Ubiquitin-specific Peptidase 8 (USP8) Regulates Endosomal Trafficking of the Epithelial Na+ Channel*

Ruifeng Zhou,Vivian R Tomkovicz,Phillip L Butler,Luis A Ochoa,Zerubbabel J Peterson,Peter M Snyder

doi:10.1074/jbc.m112.425272

Abstract

Ubiquitination plays a key role in trafficking of the epithelial Na(+) channel (ENaC). Previous work indicated that ubiquitination enhances ENaC endocytosis and sorting to lysosomes for degradation. Moreover, a defect in ubiquitination causes Liddle syndrome, an inherited form of hypertension. In this work, we identified a role for USP8 in the control of ENaC ubiquitination and trafficking. USP8 increased ENaC current in Xenopus oocytes and collecting duct epithelia and enhanced ENaC abundance at the cell surface in HEK 293 cells. This resulted from altered endocytic sorting; USP8 abolished ENaC degradation in the endocytic pathway, but it had no effect on ENaC endocytosis. USP8 interacted with ENaC, as detected by co-immunoprecipitation, and it deubiquitinated ENaC. Consistent with a functional role for deubiquitination, mutation of the cytoplasmic lysines of ENaC reduced the effect of USP8 on ENaC cell surface abundance. In contrast to USP8, USP2-45 increased ENaC surface abundance by reducing endocytosis but not degradation. Thus, USP8 and USP2-45 selectively modulate ENaC trafficking at different steps in the endocytic pathway. Together with previous work, the data indicate that the ubiquitination state of ENaC is critical for the regulation of epithelial Na(+) absorption.

Highlights

Ubiquitination controls trafficking of the epithelial Naϩ channel (ENaC) in the endocytic pathway
We found that USP8 increased ENaC current by almost 2-fold
USP8 Does Not Alter ENaC Endocytosis—In previous work, we found that ubiquitination controls ENaC cell surface expression at two distinct steps in the endocytic pathway; ubiquitination increases both ENaC endocytosis and lysosomal targeting for degradation [11, 17]

Summary

Background

Ubiquitination controls trafficking of the epithelial Naϩ channel (ENaC) in the endocytic pathway. Previous work indicated that ubiquitination enhances ENaC endocytosis and sorting to lysosomes for degradation. Consistent with a functional role for deubiquitination, mutation of the cytoplasmic lysines of ENaC reduced the effect of USP8 on ENaC cell surface abundance. Together with STAM (signal-transducing adaptor molecule), Hrs forms a complex called ESCRT-0 [18, 19] Disruption of this complex reduces ENaC degradation and increases its recycling to the cell surface [20]. USP2-45 (ubiquitin-specific peptidase 2-45) is an aldosterone-induced protein that increases ENaC current by enhancing its abundance at the cell surface [21,22,23,24]. Our goal in this work was to test whether USP8 modulates ENaC trafficking and to determine which trafficking steps are modulated by DUBs

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION