SGK Regulation of Epithelial Sodium Transport

描述（由申请方提供）：激素调节的致密上皮细胞中的Na转运对于维持哺乳动物的循环容量和调节离子环境至关重要。SGK 1是一种醛固酮调节的PI 3激酶依赖性丝氨酸-苏氨酸激酶，通过其对上皮Na通道（ENaC）的运输（致密上皮中Na+重吸收的限速步骤）的影响，在介导Na转运的激素调节中发挥重要作用。SGK 1磷酸化并抑制Nedd 4 -2，Nedd 4 -2是一种泛素连接酶，主要通过改变其运输和降解来抑制ENaC，从而导致ENaC在质膜中积累。我们最近发现，SGK 1介导的Nedd 4 -2磷酸化触发了14-3-3家族伴侣蛋白的募集。这些观察和其他最近的研究提出了以下假设：1）与磷酸肌醇脂质的相互作用激活SGK 1并帮助将其引导到适当的细胞位置以抑制Nedd 4 -2并刺激ENaC; 2）SGK 1通过刺激其与伴侣蛋白抑制剂14-3-3sigma或另一种14-3-3蛋白的相互作用来抑制Nedd 4 -2; 3）Nedd 4 -2和SGK 1的相反作用表现在ENaC内吞和内体定位的变化。我们提出以下目标：1）确定SGK 1与磷酸肌醇相互作用的功能作用和物理基础。我们将使用固定在硝酸纤维素膜上的磷脂酰肌醇脂质，并将其掺入脂质体中，来确定哪些磷脂酰肌醇与SGK 1相互作用。我们将通过研究SGK 1突变体对ENaC介导的Na+电流和体外激酶活性的影响，确定磷酸肌醇结合和这种新结构域的功能之间的关系。我们将确定SGK 1磷酸肌醇相互作用结构域在响应PI 3 K激活将SGK 1靶向特定细胞隔室中的作用。2)确定14-3-3蛋白在抑制Nedd 4 -2中的作用。我们将测试SGK 1在Nedd 4 -2突变体（其14-3-3相互作用基序被破坏）存在下刺激ENaC介导的Na+电流的能力。我们将确定哪些14-3-3亚型在肾CD和培养的CCD细胞中表达，并在免疫共沉淀试验中使用亚型特异性抗体与培养的CCD细胞中的Nedd 4 -2相互作用。我们将确定使用siRNA消除特定14-3-3亚型的表达对醛固酮刺激的Na+转运的影响。3)确定SGK 1和Nedd 4 -2对ENaC运输的影响，并表征这种影响的机制基础。我们将使用表位标记的转染ENaC的活细胞染色来检查SGK 1和Nedd 4 -2对ENaC质膜定位的影响。我们将通过检查抑制剂和显性负动力蛋白对细胞表面ENaC的影响来确定胞吐和胞吞在调节ENaC运输中的作用，并通过模型CCD细胞（mpkCCD）中内源性ENaC的生物素化来检查ENaC电流和顶端运输的相关性。我们将建立检查ENaC后内吞作用的系统。

DESCRIPTION (provided by applicant): 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 phosphorylate 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 systems for examining ENaC post-endocytosis.