Nedd4-2 Catalyzes Ubiquitination and Degradation of Cell Surface ENaC

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 alpha-, beta-, and gammaENaC. However, little is known about the mechanism by which Nedd4-2 regulates ENaC surface expression. Here we found that Nedd4-2 catalyzes ubiquitination of alpha-, beta-, and gammaENaC; 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.

Epithelial Na + absorption is regulated by Nedd4-2, an E3 ubiquitin ligase that reduces expression of the epithelial Na + channel (ENaC) at the cell surface. Defects in this regulation cause Liddle's syndrome, an inherited form of hypertension. Previous work found that Nedd4-2 binds to ENaC via PY motifs located in the C-termini of α, β, and γENaC. However, little is known about the mechanism by which Nedd4-2 regulates ENaC surface expression.
Here we found that Nedd4-2 catalyzes ubiquitination of α, β, and γENaC; Nedd4-2 overexpression increased ubiquitination whereas Nedd4-2 siRNA decreased ubiquitination. Although Nedd4-2 increased both mono/oligo-ubiquitinated and multiu b i q u i t i n a t e d f o r m s o f E N a C , monoubiquitination was sufficient for Nedd4-2 to reduce ENaC surface expression and reduce ENaC current. Ubiquitination was disrupted by Liddle's 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 hence, reduces epithelial Na + transport.
The epithelial Na + channel (ENaC) 1 functions in Na + transport across epithelia in the kidney collecting duct and connecting tubule, lung, and distal colon, where it plays a critical role in Na + homeostasis. The channel is composed of three homologous subunits (α, β , and γ ENaC) (reviewed in (1,2)). Mutations in β and γENaC cause Liddle's syndrome, an inherited form of hypertension (3). Moreover, most of the known genetic causes of hypertension are caused by defects in ENaC regulation. Defective ENaC regulation may also contribute to lung disease in cystic fibrosis (4). Thus, understanding the mechanisms that regulate ENaC may provide new insights into the pathogenesis of hypertension, cystic fibrosis, and other diseases of Na + homeostasis.
ENaC is regulated in large part by mechanisms that control its expression at the apical membrane of epithelia. Several findings have implicated an important role for Nedd4-2, an E3 ubiquitin-protein ligase. First, Nedd4-2 and ENaC interact through the binding of PY motifs (PPPxYxxL) located in the C-termini of α, β, and γENaC to multiple WW domains in Nedd4-2 (5). Importantly, ENaC mutations that disrupt this interaction cause Liddle's syndrome by increasing ENaC surface expression (6)(7)(8). Second, Nedd4-2 overexpression decreases ENaC current by reducing its expression at the cell surface (8,9). Third, silencing of endogenous Nedd4-2 by RNA interference increases ENaC current (10). Finally, aldosterone and vasopressin regulate ENaC in part by inducing phosphorylation of Nedd4-2 (via serum and glucocorticoid-induced kinase and PKA, respectively), which decreases Nedd4-2 binding to ENaC (9,11,12).
However, critical questions remain about the mechanism by which Nedd4-2 regulates ENaC. First, it is not known if Nedd4-2 regulates ENaC directly by catalyzing ubiquitination of one or more ENaC subunit, or indirectly by catalyzing ubiquitination of an accessory protein. Staub, et al. reported that α and γENaC are substrates for ubiquitination, and that mutation of lysines at the N-termini of these subunits increased ENaC surface expression (13). More recent work suggests that βENaC might also be a substrate for ubiquitination (14,15). However, it is not known if Nedd4-2 or other ubiquitin ligases catalyze ENaC ubiquitination. Second, the cellular location at which Nedd4-2 binds to and regulates ENaC has not been identified. Nedd4-2 could interact with ENaC in the biosynthetic pathway and block it's trafficking to the cell surface. Alternatively, Nedd4-2 could interact with ENaC at the cell surface and increase its endocytosis and/or degradation. In this work, our goal was to test the hypothesis that Nedd4-2 binds to ENaC and catalyzes its ubiquitination at the cell surface, which targets this pool of channels for degradation.
The cells were maintained in Dulbecco's modified Eagle's medium containing 10 µ M amiloride.
In some experiments, unbiotinylated ENaC subunits were immunoprecipitated from either the NeutrAvidin supernatant or from the total cellular lysate.
Cell Surface Ubiquitination-HEK 293T cells transfected with or without α, β-FLAG, and γENaC, Nedd4-2, and ubiquitin-HA were biotinylated at 4 ˚C (to prevent protein trafficking) as above, or not biotinylated as control. Following solubilization in 1% Triton X-100 lysis buffer, βENaC was immunoprecipitated (anti-FLAG M 2 affinity gel) from 800 µg of cell lysate. βENaC was eluted from the gel by incubating with SDS-PAGE sample buffer (100 mM dithiothreitol, 20% glycerol, 100 mM Tris-Cl, pH 6.8, and 4% SDS) at 95 ˚C for 5 min. The supernatant was diluted with 11 volumes of lysis buffer and biotinylated β ENaC was isolated by incubation with immobilized NeutrAvidin beads and separated by SDS-PAGE. Ubiquitinated βENaC at the cell surface was detected by immunoblot (anti-HA antibody, 1:2000).

