Nedd4-2 Catalyzes Ubiquitination and Degradation of Cell

Epithelial Na absorption is regulated by Nedd4-2, an E3ubiquitin-proteinligasethatreducesexpressionoftheepithelialNa channelENaCatthecellsurface.DefectsinthisregulationcauseLiddlesyndrome,aninheritedformofhypertension.Pre-vious work found that Nedd4-2 binds to ENaC via PY motifslocatedintheCterminiof -, -,and ENaC.However,littleisknown about the mechanism by which Nedd4-2 regulatesENaC surface expression. Here we found thatNedd4-2 cata-lyzesubiquitinationof -, -,and ENaC;Nedd4-2overexpres-sion increased ubiquitination, whereas Nedd4-2 silencingdecreased ubiquitination. Although Nedd4-2 increased bothmono/oligoubiquitinated and multiubiquitinated forms ofENaC, monoubiquitination was sufficient for Nedd4-2 toreduce ENaC surface expression and reduce ENaC current.Ubiquitination was disrupted by Liddlesyndrome-associatedmutations in ENaC or mutation of the catalytic HECT domainin Nedd4-2. Several findings suggest that the interactionbetweenNedd4-2andENaCislocalizedtothecellsurface.First,Nedd4-2boundtoapopulationofENaCatthecellsurface.Sec-ond, Nedd4-2 catalyzed ubiquitination of cell surface ENaC.Third,Nedd4-2selectivelyreducedENaCexpressionatthecellsurface but did not alter the quantity of immature ENaC in thebiosynthetic pathway. Finally, Nedd4-2 induced degradation ofthecellsurfacepoolofENaC.Together,thedatasuggestamodelin which Nedd4-2 binds to and ubiquitinates ENaC at the cellsurface, which targets surface ENaC for degradation, and thus,reduces epithelial Na transport.