Journal
AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY
Volume 301, Issue 1, Pages F189-F196Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajprenal.00487.2010
Keywords
aldosterone; epithelial sodium channel; deubiquitylation
Categories
Funding
- Swiss National Science Foundation [31003A_125422/1, IZK0Z3-125255]
- Leducq Foundation, Paris (Transatlantic Network on Hypertension)
- Swiss Kidney Foundation
- [Transregio Sonderforschungsbereich Konstanz-Zurich (TR SFB 11)]
- Swiss National Science Foundation (SNF) [IZK0Z3-125255, 31003A_125422] Funding Source: Swiss National Science Foundation (SNF)
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Oberfeld B, Ruffieux-Daidie D, Vitagliano JJ, Pos KM, Verrey F, Staub O. Ubiquitin-specific protease 2-45 (Usp2-45) binds to epithelial Na+ channel (ENaC)-ubiquitylating enzyme Nedd4-2. Am J Physiol Renal Physiol 301: F189-F196, 2011. First published April 6, 2011; 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 alpha-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.
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