Participation of the ubiquitin-conjugating enzyme UBE2E3 in Nedd4-2-dependent regulation of the epithelial Na+ channel.
The epithelial Na+ channel (ENaC) is a heteromeric protein complex playing a fundamental role in Na+ homeostasis and blood pressure regulation. Specific mutations inactivating PY motifs in ENaC C termini cause Liddle's syndrome, an inherited form of hypertension. Previously we showed that these PY motifs serve as binding sites for the E3 enzyme Nedd4-2, implying ubiquitination as a regulatory mechanism of ENaC. Ubiquitination involves the sequential action of E1, E2, and E3 enzymes. Here we identify the E2 enzyme UBE2E3, which acts in concert with Nedd4-2, and show by coimmunoprecipitation that UBE2E3 and Nedd4-2 interact together. In Xenopus laevis oocytes, UBE2E3 reduces ENaC activity marginally, consistent with Nedd4-2 being the rate-limiting factor in this process, whereas a catalytically inactive mutant of UBE2E3 (UBE2E3-CS) causes elevated ENaC activity by increasing cell surface expression. No additive effect is observed when UBE2E3-CS is coexpressed with an inactive Nedd4-2 mutant, and the stimulatory role of UBE2E3-CS depends on the integrity of the PY motifs (Nedd4-2 binding sites) and the ubiquitination sites on ENaC. In renal mpkCCD(cl4) cells, displaying ENaC-dependent transepithelial Na+ transport, Nedd4-2 and UBE2E3 can be coimmunoprecipitated and overexpression of UBE2E3 affects Na+ transport, corroborating the concept of a concerted action of UBE2E3 and Nedd4-2 in ENaC regulation.
Department of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland.
Christophe
C
Debonneville
Olivier
O
Staub
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D056827:Endosomal Sorting Complexes Required for Transport; D004847:Epithelial Cells / Q000378:metabolism; D053503:Epithelial Sodium Channels; D005260:Female; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D000075702:Nedd4 Ubiquitin Protein Ligases; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins; D014982:Xenopus laevis
D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D011994:Recombinant Proteins; D015222:Sodium Channels; D029867:Xenopus Proteins; D044763:Ubiquitin-Conjugating Enzymes; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618482:Nedd4 protein, Xenopus; C000618461:Nedd4l protein, mouse; C000618460:nedd4l protein, Xenopus; D044767:Ubiquitin-Protein Ligases