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

J Biol Chem. 2011 Oct 28;286(43):37830-40. doi: 10.1074/jbc.M111.293233. Epub 2011 Sep 7.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins / genetics
  • 14-3-3 Proteins / metabolism*
  • Aldosterone / genetics
  • Aldosterone / metabolism
  • Amino Acid Motifs
  • Animals
  • Endosomal Sorting Complexes Required for Transport / genetics
  • Endosomal Sorting Complexes Required for Transport / metabolism*
  • Epithelial Sodium Channels / biosynthesis
  • Gene Expression Regulation / physiology*
  • HEK293 Cells
  • Humans
  • Immediate-Early Proteins / genetics
  • Immediate-Early Proteins / metabolism*
  • Mice
  • Mutation
  • Nedd4 Ubiquitin Protein Ligases
  • Neural Stem Cells / cytology
  • Neural Stem Cells / metabolism*
  • Phosphorylation / physiology
  • Protein Multimerization / physiology
  • Protein Stability
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*

Substances

  • 14-3-3 Proteins
  • Endosomal Sorting Complexes Required for Transport
  • Epithelial Sodium Channels
  • Immediate-Early Proteins
  • YWHAE protein, human
  • Aldosterone
  • Nedd4 Ubiquitin Protein Ligases
  • Nedd4 protein, human
  • Nedd4L protein, human
  • Nedd4l protein, mouse
  • Ubiquitin-Protein Ligases
  • Protein-Serine-Threonine Kinases
  • serum-glucocorticoid regulated kinase