title: 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.
abstract: 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 14-3-3ε 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.
authors: Division of Nephrology, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305, USA.
authors: Sindhu
authors: S
authors: Chandran
authors: Hui
authors: H
authors: Li
authors: Wuxing
authors: W
authors: Dong
authors: Karolina
authors: K
authors: Krasinska
authors: Chris
authors: C
authors: Adams
authors: Ludmila
authors: L
authors: Alexandrova
authors: Allis
authors: A
authors: Chien
authors: Kenneth R
authors: KR
authors: Hallows
authors: Vivek
authors: V
authors: Bhalla
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D000450:Aldosterone / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D053503:Epithelial Sodium Channels / Q000096:biosynthesis; D005786:Gene Expression Regulation / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D000075702:Nedd4 Ubiquitin Protein Ligases; D058953:Neural Stem Cells / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D055503:Protein Multimerization / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D000099184:Serum-Glucocorticoid Regulated Kinases
chemical_list: D048948:14-3-3 Proteins; D000450:Aldosterone; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D017874:Immediate-Early Proteins; D000075702:Nedd4 Ubiquitin Protein Ligases; D017346:Protein Serine-Threonine Kinases; D044767:Ubiquitin-Protein Ligases; D000099184:Serum-Glucocorticoid Regulated Kinases; C482983:YWHAE protein, human; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618461:Nedd4l protein, mouse