亚基的过表达 减弱培养的心肌细胞中的β-肾上腺素能反应。 这一建议的优点是将采用一种综合办法， 利用病毒基因转移，生物化学，细胞生物学，电压钳， 细胞内Ca 2+和心脏蛋白质的高分辨率成像。基于 根据初步数据，该项目将确定重要的监管 在心肌细胞的局部战略位点的机制。成功完成 计划中的工作将对分子过程产生新的见解 在病理学上观察到的潜在的基本信号变化 心力衰竭和心脏衰老的过程。

DESCRIPTION (provided by applicant): Many investigations have helped define the molecular elements underlying the major Ca2+ signaling cycle in heart known as the excitation-contraction (EC) coupling cascade. Since phosphorylation reactions have been a preeminent focus of regulatory mechanisms of this pathway, protein phosphatases are under-appreciated enzymes in cardiac signaling. The PI has surprising new preliminary results that inhibition of serine/threonine phosphatases, PP1 and PP2A, leads to a rapid delocalization of a transverse-tubule-associated cytoskeletal protein that is accompanied by changes in EC coupling, ion channel activity and contractile events. These results are consistent with the emerging theme that intracellular signaling specificity includes the intimate association of kinases and phosphatases with specific subcellular sites that controls the local dynamic balance of protein phosphorylation. This hypothesis will be explored from a new perspective by identifying the role of spatial segregation of protein phosphatase 2A (PP2A) in signaling in cardiac myocytes. The project is organized around three inter-related aims. (1) What are the physiological properties of local events in the excitation-contraction coupling cascade that are regulated by phosphatases? Fundamental properties of cardiac myocytes regulated by protein phosphatases will be characterized. (2) How does subcellular targeting of PP2A regulate Ca2+ signaling and excitation contraction coupling in heart cells? New viral gene transfer methods will be used to overexpress targeting subunits of PP2A. The functional impact will be assessed. (3) How does subcellular targeting of PP2A regulate signal transduction pathways in cardiac myocytes? These experiments will capitalize on a new discovery by the PI that the overexpression of a PP2A targeting subunit blunts beta-adrenergic responsiveness in cultured cardiac myocytes. A strength of this proposal is that an integrated approach will be used that exploits viral gene transfer, biochemistry, cell biology, voltage-clamp and high resolution imaging of intracellular Ca2+ and cardiac proteins. Based on the preliminary data presented, this project will define important regulatory mechanisms at local strategic sites in cardiac cells. Successful completion of the planned work will yield new insights into the molecular processes underlying fundamental signaling changes that are seen in pathological processes of heart failure and the senescent heart.