title: Ubiquitin-specific protease 2-45 (Usp2-45) binds to epithelial Na+ channel (ENaC)-ubiquitylating enzyme Nedd4-2.
abstract: Regulation of the epithelial Na(+) channel (ENaC) by ubiquitylation is controlled by the activity of two counteracting enzymes, the E3 ubiquitin-protein ligase Nedd4-2 (mouse ortholog of human Nedd4L) and the ubiquitin-specific protease Usp2-45. Previously, Usp2-45 was shown to decrease ubiquitylation and to increase surface function of ENaC in Xenopus laevis oocytes, whereas the splice variant Usp2-69, which has a different N-terminal domain, was inactive toward ENaC. It is shown here that the catalytic core of Usp2 lacking the N-terminal domain has a reduced ability relative to Usp2-45 to enhance ENaC activity in Xenopus oocytes. In contrast, its catalytic activity toward the artificial substrate ubiquitin-AMC is fully maintained. The interaction of Usp2-45 with ENaC exogenously expressed in HEK293 cells was tested by coimmunoprecipitation. The data indicate that different combinations of ENaC subunits, as well as the α-ENaC cytoplasmic N-terminal but not C-terminal domain, coprecipitate with Usp2-45. This interaction is decreased but not abolished when the cytoplasmic ubiquitylation sites of ENaC are mutated. Importantly, coimmunoprecipitation in HEK293 cells and GST pull-down of purified recombinant proteins show that both the catalytic domain and the N-terminal tail of Usp2-45 physically interact with the HECT domain of Nedd4-2. Taken together, the data support the conclusion that Usp2-45 action on ENaC is promoted by various interactions, including through binding to Nedd4-2 that is suggested to position Usp2-45 favorably for ENaC deubiquitylation.
authors.affiliation: Institute of Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Oberfeld
authors.forename: Dorothée
authors.initials: D
authors.lastname: Ruffieux-Daidié
authors.forename: Jean-Jacques
authors.initials: JJ
authors.lastname: Vitagliano
authors.forename: Klaas Martinus
authors.initials: KM
authors.lastname: Pos
authors.forename: François
authors.initials: F
authors.lastname: Verrey
authors.forename: Olivier
authors.initials: O
authors.lastname: Staub
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D004247:DNA / Q000235:genetics; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D007700:Kinetics; D051379:Mice; D000075702:Nedd4 Ubiquitin Protein Ligases; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D043222:Ubiquitin Thiolesterase; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins; D014982:Xenopus laevis
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D004247:DNA; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618482:Nedd4 protein, Xenopus; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618461:Nedd4l protein, mouse; C000618460:nedd4l protein, Xenopus; D044767:Ubiquitin-Protein Ligases; D010450:Endopeptidases; C108665:USP2 protein, human; D043222:Ubiquitin Thiolesterase; D005978:Glutathione