SGK Regulation of Epithelial Sodium Transport

描述（由申请方提供）：激素调节的肾小管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是上皮Na+转运的最深入研究的调节剂之一，并且由于其在介导醛固酮对上皮钠通道（ENaC）的作用中的重要性而被特别认识。SGK1受双重控制：其表达受醛固酮通过影响基因转录控制，其活性受涉及激酶级联网络的磷酸化控制。最近的证据表明，哺乳动物雷帕霉素靶蛋白（mTOR）是介导SGK 1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发生物理相互作用，这对于丝氨酸422（S422）处的网关磷酸化至关重要。我们进一步假设，SGK1与这种含有mTOR的复合物一起被募集到另一种蛋白质复合物中，其中SGK1通过磷酸化靶点如Nedd4 - 2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的物理缔合和磷酸化的机制基础。有两种含mTOR的多蛋白复合物，mTORC 1和mTORC 2，它们具有不同的组分和底物，并调节不同的细胞过程。我们将：A）使用酵母双杂交测定和体外相互作用检查单个mT0RC 1和2组分与SGK1的物理相互作用。B）鉴定SGK 1和mSin1内介导其相互作用的结构域和特定氨基酸。C）确定mSinl和SGKl之间的物理相互作用是否是SGKl被mTORC 2磷酸化所必需的。D）检查SGK1亲属、Akt、SGK2和SGK3的mTORC2调节。2：表征mTOR和SGKl对培养的皮质集合管（CCD）细胞中的ENaC的功能作用。这一目标将建立上述特征的物理相互作用和SGK1修饰在控制培养细胞中ENaC介导的Na+转运中的功能作用。我们将：A）使用mTOR和SGK1的小分子抑制剂的组合和遗传操作，评估SGK1作为CCD细胞系中ENaC的mTOR依赖性活化的介体的功能作用。B）通过表征突变体对SGK1磷酸化和ENaC电流的影响来评估SGK1与mTORC 2相互作用的功能意义。C）表征细胞条件和激素环境对SGK1和ENaC的mTOR依赖性活化的影响。3：表征mTOR和SGKl在体内Na+稳态中的作用。为了将这些发现推进到天然组织中，我们将：A）检查mTOR激活剂和抑制剂对野生型和SGK1缺失小鼠中Na+平衡、血压和ENaC亚细胞定位的影响。B）检查从醛固酮处理的小鼠收获的分离的灌注CCD中阿米洛利可激发的跨上皮Na+电流的mTOR调节。C）使用膜片钳检查mTOR在调节分离的CCD中的阿米洛利可诱发电流中的作用。

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.