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）对于控制远端肾单位的钠重吸收至关重要。 ENaC 调节对于钠平衡和细胞外液容量的整体控制以及血压的控制至关重要。 ENaC 失调是某些形式原发性高血压的基础，原发性高血压是一种常见疾病，也是心血管疾病发病和死亡的主要原因。然而，ENaC 调节的分子机制尚不完全清楚。 这项工作旨在明确肾上皮细胞中 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从顶膜回收 ENaC 的机制上。相比之下，我们对促进 ENaC 向顶端细胞表面受调节的正向运输的机制的了解并不完整。具体而言，顶膜 ENaC 回收和胞吐作用中涉及的磷酸化依赖性步骤和蛋白质相互作用尚未阐明。 根据我们之前的发现，14-3-3 蛋白是调节 ENaC 运输的磷蛋白的重要稳定剂。 14-3-3 结合蛋白的亲和捕获与定量蛋白质组学分析相结合，为我们提供了调节极化肾上皮中 ENaC 运输的候选药物。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，蛋白激酶可响应醛固酮和加压素调节 ENaC 运输。 首先，我们将定义 AS160 的作用机制，AS160 是一种新鉴定的 14-3-3 结合蛋白，也是醛固酮介导的 ENaC 运输的磷酸化依赖性调节剂。使用生化和功能测定，我们将测试以下假设：AS160 在基础条件下稳定细胞内区室中的 ENaC，并允许 ENaC 运输至顶膜以响应其磷酸化。其次，我们将确定 TBC1D1（通过 14-3-3 亲和捕获识别的新靶点）是否会响应加压素/PKA 刺激而调节 ENaC 运输。 这项工作有望揭示控制顶端 ENaC 密度的新机制，并确定治疗钠转运上皮细胞盐水平衡疾病的新靶点。 公共健康相关性：这项名为“上皮钠通道 (ENaC) 运输的磷酸化依赖性调节”的研究将研究磷酸化依赖性 14-3-3 结合蛋白是否稳定顶端 ENaC 运输途径中的多个步骤，以及它们的结合是否伙伴 AS160 (TBC1D4) 和 TBC1D1 是介导醛固酮和醛固酮作用的蛋白激酶的关键底物。加压素。通过这项研究，我们将加深对 ENaC 调节分子机制的理解，其结果将进一步深入了解钠平衡、血容量以及血压的控制。