Article: article from journal or magazin.
Ubiquitin-specific protease 2-45 (Usp2-45) binds to epithelial Na+ channel (ENaC)-ubiquitylating enzyme Nedd4-2.
American Journal of Physiology. Renal Physiology
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.
Animals, Blotting, Western, Catalysis, DNA/genetics, Endopeptidases/biosynthesis, Endopeptidases/genetics, Endosomal Sorting Complexes Required for Transport/biosynthesis, Endosomal Sorting Complexes Required for Transport/genetics, Epithelial Sodium Channel/metabolism, Escherichia coli/metabolism, Glutathione/metabolism, HEK293 Cells, Humans, Immunoprecipitation, Kinetics, Mice, Oocytes/metabolism, Patch-Clamp Techniques, Protein Binding, Recombinant Proteins/chemistry, Recombinant Proteins/metabolism, Transfection, Ubiquitin-Protein Ligases/biosynthesis, Ubiquitin-Protein Ligases/genetics, Xenopus laevis
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