首页> 外文期刊>The Journal of biological chemistry >Neural Precursor Cell-expressed Developmentally Down-regulated Protein 4-2 (Nedd4-2) Regulation by 14-3-3 Protein Binding at Canonical Serum and Glucocorticoid Kinase 1 (SGK1) Phosphorylation Sites
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Neural Precursor Cell-expressed Developmentally Down-regulated Protein 4-2 (Nedd4-2) Regulation by 14-3-3 Protein Binding at Canonical Serum and Glucocorticoid Kinase 1 (SGK1) Phosphorylation Sites

机译:神经前体细胞表达显影下调蛋白4-2(NEDD4-2)调节在规范血清和糖皮质激素激酶1(SGK1)磷酸化位点上的14-3-3蛋白结合

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Regulation of epithelial Na+ channel (ENaC)-mediated transport in the distal nephron is a critical determinant of blood pressure in humans. Aldosterone via serum and glucocorticoid kinase 1 (SGK1) stimulates ENaC by phosphorylation of the E3 ubiquitin ligase Nedd4-2, which induces interaction with 14-3-3 proteins. However, the mechanisms of SGK1- and 14-3-3-mediated regulation of Nedd4-2 are unclear. There are three canonical SGK1 target sites on Nedd4-2 that overlap phosphorylation-dependent 14-3-3 interaction motifs. Two of these are termed “minor,” and one is termed “major,” based on weak or strong binding to 14-3-3 proteins, respectively. By mass spectrometry, we found that aldosterone significantly stimulates phosphorylation of a minor, relative to the major, 14-3-3 binding site on Nedd4-2. Phosphorylation-deficient minor site Nedd4-2 mutants bound less 14-3-3 than did wild-type (WT) Nedd4-2, and minor site Nedd4-2 mutations were sufficient to inhibit SGK1 stimulation of ENaC cell surface expression. As measured by pulse-chase and cycloheximide chase assays, a major binding site Nedd4-2 mutant had a shorter cellular half-life than WT Nedd4-2, but this property was not dependent on binding to 14-3-3. Additionally, a dimerization-deficient 14-3-3? mutant failed to bind Nedd4-2. We conclude that whereas phosphorylation at the Nedd4-2 major site is important for interaction with 14-3-3 dimers, minor site phosphorylation by SGK1 may be the relevant molecular switch that stabilizes Nedd4-2 interaction with 14-3-3 and thus promotes ENaC cell surface expression. We also propose that major site phosphorylation promotes cellular Nedd4-2 protein stability, which potentially represents a novel form of regulation for turnover of E3 ubiquitin ligases.
机译:远端肾上的上皮NA +通道(ENAC)介断的运输的调节是人类血压的关键决定因素。通过血清和糖皮质激素激酶1(SGK1)醛固酮通过E3泛素连接酶NEDD4-2的磷酸化刺激ENAC,其诱导与14-3-3蛋白的相互作用。然而,SGK1-和14-3-3介导的NEDD4-2调节的机制尚不清楚。 NEDD4-2上有三个规范SGK1靶位点,其重叠磷酸化依赖性的14-3-3相互作用。其中两种被称为“次要”,并且一个基于弱或强的结合14-3-3蛋白被称为“主要”。通过质谱,我们发现醛固酮显着刺激了在NEDD4-2上的主要14-3-3个结合位点的次要的磷酸化。磷酸化缺陷次次突变位点小于4-3-3的突变体比野生型(WT)NEDD4-2,而次要的位点NEDD4-2突变足以抑制ENAC细胞表面表达的SGK1刺激。通过脉冲序列和环己酰亚胺追踪测定来测量,主要的结合位点NEDD4-2突变体具有比WT NEDD4-2更短的细胞半衰期,但该性质不依赖于结合至14-3-3。另外,减少缺陷14-3-3?突变体未能结合NEDD4-2。我们得出结论,当NEDD4-2主要部位的磷酸化对于与14-3-3二聚体的相互作用是重要的,SGK1的次要部位磷酸化可以是稳定NEDD4-2与14-3-3的相互作用的相关分子开关,从而促进enac细胞表面表达。我们还提出主要部位磷酸化促进细胞NEDD4-2蛋白质稳定性,其可能代表E3泛素连接酶的成交量的新形式。

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