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Molecular and Cellular Biology, March 2004, p. 2397-2409, Vol. 24, No. 6
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.6.2397-2409.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Participation of the Ubiquitin-Conjugating Enzyme UBE2E3 in Nedd4-2-Dependent Regulation of the Epithelial Na+ Channel

Christophe Debonneville and Olivier Staub*

Department of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland

Received 14 May 2003/ Returned for modification 17 July 2003/ Accepted 13 December 2003

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 mpkCCDcl4 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.

* Corresponding author. Mailing address: Department of Pharmacology and Toxicology, University of Lausanne, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland. Phone: 41-21-692-5407. Fax: 41-21-692-5355. E-mail: olivier.staub{at}ipharm.unil.ch.

Molecular and Cellular Biology, March 2004, p. 2397-2409, Vol. 24, No. 6
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.6.2397-2409.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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