Direct modulation of PKA signal termination by PDE and Calcineurin

项目摘要 为了传递信息，交流既需要信号，也需要沉默。在生物学中，这是 通过动态和可逆的信号酶实现，允许自身被激活和 已停用。作为一种典型的信号转导通路，蛋白激酶A(PKA)被发现同时被激活 并在几秒钟内失活，这对于它在关键生理过程中的作用是必不可少的，例如 调节心脏的收缩能力和胰腺对胰岛素的振荡释放。PKA在以下情况下处于非活动状态 催化(C)和调节(R)亚单位在封闭的确认中结合，并且PKA由 第二信使环腺苷单磷酸cAMP与调节亚基的结合 催化亚单位的释放。虽然曾假设cAMP的平衡结合将是 与PKA的激活和失活相称，cAMP的解离与调控 在体外，调节亚基和催化亚基的亚基和重新结合太慢了，不能与FAST相一致 在细胞中观察到的失活。 在这项研究中提出的工作将检验PKA失活需要 磷酸二酯酶(PDE)直接清除cAMP，并通过 钙调神经磷酸酶(Calcineurin，CN.)这一假设将通过以下两个具体目标进行检验： 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进行测量。 第二、体外和活细胞定量的PKA活性将被用来评估PKA RII的作用 PKA失活过程中CN的去磷酸化作用。此外，PKA依赖的活体细胞观察 线粒体形态的变化将使我们能够表征线粒体下游的功能影响 用CN使RII去磷酸化。 这些研究的结果将回答PKA信号动力学领域中一个长期存在的问题。

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.