Phosphatases and Local signaling in Heart

描述(由申请人提供)：许多调查都有助于确定 心脏中主要的钙信号循环的分子元件被称为 激发-收缩(EC)耦合级联。自磷酸化以来 反应一直是这一调控机制的一个突出焦点 途径，蛋白磷酸酶是心脏中被低估的酶 发信号。PI有令人惊讶的新的初步结果，即抑制 丝氨酸/苏氨酸磷酸酶，PP1和PP2A，导致快速去局部化 一种与横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 结果与新兴的主题一致，即细胞内信号 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置 磷酸化。我们将从一个新的角度来探讨这一假说 鉴定蛋白磷酸酶2A(PP2A)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进行组织。(1)什么是 兴奋-收缩耦合中局部事件的生理特性 受磷酸酶调控的级联反应？心脏的基本特性 由蛋白磷酸酶调节的肌细胞将被描述。(2)如何 PP2A亚细胞靶向调控钙信号和兴奋 心肌细胞的收缩偶联？新的病毒基因转移方法将是 用于过度表达PP2A的靶向亚基。对功能的影响将是 评估过了。(3)PP2A的亚细胞靶向性如何调节信号 心肌细胞中的信号转导途径？这些实验将利用 PI的一个新发现：PP2A靶向亚基的过度表达 钝化培养的心肌细胞的β-肾上腺素能反应。 这项提议的一个优点是，将使用一种综合方法， 利用病毒基因转移、生物化学、细胞生物学、电压钳和 细胞内钙离子和心肌蛋白质的高分辨率成像。基于 提供的初步数据，该项目将定义重要的监管 心肌细胞中局部战略位置的机制。成功完成 计划中的工作将对分子过程产生新的见解 在病理性疾病中看到的潜在的基本信号变化 心力衰竭的过程和衰老的心脏。

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.