Phosphopeptide screen uncovers novel phosphorylation sites of Nedd4-2 that potentiate its inhibition of the epithelial Na+ channel

J Biol Chem. 2010 Jul 9;285(28):21671-8. doi: 10.1074/jbc.M109.084731. Epub 2010 May 13.

Abstract

The E3 ubiquitin ligase Nedd4-2 regulates several ion transport proteins, including the epithelial Na(+) channel (ENaC). Nedd4-2 decreases apical membrane expression and activity of ENaC. Although it is subject to tight hormonal control, the mechanistic basis of Nedd4-2 regulation remains poorly understood. To characterize regulatory inputs to Nedd4-2 function, we screened for novel sites of Nedd4-2 phosphorylation using tandem mass spectrometry. Three of seven identified Xenopus Nedd4-2 Ser/Thr phosphorylation sites corresponded to previously identified target sites for SGK1, whereas four were novel, including Ser-293, which matched the consensus for a MAPK target sequence. Further in vitro and in vivo phosphorylation experiments revealed that Nedd4-2 serves as a target of JNK1, but not of p38 MAPK or ERK1/2. Additional rounds of tandem mass spectrometry identified two other phosphorylated residues within Nedd4-2, including Thr-899, which is present within the catalytic domain. Nedd4-2 with mutations at these sites had markedly inhibited JNK1-dependent phosphorylation, virtually no ENaC inhibitory activity, and significantly reduced ubiquitin ligase activity. These data identify phosphorylatable residues that activate Nedd4-2 and may work together with residues targeted by inhibitory kinases (e.g. SGK1 and protein kinase A) to govern Nedd4-2 regulation of epithelial ion transport.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Catalytic Domain
  • Endosomal Sorting Complexes Required for Transport / chemistry*
  • Epithelial Sodium Channels / chemistry*
  • Humans
  • Immediate-Early Proteins / metabolism
  • Mice
  • Mitogen-Activated Protein Kinase 8 / metabolism
  • Molecular Sequence Data
  • Nedd4 Ubiquitin Protein Ligases
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism
  • Recombinant Proteins / chemistry
  • Tandem Mass Spectrometry / methods
  • Ubiquitin / chemistry
  • Ubiquitin-Protein Ligases / chemistry*
  • Xenopus
  • Xenopus Proteins

Substances

  • Endosomal Sorting Complexes Required for Transport
  • Epithelial Sodium Channels
  • Immediate-Early Proteins
  • Recombinant Proteins
  • Ubiquitin
  • Xenopus Proteins
  • Nedd4 Ubiquitin Protein Ligases
  • Nedd4 protein, Xenopus
  • Nedd4 protein, human
  • Nedd4L protein, human
  • Nedd4l protein, mouse
  • nedd4l protein, Xenopus
  • Ubiquitin-Protein Ligases
  • Protein-Serine-Threonine Kinases
  • serum-glucocorticoid regulated kinase
  • Mitogen-Activated Protein Kinase 8