Nedd4-2 Catalyzes Ubiquitination and Degradation of Cell Surface ENaC

Abstract

Epithelial Na؉ absorption is regulated by Nedd4-2, an E3 ubiquitin-protein ligase that reduces expression of the epithelial Na ؉ channel ENaC at the cell surface. Defects in this regulation cause Liddle syndrome, an inherited form of hypertension. Previous work found that Nedd4-2 binds to ENaC via PY motifs located in the C termini of ␣-, ␤-, and ␥ENaC. However, little is known about the mechanism by which Nedd4-2 regulates ENaC surface expression. Here we found that Nedd4-2 catalyzes ubiquitination of ␣-, ␤-, and ␥ENaC; Nedd4-2 overexpression increased ubiquitination, whereas Nedd4-2 silencing decreased ubiquitination. Although Nedd4-2 increased both mono/oligoubiquitinated and multiubiquitinated forms of ENaC, monoubiquitination was sufficient for Nedd4-2 to reduce ENaC surface expression and reduce ENaC current. Ubiquitination was disrupted by Liddle syndrome-associated mutations in ENaC or mutation of the catalytic HECT domain in Nedd4-2. Several findings suggest that the interaction between Nedd4-2 and ENaC is localized to the cell surface. First, Nedd4-2 bound to a population of ENaC at the cell surface. Second, Nedd4-2 catalyzed ubiquitination of cell surface ENaC. Third, Nedd4-2 selectively reduced ENaC expression at the cell surface but did not alter the quantity of immature ENaC in the biosynthetic pathway. Finally, Nedd4-2 induced degradation of the cell surface pool of ENaC. Together, the data suggest a model in which Nedd4-2 binds to and ubiquitinates ENaC at the cell surface, which targets surface ENaC for degradation, and thus, reduces epithelial Na ؉ transport.

Role of Epithelial Sodium Channels and Their Regulators in Hypertension

“…Nedd4-2-dependent ubiquitination of ENaC subunits targets channels for internalization from the plasma membrane (10). Once internalized, channels may be targeted for degradation (11)(12)(13). Alternatively, the release of ubiquitin by deubiquitinating enzymes allows channels to recycle to the plasma membrane (14).…”

Epithelial sodium channel regulated by differential composition of a signaling complex

“…Disorders of ENaC expression or regulation result in severe disease phenotypes in humans (1,2). Two pleiotropic signaling pathways are implicated in regulating ENaC trafficking and activity: phosphatidylinositide 3Ј-kinase (PI3K)-dependent signals stimulate the AGC family kinase serum-and glucocorticoid-induced kinase 1 (SGK1), which phosphorylates and inhibits the HECT-domain ubiquitin ligase Nedd4-2; Nedd4-2 in turn physically interacts with and ubiquitinates the channel, reducing its recycling to the plasma membrane and stimulating its internalization and degradation (1,3,4). It is interesting to note that the PI3K pathway, and SGK1, in particular, affect a variety of other signaling molecules and transcription factors to markedly influence distinct cellular processes such as proliferation, apoptosis, and glucose metabolism.…”

Regulation of Epithelial Sodium Channel Trafficking by Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9)

“…Mutations were generated by site-directed mutagenesis (QuikChange; Stratagene). ␣ENaC-FLAG, ␤ENaC-FLAG, and ␥ENaC-FLAG were generated by insertion of a FLAG epitope (DYKDDDDK) at the C terminus (5,41). Human PCSK9-V5 was a generous gift from Nabil Seidah (24), and Nedd4-2-HA was generated as described (42).…”

“…To measure the rate of degradation of the cell surface fraction of ENaC, HEK 293 cells transfected with ␣ENaC-FLAG, ␤ENaC, and ␥ENaC with PCSK9 or GFP were biotinylated on ice and then incubated at 37°C for 0 -120 min (5,41). Biotinylated ␣ENaC-FLAG was isolated using NeutrAvidin-agarose, detected by immunoblot (anti-FLAG M2-peroxidase-conjugated antibody), and quantitated by densitometry.…”

“…To test for interactions between ENaC and PCSK9, the cells were transfected with ␣ENaC, ␤ENaC, and ␥ENaC (individually or together, 1 g each) and PCSK9-V5 or GFP (1 or 3 g) using Lipofectamine2000 (Invitrogen) (5,41). One ENaC subunit contained a FLAG epitope.…”