SGK Regulation of Epithelial Sodium Transport

描述(由申请人提供)：激素调节的钠在肾小管中的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是研究最多的上皮钠转运调节因子之一，在调节醛固酮对上皮钠通道(ENaC)的作用中具有重要作用。SGK1处于双重调控之下：其表达受醛固酮控制，通过影响基因转录，其活性受磷酸化调控，涉及一系列的激酶级联反应。最近的证据表明，哺乳动物的雷帕霉素靶标(MTOR)是介导SGK1激活所需的一个关键的“门户”磷酸化事件的激酶。我们假设SGK1与含有mTOR的多蛋白复合体的特定成分(S)物理上相互作用，mTOR是丝氨酸422处的网关磷酸化所必需的(S422)。我们进一步假设SGK1和这个含有mTOR的复合体被招募到另一个蛋白质复合体，在那里SGK1通过磷酸化Nedd4-2等靶标来调节ENaC。考虑到这些假设，我们这次竞争性更新的具体目标是：1：确定mTOR与SGK1的物理结合和磷酸化的机制基础。有两个含有mTOR的多蛋白复合体，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来检验单个mTORC1&2组分与SGK1的物理相互作用。B)确定SGK1和mSin1中调节它们相互作用的结构域和特定氨基酸。C)确定mSin1和SGK1之间的物理相互作用是否需要使SGK1被mTORC2磷酸化。D)研究SGK1近亲Akt、SGK2和SGK3的mTORC2调控。2：研究mTOR和SGK1对培养的大脑皮层集合管细胞ENaC的作用。这一目标将确定上述物理相互作用和SGK1修饰在控制ENaC介导的培养细胞钠转运中的功能作用。我们将：a)使用mTOR和SGK1的小分子抑制剂和基因操作的组合，评估SGK1作为mTOR依赖的ENaC激活的媒介的功能作用。B)通过表征突变体对SGK1磷酸化和ENaC电流的影响，评估SGK1与mTORC2相互作用的功能意义。C)描述细胞条件和激素环境对mTOR依赖的SGK1和ENaC激活的影响。3.研究mTOR和SGK1在体内钠稳态中的作用。为了将这些发现推广到天然组织中，我们将：a)检测mTOR激活剂和抑制剂对野生型和SGK1缺失小鼠的钠平衡、血压和ENaC亚细胞定位的影响。B)检测从经醛固酮处理的小鼠获取的分离的灌流CCD上可抑制的跨跨上皮钠电流的mTOR调节。C)用膜片钳检测mTOR在调节阿米洛利抑制电流中的作用。

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.