SGK Regulation of Epithelial Sodium Transport

激素调节的紧密上皮钠转运对维持循环容量和调节 哺乳动物体内的离子环境。SGK1是一种受醛固酮调节的PI3依赖的丝氨酸苏氨酸激酶，它 通过对上皮细胞转运的影响，在调节激素对钠转运的调节中发挥重要作用 钠通道(ENaC)，紧致上皮细胞重吸收Na+的限速步骤。SGK1磷酸化并抑制 Nedd4-2，一种泛素连接酶，主要通过改变ENaC的运输和降解来抑制ENaC，从而导致ENaC 在质膜中积累。我们最近发现SGK1介导的Nedd4-2的磷酸化触发 招募14-3-3家族的伴侣蛋白。这些观察和其他最近的研究表明 以下假设：1)与肌醇磷脂的相互作用激活SGK1，并帮助其导向适当的 抑制Nedd4-2和刺激ENaC的细胞定位；2)SGK1通过刺激其与ENaC的相互作用来抑制Nedd4-2 伴侣抑制剂14-3-3sigma或其他14-3-3蛋白；3)Nedd4-2和SGK1的相反作用是 主要表现为ENaC内吞作用和内体定位的改变。我们提出了以下目标：1)确定 SGK1与肌醇磷脂相互作用的功能作用和物理基础。我们将确定哪一个 通过将肌醇磷脂固定在硝酸纤维素膜上，肌醇磷脂与SGK1相互作用 转化为脂质体。我们将通过以下方法确定磷脂酰肌醇结合和这个新结构域功能的关系 检测SGK1突变体对ENaC介导的钠电流和体外激活酶活性的影响。我们将决定 SGK1磷脂酰肌醇相互作用结构域在靶向SGK1特定细胞室中的作用 对PI3K激活的反应。2)确定14-3-3蛋白在抑制Nedd4-2中的作用。我们将测试他们的能力 SGK1在具有14-3-3相互作用的Nedd4-2突变体中刺激ENaC介导的钠电流 主旨被破坏了。我们将确定哪些14-3-3亚型在肾脏CD和培养的CD细胞中表达，以及 在免疫共沉淀试验中使用异构体特异性抗体与培养的CD细胞中的Nedd4-2相互作用。我们会 测定消除特异性14-3-3亚型表达对醛固酮刺激的Na+转运的影响 SiRNA。3)确定SGK1和Nedd4-2对ENaC转运的影响，并表征其机制基础 这种效果。我们将使用标记表位的转基因ENaC的活细胞染色来检测SGK1和SGK1的作用 Nedd4-2对ENaC质膜定位的影响。我们将确定胞吐作用和内吞作用(S)在 通过检测抑制物和显性负动力蛋白对细胞表面ENaC的影响来调节ENaC的运输， 并在一个模型中通过内源性ENaC的生物素化来检验ENaC电流和心尖运输的相关性 Ccd细胞(MpkCCD)。我们将建立ENaC内吞后检测系统。

Hormone-regulated Na transport in tight epithelia is essential for maintenance of circulatory volume and regulation of the ionic milieu in mammals. SGK1 is an aldosterone-regulated PI3-kinase-dependent serine-threonine kinase that plays an important role in mediating hormonal regulation of Na transport through its effects on trafficking of the epithelial Na channel (ENaC), the rate limiting step in Na+ reabsorption in tight epithelia. SGK1 phosphorylates and inhibits Nedd4-2, a ubiquitin ligase that inhibits ENaC primarily by altering its trafficking and degradation, thus leading to ENaC accumulation in the plasma membrane. We recently found that SGK1-mediated phosphorylation of Nedd4-2 triggers recruitment of a chaperone protein of the 14-3-3 family. These observations and other recent studies suggest the following hypotheses: 1) Interactions with phosphoinositide lipids activate SGK1 and help direct it to the appropriate cellular location to inhibit Nedd4-2 and stimulate ENaC; 2) SGK1 inhibits Nedd4-2 by stimulating its interaction with the chaperone inhibitor 14-3-3sigma, or another 14-3-3 protein; 3) The opposing effects of Nedd4-2 and SGK1 are manifested in changes in ENaC endocytosis and endosomal localization. We propose the following aims: 1) Determine the functional roles and physical basis of SGK1 interactions with phosphoinositides. We will determine which phosphoinositides interact with SGK1 using phosphoinositide lipids fixed to nitrocellulose membranes, and incorporated into liposomes. We will determine the relationship of phosphoinositide binding and function of this novel domain by examining the effect of SGK1 mutants on ENaC-mediated Na+ currents and on in vitro kinase activity. We will determine the role of the SGK1 phosphoinositide interaction domain in targeting SGK1 to specific cellular compartments in response to PI3K activation. 2) Determine the role of 14-3-3 proteins in inhibiting Nedd4-2. We will test the ability of SGK1 to stimulate ENaC-mediated Na+ currents in the presence of Nedd4-2 mutants that have their 14-3-3 interaction motif disrupted. We will determine which 14-3-3 isoforms are expressed in kidney CD and in cultured CCD cells, and interact with Nedd4-2 in cultured CCD cells using isoform specific antibodies in coimmunoprecipitation assays. We will determine the effect on aldosterone-stimulated Na+ transport of eliminating expression of specific 14-3-3 isoforms using siRNA. 3) Determine the effect of SGK1 and Nedd4-2 on ENaC trafficking, and characterize the mechanistic basis of this effect. We will use live cell staining of epitope-tagged transfected ENaC to examine the effects of SGK1 and Nedd4-2 on plasma membrane localization of ENaC. We will determine the role(s) of exocytosis and endocytosis in regulated ENaC trafficking by examining the effects of inhibitors and dominant negative dynamin on cell surface ENaC, and examine the correlation of ENaC currents and apical trafficking by biotinylation of endogenous ENaC in a model CCD cell (mpkCCD). We will establish systemsfor examining ENaC post-endocytosis.