SGK Regulation of Epithelial Sodium Transport

激素调节的致密上皮Na转运对于维持循环容量和调节细胞内Na+浓度是必不可少的。 哺乳动物体内的离子环境。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， 通过影响上皮细胞的运输，在介导钠转运的激素调节中起重要作用。 Na通道（ENaC）是致密上皮细胞Na+重吸收的限速步骤。SGK 1磷酸化并抑制 Nedd 4 -2，一种泛素连接酶，主要通过改变其运输和降解来抑制ENaC，从而导致ENaC 积累在质膜上。我们最近发现，SGK 1介导的Nedd 4 -2磷酸化触发 募集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 细胞定位以抑制Nedd 4 -2并刺激ENaC; 2）SGK 1通过刺激Nedd 4 - 2与ENaC的相互作用来抑制Nedd 4 -2。 伴侣蛋白抑制剂14-3-3sigma或另一种14-3-3蛋白; 3）Nedd 4 -2和SGK 1的相反作用是 表现为ENaC内吞作用和内体定位的变化。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。我们将决定 磷酸肌醇与SGK 1相互作用，使用磷酸肌醇脂质固定在硝酸纤维素膜上，并掺入 转化为脂质体。我们将确定磷脂酰肌醇结合和这个新结构域的功能的关系， 检查SGK 1突变体对ENaC介导的Na+电流和体外激酶活性的影响。我们将确定 SGK 1磷酸肌醇相互作用结构域在将SGK 1靶向特定细胞区室中的作用， 对PI 3 K激活的反应。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在具有14-3-3相互作用的Nedd 4 -2突变体存在下刺激ENaC介导的Na+电流 主题被打乱了我们将确定哪些14-3-3同种型在肾CD和培养的CCD细胞中表达， 在免疫共沉淀试验中使用同种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 测定消除特异性14-3-3同种型表达对醛固酮刺激的Na+转运的影响， siRNA。3)确定SGK 1和Nedd 4 -2对ENaC运输的影响，并表征ENaC运输的机制基础。 这种效果。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。我们将确定胞吐和胞吞作用在 通过检查抑制剂和显性负性发动蛋白对细胞表面ENaC的影响来调节ENaC的运输， 并在模型中通过内源性ENaC的生物素化检查ENaC电流和顶端运输的相关性 CCD单元（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.