Phosphorylation-dependent regulation of epithelial sodium channel (ENaC) traffick

上皮钠通道 (ENaC) 运输的磷酸化依赖性调节

• 批准号: 8234159
• 负责人: Xiubin Liang
• 金额: $ 8.09万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8234159
• 关键词: 14-3-3 Proteins; Address; Affect; Affinity; Aldosterone; Apical; Attention; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Blood Pressure; Blood Volume; Cardiovascular system; Cell surface; Cells; Consensus; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis; Data; Disease; Distal; Duct (organ) structure; Ductal Epithelium; Endocytosis; Environment; Epithelial; Epithelial Cells; Epithelium; Equilibrium; Essential Hypertension; Event; Exocytosis; Extracellular Fluid; Forskolin; GTPase-Activating Proteins; Goals; Health; Hormones; Hypertension; Insulin; Kidney; Knowledge; Link; Mediating; Membrane Protein Traffic; Mentors; Molecular; Morbidity - disease rate; Nephrons; Nephrosis; Pathway interactions; Phosphoproteins; Phosphorylation; Phosphorylation Site; Probability; Protein Kinase; Proteins; Proteomics; Publishing; Pulmonary Hypertension; Records; Recycling; Regulation; Regulatory Pathway; Research; Research Personnel; Retrieval; Role; Signal Transduction; Site; Sodium; Sodium Channel; Sodium Chloride; Surface; Testing; Training; Universities; Vasopressins; Water; Work; airway epithelium; apical membrane; base; career; density; epithelial Na+ channel; genetic regulatory protein; human disease; in vivo; insight; mortality; mutant; novel; rab GTP-Binding Proteins; renal epithelium; response; trafficking

DESCRIPTION (provided by applicant): The epithelial sodium channel (ENaC) is of fundamental importance in the control of sodium reabsorption in the distal nephron. ENaC regulation is critical for the overall control of sodium balance and extracellular fluid volume, and thereby of blood pressure. Dysregulation of ENaC underlies some forms of essential hypertension - a common condition and a major cause of cardiovascular morbidity and mortality. However, the molecular mechanisms of ENaC regulation are not completely understood. This work aims to define the phosphorylation-dependent regulation of ENaC trafficking in renal epithelia. Nearly all research in this field has focused on the mechanisms that govern ENaC retrieval from the apical membrane. By contrast, our knowledge of the mechanisms that promote the regulated forward trafficking of ENaC to the apical cell surface is incomplete. Specifically, the phosphorylation-dependent steps and protein interactions involved in apical membrane ENaC recycling and exocytosis have not been elucidated. Based on our prior findings, 14-3-3 proteins are essential stabilizers of the phosphoproteins that regulate ENaC trafficking. Affinity capture of 14-3-3 binding proteins, combined with quantitative proteomic analysis, has given us candidates for the regulation of ENaC traffic in polarized renal epithelia. With biochemical and functional assays, we will evaluate the 14-3-3 binding phosphoproteins that impact the apical ENaC trafficking pathway. Our preliminary data has indicated that the Rab-GAP proteins, AS160 (TBC1D4) and TBC1D1, are key substrates for the protein kinases that regulate ENaC trafficking in response to aldosterone and vasopressin. To begin, we will define the mechanism of action of AS160, a newly identified 14-3-3 binding protein and phosphorylation-dependent regulator of aldosterone-mediated ENaC trafficking. Using biochemical and functional assays, we will test the hypothesis that AS160 stabilizes ENaC within intracellular compartments under basal conditions, and permits ENaC trafficking to the apical membrane in response to its phosphorylation. Second, we will determine whether TBC1D1, a new target identified by 14-3-3 affinity capture, regulates ENaC trafficking in response to vasopressin/PKA stimulation. This work is expected to reveal new mechanisms for the control of apical ENaC density, and identify novel targets for the treatment of diseases of salt and water balance in sodium transporting epithelia. PUBLIC HEALTH RELEVANCE: This study, titled "Phosphorylation-dependent regulation of epithelial sodium channel (ENaC) trafficking", will investigate whether phosphorylation-dependent 14-3-3 binding proteins stabilize multiple steps in the apical ENaC trafficking pathway, and whether their binding partners, AS160 (TBC1D4) and TBC1D1, are key substrates for the protein kinases that mediate the actions of aldosterone and vasopressin. Through this study, we will advance our understanding of molecular mechanisms of ENaC regulation and the results will provide further insight into the control of sodium balance, blood volume and thereby of blood pressure.

描述（由申请人提供）：上皮钠通道（ENAC）对于控制远端肾肾上腺钠重吸收的基本意义。 ENAC调节对于钠平衡和细胞外流体体积的总体控制至关重要，因此至关重要。 ENAC的失调是某些形式的基本高血压 - 一种常见的状况和心血管发病率和死亡率的主要原因。但是，尚未完全了解ENAC调节的分子机制。 这项工作旨在定义肾上皮中ENAC运输的磷酸化依赖性调节。该领域的几乎所有研究都集中在控制着根尖膜中ENAC检索的机制上。相比之下，我们对促进ENAC向根尖细胞表面进行调节的正向运输的机制的了解是不完整的。具体而言，尚未阐明磷酸化依赖性的步骤和蛋白质相互作用，尚未阐明与顶膜ENAC回收和胞吐作用有关的蛋白质相互作用。 根据我们先前的发现，14-3-3蛋白是调节ENAC运输的磷蛋白的必要稳定剂。 14-3-3结合蛋白的亲和力捕获，再加上定量蛋白质组学分析，使我们为极化肾上皮中的ENAC交通调节提供了候选者。通过生化和功能测定，我们将评估影响顶端ENAC运输途径的14-3-3结合磷蛋白。我们的初步数据表明，RAB-GAP蛋白AS160（TBC1D4）和TBC1D1是蛋白激酶的关键底物，这些蛋白激酶会调节ENAC运输以响应醛固酮和血管加压素。 首先，我们将定义AS160的作用机理，AS160是一种新鉴定的14-3-3结合蛋白和磷酸化依赖性醛固酮介导的ENAC运输的调节剂。使用生化和功能测定法，我们将测试以下假设：AS160在基础条件下稳定在细胞内室内的ENAC，并允许ENAC运输以响应其磷酸化的响应。其次，我们将确定通过14-3-3亲和力捕获确定的新目标TBC1D1是否会根据加压素/PKA刺激来调节ENAC运输。 预计这项工作将揭示控制顶部ENAC密度的新机制，并确定用于治疗钠和水平衡疾病的新目标。 公共卫生相关性：这项研究的标题为“上皮钠通道（ENAC）运输的磷酸化依赖性调节”，将研究是否依赖于磷酸化的14-3-3结合蛋白是否稳定了顶端ENAC运输中的多个步骤介导醛固酮和加压素的作用。通过这项研究，我们将提高人们对ENAC调节分子机制的理解，结果将进一步了解控制钠平衡，血容量以及因此血压的控制。