Direct modulation of PKA signal termination by PDE and Calcineurin

项目摘要 为了传递信息，通信需要信号和沉默。在生物学中， 通过动态和可逆的信号酶实现，使其自身被激活， 灭活蛋白激酶A（Protein Kinase A，PKA）作为一种典型的信号激酶，在细胞内被激活， 并在几秒钟内失活，这对于它在关键生理过程中的作用至关重要， 调节心脏收缩力和胰腺振荡释放胰岛素。PKA是不活跃的，当它 催化（C）和调节（R）亚基以封闭的构象结合，PKA被 第二信使环磷酸腺苷（cAMP）与调节亚单位的结合， 催化亚基的释放。虽然已经假设cAMP的平衡结合将是 与PKA的激活和失活相当，cAMP从调节蛋白的解离， 调节亚基和催化亚基的重组在体外太慢而不能与快速重组相一致。 在细胞中观察到失活。 在这项研究中提出的工作将检查PKA的失活需要的假设， 磷酸二酯酶直接清除cAMP，调节亚基的去磷酸化， 磷酸酶钙调磷酸酶，CN.将以下列两个具体目标检验这一假设： 首先，破坏PDE直接降解与PKA结合的cAMP的能力的动力学效应将是： 在分子和细胞水平上测量。 第二，PKA活性的体外和活细胞定量将用于评估PKA RII的作用。 在PKA失活中通过CN去磷酸化。此外，PKA依赖性的活细胞观察 线粒体形态学的变化将使我们能够表征 RII通过CN去磷酸化。 这些研究的结果将回答PKA信号动力学领域长期存在的问题。

Project Summary In order to convey information, communication requires both signal and silence. In biology this is achieved through signaling enzymes that are dynamic and reversible, allowing itself to be both activated and inactivated. As a prototypical signaling kinase, Protein Kinase A, PKA, has been observed to be both activated and inactivated in a matter of seconds, which is essential for its roles in key physiological processes such as regulating heart contractility and the oscillatory release of insulin by the pancreas. PKA is inactive when its catalytic (C) and regulatory (R) subunits are bound in a closed confirmation and PKA is activated by the binding of the second messenger cyclic adenosine monophosphate, cAMP, to the regulatory subunit prompting the release of the catalytic subunit. While it had been assumed that equilibrium binding of cAMP would be commensurate with the activation and inactivation of PKA, both the dissociation of cAMP from the regulatory subunit and re-association of the regulatory and catalytic subunits are far too slow in vitro to agree with the fast inactivation observed in cells. The work proposed in this fellowship will examine the hypothesis that the inactivation of PKA requires the direct removal of cAMP by phosphodiesterases, PDEs, and the dephosphorylation of the regulatory subunit by the phosphatase Calcineurin, CN. This hypothesis will be tested with the following two specific aims:  First, the kinetic effects of disrupting the ability of PDE to directly degrade cAMP bound to PKA will be measured at both the molecular and cellular levels.  Second, in vitro and live-cell quantification of PKA activity will be used to evaluate the role of PKA RII dephosphorylation by CN in PKA inactivation. Furthermore, live-cell observation of PKA dependent changes in mitochondria morphology will allow us to characterize the downstream functional effects of RII dephosphorylation by CN. The results from these studies will answer a long standing question in the field of PKA signaling dynamics.