Local Signaling in Diabetic Comorbidities

 描述(申请人提供)：信号转导通路的空间和时间组织影响细胞内事件的精确度和保真度。我们已经发现了一系列名为A-Kinase锚定蛋白(AKAP)的非催化调节元件，它们将不同的钙和cAMP反应蛋白激酶和磷酸酶组合在一起，以定制效应蛋白的调节。该应用侧重于一种称为AKAP79/150的多价锚定蛋白的分子病理生理学(AKAP79是人型，AKAP150是小鼠的同源基因)。我们发现了该蛋白，并确定了蛋白激酶A(PKA)、磷酸酶PP2B和蛋白激酶C(PKC)的传统异构体的结合部位。生理学研究表明，AKAP79/150酶积极参与控制血糖的动态平衡，并协调胰岛素的某些额外方面的作用。这项应用是围绕着与锚定的PKC在控制动脉收缩和高血压的信号事件中的作用有关的令人兴奋的数据而开发的。这种病理状态背后的分子事件包括通过离子通道的异常钙内流，该离子通道部分由AKAP79/150相关的PKC控制。有待检验的假设是，操纵锚定的PKC可以提供一定程度的血管益处，以缓解糖尿病引起的高血压。提出了两个具体的目标：目标1：单个AKAP79复合体是如何配置的？我们将利用三种前沿方法：a)单分子下拉光漂白(SiMPull)计算单个锚定蛋白上的酶组合范围，b)单粒子电子显微镜(EM)获得高阶AKAP79组装的近原子结构，以及c)血管平滑肌中天然AKAP-酶复合体的超分辨率成像。目的2：PKC锚定是否控制糖尿病相关高血压？65%的糖尿病患者会患高血压。我们的AKAP150-/-和AKAP150-/-ΔPKC敲门小鼠表现出较低的基础血压，表明AKAP相关的PKC参与了血管张力的控制。目的2将测试这种蛋白-蛋白相互作用蛋白的选择性破坏是否会降低糖尿病高血压小鼠模型的血管张力。

 DESCRIPTION (provided by applicant): The spatial and temporal organization of signal transduction pathways influences the precision and fidelity of intracellular events. We have discovered a family of non-catalytic regulatory elements called A-Kinase Anchoring Proteins (AKAPs) that bring together different combinations of calcium- and cAMP-responsive protein kinases and phosphatases to customize the regulation of effector proteins. This application focuses on the molecular pathophysiology of a multivalent anchoring protein known as AKAP79/150 (AKAP79 is the human form, AKAP150 is the murine ortholog). We discovered this protein and defined binding sites for protein kinase A (PKA), the phosphatase PP2B and conventional isoforms of protein kinase C (PKC). Physiological studies have demonstrated that AKAP79/150-tethered enzymes actively participate in the control of glucose homeostasis and coordinate certain extra pancreatic aspects of insulin action. This application is developed around exciting data pertaining to the role of anchored PKC in signaling events that govern arterial constriction and hypertension. Molecular events underlying this pathological state include aberrant calcium influx through ion channels that are controlled, in part, by AKAP79/150-associated PKC. The hypothesis to be tested is that manipulation of anchored PKC affords a measure of vascular benefit to alleviate diabetes-induced hypertension. Two specific aims are proposed: Aim 1: How are individual AKAP79 complexes configured? We will harness three cutting-edge approaches: a) single-molecule pull-down photobleaching (SiMPull) to calculate the range of enzyme combinations on individual anchoring proteins, b) single-particle electron microscopy (EM) to obtain near-atomic structures of higher-order AKAP79 assemblies and c) super resolution imaging of native AKAP-enzyme complexes in vascular smooth muscle. Aim 2: Does PKC-anchoring govern diabetes-related hypertension? Sixty-five percent of diabetics develop hypertension. Our AKAP150-/- and AKAP150ΔPKC knockin mice exhibit lower basal blood pressure suggesting that AKAP-associated PKC participates in the control of vascular tone. Aim 2 will test if selective disruption of this protein-protein interactin reduces vascular tone in mouse models of diabetic hypertension.