Ubiquitin-specific protease 2-45 (Usp2-45) binds to epithelial Na+ channel (ENaC)-ubiquitylating enzyme Nedd4-2

• Published in: American journal of physiology. Renal physiology 2011-07, Vol.301 (1), p.F189-F196
• Main Authors: Oberfeld, Benjamin, Ruffieux-Daidié, Dorothée, Vitagliano, Jean-Jacques, Pos, Klaas Martinus, Verrey, François, Staub, Olivier
• Format: Article
• Language: English
• Subjects: Animals; Blotting, Western; Catalysis; DNA - genetics; Endopeptidases - biosynthesis; Endopeptidases - genetics; Endopeptidases - metabolism; Endosomal Sorting Complexes Required for Transport - biosynthesis; Endosomal Sorting Complexes Required for Transport - genetics; Endosomal Sorting Complexes Required for Transport - metabolism; Enzymes; Epithelial Sodium Channels - metabolism; Escherichia coli - metabolism; Frogs; Glutathione - metabolism; HEK293 Cells; Humans; Immunoprecipitation; Kinetics; Mice; Nedd4 Ubiquitin Protein Ligases; Oocytes - metabolism; Patch-Clamp Techniques; Physiology; Proteases; Protein Binding; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sodium; Transfection; Ubiquitin-Protein Ligases - biosynthesis; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Xenopus laevis; Xenopus Proteins
• ISSN: 1931-857X; 1522-1466
• DOI: 10.1152/ajprenal.00487.2010

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.