ANCHORED PROTEIN KINASE A IN SIGNAL TRANSDUCTION

这项研究计划的一个主要目标是发现分子， 控制组装、调节和生理功能的机制 抑制cAMP作用的远端信号传导模块的功能， 包括锚定蛋白激酶A（PKA）同种型。 我们已经发现 一种新的A激酶锚蛋白，AKAP-KL，其（a）与 PKA调节的K通道蛋白（ROMK），位于 （B）促进上皮细胞的磷酸化/活化， 完整细胞中的通道。 分子和细胞机制， AKAP-KL偶联在极化细胞基底外侧表面产生的cAMP， 细胞在远端开放通道，顶膜将 阐明。AKAP-KL的结构特性，介导的 PKAII的调节亚基（RII）的结合和靶向， AKAP-KL RII复合物与特定细胞内位点的对接将 通过诱变，转染/表达， 生化分析和共聚焦显微镜。生理作用 在AKAP-KL中的预期靶向和束缚结构域，以抑制或 促进爪蟾卵母细胞K+通道开放。AKAP的确切作用- KL PKAII复合物在跨上皮、cAMP介导的信号传导中将 通过改变锚定的浓度和定位来建立 PKAII在完整的，高度极化的上皮细胞中， 肾小管（LLC细胞）和皮质集合管（MDCK）区域 肾单位ROMK磷酸化速率和水平的测定 和顶膜的激活（作为激素的功能 在基底外侧质膜处的浓度）将提供新的 对捕获弥散cAMP信号的机制的深入了解， 放大并精确地聚焦在基底效应器上， 跨上皮调节。I型PKAs介导的信号传导也可能 通过目标多样化。 然而，我们对高... 亲和RI锚蛋白。 我们发现并克隆了一个cDNA 在C. elegans（AKAPce）。我们将系统地 确定生物化学性质，结构-功能关系， 细胞特异性和发育模式的表达和生理 AKAPce的功能。知识的性质和功能 AKAPce将促进哺乳动物RI AKAP的表征， 设计分子工具来操纵PKAI的位置/功能 在哺乳动物细胞中。

A major goal of this research program is to discover molecules and mechanisms that govern the assembly, regulation and physiological function of distal signaling modules that medicate cAMP action and include anchored protein kinase A (PKA) isoforms. We have discovered a novel A kinase anchor protein, AKAP-KL, that (a) is co-localized with a PKA-regulated K channel protein (ROMK) at the apical surface of epithelial cells and (b) promotes the phosphorylation/activation of the channel in intact cells. The molecular and cellular mechanisms by which AKAP-KL couples cAMP generated at the basolateral surface of a polarized cell to opening channels at the distal, apical membrane will be elucidated. The structural properties of AKAP-KL that mediate the binding of regulatory subunits (RII) of PKAII and the targeting and docking of AKAP-KL RII complexes to specific intracellular sites will be elucidated by a combination of mutagenesis, transfection/expression, biochemical analysis and confocal microscopy. The physiological roles of predicated targeting and tethering domains in AKAP-KL to suppress or promote K+ channel opening in Xenopus oocytes. The precise role of AKAP- KL PKAII complexes in trans-epithelial, cAMP-medicated signaling will be established by varying the concentration and localization of anchored PKAII in intact, highly-polarized epithelial cells that are models for tubular (LLC cells) and cortical collecting duct (MDCK) regions of the nephron. Determination of the rates and levels of ROMK phosphorylation and activation at the apical membrane (as a function of hormone concentration at the basolateral plasma membrane) will provide novel insights into the mechanism by which diffuse cAMP signals are captured, amplified and focused precisely on a substrate-effector to accomplish trans-epithelial regulation. Signaling medicated by type I PKAs may also be diversified by targeting. However, nothing is known about high- affinity RI anchor proteins. We have discovered and cloned a cDNA encoding a novel, RI AKAP in C. elegans (AKAPce). We will systematically determine the biochemical properties, structure-function relationships, cell-specific and developmental patterns of expression and physiological functions for AKAPce. Knowledge of the properties and functions of AKAPce will facilitate characterization of mammalian RI AKAPs and enable the design of molecular tools to manipulate the location/function PKAI in mammalian cells.