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SGK Regulation of Epithelial Sodium Transport

SGK 对上皮钠转运的调节

基本信息

批准号：
7751896
负责人：
DAVID PEARCE
金额：
$ 29.84万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-08-15 至 2011-09-19
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7751896
关键词：
1-Phosphatidylinositol 3-Kinase 14-3-3 Family 14-3-3 Proteins Aldosterone Antibodies Apical Behavior Binding Binding Sites Biological Assay Biotinylation Cell membrane Cell model Cell surface Cells Congestive Heart Failure Cultured Cells Data Distal Dominant-Negative Mutation Dynamin Endocytosis Epithelial Epithelium Epitopes Event Exocytosis Fab Immunoglobulins Figs - dietary Fluorescence Microscopy Hormones Hypertension Immunohistochemistry In Situ Hybridization In Vitro Insulin Antagonists Ion Transport Kidney Laboratories Learning Life Lipid Binding Lipids Liposomes Location Lysosomes Maintenance Mammals Mediating Membrane Metabolic syndrome Modeling Molecular Chaperones Monitor Morbidity - disease rate Movement N-terminal Nephrons Pathway interactions Phosphatidylinositols Phosphorylation Phosphorylation Site Phosphotransferases Play Protein Isoforms Protein-Serine-Threonine Kinases Proteins Pyroxylin Recycling Regulation Role Signal Pathway Small Interfering RNA Sodium Staining method Stains System Testing Time Ubiquitination Xenopus oocyte base epithelial Na+ channel hormone regulation inhibitor/antagonist mortality mutant novel protein protein interaction response salt sensitive scaffold trafficking ubiquitin ligase

项目摘要

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.

紧密上皮细胞中激素调节的钠转运对于维持循环量和调节哺乳动物的离子环境。 SGK1 是一种醛固酮调节的 PI3 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 Na 运输的激素调节中发挥重要作用 Na 通道 (ENaC)，紧密上皮细胞中 Na+ 重吸收的限速步骤。 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 磷酸化触发 14-3-3家族的伴侣蛋白的招募。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 SGK1 并帮助将其引导至适当的位置抑制 Nedd4-2 并刺激 ENaC 的细胞定位； 2) SGK1 通过刺激 Nedd4-2 与 Nedd4-2 的相互作用来抑制 Nedd4-2 分子伴侣抑制剂14-3-3sigma，或另一种14-3-3蛋白； 3) Nedd4-2和SGK1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 SGK1 与磷酸肌醇相互作用的功能作用和物理基础。我们将确定哪个磷酸肌醇通过固定在硝酸纤维素膜上的磷酸肌醇脂质与 SGK1 相互作用，并掺入进入脂质体。我们将通过以下方式确定磷酸肌醇结合与该新结构域功能的关系检查 SGK1 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 SGK1 磷酸肌醇相互作用结构域在将 SGK1 靶向特定细胞区室中的作用对 PI3K 激活的反应。 2)确定14-3-3蛋白在抑制Nedd4-2中的作用。我们将测试以下人员的能力 SGK1 在存在 14-3-3 相互作用的 Nedd4-2 突变体的情况下刺激 ENaC 介导的 Na+ 电流主题被打乱。我们将确定哪些 14-3-3 同工型在肾 CD 和培养的 CCD 细胞中表达，并且在共免疫沉淀测定中使用异构体特异性抗体与培养的 CCD 细胞中的 Nedd4-2 相互作用。我们将使用消除特定 14-3-3 异构体的表达来确定对醛固酮刺激的 Na+ 转运的影响 siRNA。 3)确定SGK1和Nedd4-2对ENaC运输的影响，并描述其机制基础这个效果。我们将使用表位标记的转染 ENaC 的活细胞染色来检查 SGK1 和 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在通过检查抑制剂和显性负动力对细胞表面 ENaC 的影响来调节 ENaC 运输，并通过模型中内源 ENaC 的生物素化检查 ENaC 电流与顶端运输的相关性 CCD 单元 (mpkCCD)。我们将建立检查 ENaC 内吞作用后的系统。