SGK Regulation of Epithelial Sodium Transport

SGK 对上皮钠转运的调节

• 批准号: 8335449
• 负责人: DAVID PEARCE
• 金额: $ 33.6万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-20 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8335449
• 关键词: Aldosterone; Amiloride; Amino Acids; Blood; Blood Pressure; Body Fluids; Cell Line; Cell physiology; Cells; Complex; Cultured Cells; Data; Defect; Disease; Duct (organ) structure; Edema; Electrolytes; Epithelial; Equilibrium; Event; Excretory function; Genetic Transcription; Glomerular Filtration Rate; Goals; Grant; Harvest; Health; Heart failure; Homeostasis; Hormonal; Hormones; Hypertension; In Vitro; Individual; Ions; Kidney; Kidney Failure; Knockout Mice; Learning; Light; Literature; Liver diseases; Location; Measurement; Mediating; Mediator of activation protein; Mind; Modification; Molecular; Mus; Mutagenesis; Myocardial Infarction; Output; Patients; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Probability; Process; Protein-Serine-Threonine Kinases; Proteins; Recruitment Activity; Regulation; Relative (related person); Renal tubule structure; Research; Risk; Role; Serine; Signal Transduction; Sodium; Sodium Channel; Sodium-Restricted Diet; Stroke; Tissues; Two-Hybrid System Techniques; Urine; Yeasts; apical membrane; base; blood pressure regulation; design; epithelial Na+ channel; genetic manipulation; genetic regulatory protein; hypertension treatment; in vivo; inhibitor/antagonist; kidney cell; mTOR protein; mutant; patch clamp; protein complex; response; salt sensitive; small hairpin RNA; small molecule; trafficking

DESCRIPTION (provided by applicant): Hormone-regulated Na+ transport in the kidney tubules is critical to the control of blood pressure and other vital processes in health and disease. The serine-threonine kinase SGK1 is one of the most intensively studied regulators of epithelial Na+ transport, and has been particularly recognized for its importance in mediating the effects of aldosterone on the epithelial sodium channel (ENaC). SGK1 is under dual control: its expression is controlled by aldosterone through effects on gene transcription, and its activity is controlled by phosphorylation involving a network of kinase cascades. Recent evidence has identified mammalian target of rapamycin (mTOR) as the kinase mediating a key "gateway" phosphorylation event required for SGK1 activation. We hypothesize that SGK1 physically interacts with specific component(s) of a multi-protein complex containing mTOR, which is essential for the gateway phosphorylation at serine 422 (S422). We further hypothesize that SGK1 together with this mTOR-containing complex is recruited to another protein complex, where SGK1 regulates ENaC by phosphorylating targets such as Nedd4-2. With these hypotheses in mind, our specific aims for this competing renewal are to: 1: Determine the mechanistic basis of mTOR physical association with, and phosphorylation of SGK1. There are two mTOR-containing multi-protein complexes, mTORC1 and mTORC2, which have distinct components and substrates, and regulate distinct cellular processes. We will: A) Examine the physical interactions of individual mTORC1 & 2 components with SGK1 using the yeast two hybrid assay and in vitro interaction. B) Identify domains and specific amino acids within SGK1 and mSin1, which mediate their interaction. C) Determine if physical interaction between mSin1 and SGK1 is necessary for SGK1 to be phosphorylated by mTORC2. D) Examine mTORC2 regulation of SGK1 relatives, Akt, SGK2, and SGK3. 2: Characterize the functional effects of mTOR and SGK1 on ENaC in cultured cortical collecting duct (CCD) cells. This aim will establish the functional role of the above characterized physical interactions and SGK1 modifications in controlling ENaC-mediated Na+ transport in cultured cells. We will: A) Assess the functional role of SGK1 as a mediator of mTOR-dependent activation of ENaC in a CCD cell line, using a combination of small molecule inhibitors of mTOR and SGK1, and genetic manipulation. B) Assess the functional implications of SGK1 interaction with mTORC2 by characterizing the effect of mutants on SGK1 phosphorylation and ENaC current. C) Characterize the effect of cellular conditions and hormonal milieu on mTOR-dependent activation of SGK1 and ENaC. 3: Characterize the role of mTOR and SGK1 in Na+ homeostasis in vivo. In order to advance these findings into native tissues, we will: A) Examine the effects of mTOR activators and inhibitors on Na+ balance, blood pressure, and ENaC subcellular localization in wild type and SGK1-null mice. B) Examine mTOR regulation of amiloride-inhibitable transepithelial Na+ current in isolated perfused CCD harvested from aldosterone-treated mice. C) Examine the role of mTOR in regulation of amiloride-inhibitable currents in isolated CCD using patch clamp.

描述（申请人提供）：肾小管中激素调节的Na+转运对控制血压和其他健康和疾病的重要过程至关重要。丝氨酸-苏氨酸激酶SGK1是研究最深入的上皮Na+运输调节因子之一，它在调节醛固酮对上皮钠通道（ENaC）的影响方面的重要性得到了特别的认可。SGK1受到双重控制：醛固酮通过影响基因转录来控制其表达，而其活性则通过涉及激酶级联网络的磷酸化来控制。最近的证据已经确定哺乳动物雷帕霉素靶点（mTOR）是介导SGK1激活所需的关键“门户”磷酸化事件的激酶。我们假设SGK1与含有mTOR的多蛋白复合物的特定组分相互作用，mTOR对于丝氨酸422 （S422）的入口磷酸化至关重要。我们进一步假设SGK1与这种含mtor复合物一起被募集到另一种蛋白质复合物中，其中SGK1通过磷酸化Nedd4-2等靶点来调节ENaC。考虑到这些假设，我们对这种竞争性更新的具体目标是：1：确定mTOR与SGK1物理关联和磷酸化的机制基础。有两种含有mtor的多蛋白复合物mTORC1和mTORC2，它们具有不同的成分和底物，并调节不同的细胞过程。我们将：A)使用酵母双杂交试验和体外相互作用检查单个mTORC1和2组分与SGK1的物理相互作用。B)鉴定SGK1和mSin1中介导它们相互作用的结构域和特定氨基酸。C)确定mTORC2磷酸化SGK1是否需要mSin1和SGK1之间的物理相互作用。D)检测mTORC2对SGK1亲属、Akt、SGK2和SGK3的调控。2：表征mTOR和SGK1对体外培养的皮质集管（CCD）细胞ENaC的功能影响。本研究旨在确定上述物理相互作用和SGK1修饰在控制培养细胞中enact介导的Na+转运中的功能作用。我们将：A)评估SGK1作为mTOR依赖性ENaC在CCD细胞系中激活的中介的功能作用，使用mTOR和SGK1的小分子抑制剂和遗传操作的组合。B)通过表征突变体对SGK1磷酸化和ENaC电流的影响，评估SGK1与mTORC2相互作用的功能意义。C)表征细胞条件和激素环境对mtor依赖性SGK1和ENaC激活的影响。3：表征mTOR和SGK1在体内Na+稳态中的作用。为了将这些发现推进到原生组织中，我们将：A)在野生型和sgk1缺失的小鼠中检测mTOR激活剂和抑制剂对Na+平衡、血压和ENaC亚细胞定位的影响。B)在醛固酮处理小鼠的离体灌注CCD中检测mTOR对阿米洛利抑制的上皮Na+电流的调节。C)使用膜片钳检查mTOR在隔离CCD中调节阿米洛利抑制电流中的作用。