N e d d 4 -2 C a t a l y z e s E N a C Ubiquitination-
To determine which ENaC subunits are substrates for ubiquitination, we cotransfected HEK 293T cells with α, β, and γENaC (one of the subunits contained a FLAG epitope at its C-terminus) with or without ubiquitin containing an HA epitope (Ub-HA). In order to observe ubiquitinated ENaC subunits, the cells were treated with ALLN to prevent proteasomal degradation. Total α, β, and γENaC were detected by immunoprecipitation followed by immunoblot (Fig. 1, bottom). Ubiquitinated E N a C s u b u n i t s w e r e i s o l a t e d b y immunoprecipitation of ubiquitin (anti-HA) and detected by immunoblot (anti-FLAG). We detected ubiquitinated α, β, and γENaC in cells cotransfected with Ub-HA, but not in cells transfected separately with either ENaC or Ub-HA (Fig. 1, top). Interestingly, we detected at least two different ubiquitinated forms of each subunit; faster migrating bands likely represent ENaC subunits with one or a few ubiquitins attached (monoubiquitinated or oligoubiquitinated), whereas slower migrating bands are multiubiquitinated (polyubiquitinated or polymonoubiquitinated) ENaC.
To test if Nedd4-2 catalyzes ubiquitination of one or more ENaC subunit, we used two strategies. First, we overexpressed Nedd4-2. In these experiments, ALLN was omitted to reduce basal levels of ubiquitination. Fig. 2A-C shows representative immunoblots and Fig. 2D-F shows quantitation of multi-ubiquitinated (left panel) and mono/oligoubiquitinated ENaC subunits (right panel) Nedd4-2 significantly increased ubiquitination of α, β, and γENaC (compare lanes 1 and 2 in Fig. 2A-C). Moreover, Nedd4-2 increased both the faster and slower migrating forms. As controls for specificity, we did not detect ubiquitinated proteins in cells lacking ENaC ( Fig. 2A  As a second strategy, we silenced expression of endogenous Nedd4-2 with Nedd4-2 siRNA. We characterized this siRNA in previous work; it selectively decreased Nedd4-2 protein levels (but not the related E3 ligase Nedd4), and it increased ENaC current in epithelia (10). Here we found that Nedd4-2 siRNA decreased ubiquitination of αENaC ( Fig. 3A and 3B).

Nedd4-2 HECT Domain and ENaC PY M o t i f s a r e R e q u i r e d f o r E N a C Ubiquitination-
The C-terminus of Nedd4-2 contains a HECT domain. Through the binding of ubiquitin to Cys-821, this domain catalyzes ubiquitination of target proteins. Previous work indicates that the HECT domain is required for Nedd4-2 to inhibit ENaC (5,20). Mutation of Cys-821 to Ala abolished Nedd4-2-mediated ubiquitination of α and β ENaC, as well as the slower migrating form of γENaC ( Fig. 2A-C, compare lanes 2 and 3, quantified in Fig. 2D-F). Interestingly, the mutant Nedd4-2 increased the faster migrating mono-or oligoubiquitinated form of γENaC (compared to the group without Nedd4-2), although much less than wild-type Nedd4-2 ( Fig. 2C and 2F). Thus, the catalytic activity of the HECT domain is required for Nedd4-2 to induce ubiquitination of α, β, and the slower migrating form of γ ENaC, but not mono/oligoubiquitinated γENaC.
Nedd4-2 binds to ENaC via PY motifs located in the cytoplasmic C-terminus of each ENaC subunit. Mutation of these motifs prevents Nedd4-2 from inhibiting ENaC (5). To test if binding is required for ubiquitination, we disrupted the PY motifs. Deletion of this motif in βENaC by a Liddle's syndrome mutation (R566X, "β L ") reduced Nedd4-2-induced ubiquitination of α and γENaC (Figs. 2A and 2C, compare lanes 2 and 4, quantitated in 2D and 2F). Mutation of the PY motif (Y620A, "β Y-A ") produced a minimal decrease in the faster migrating form of ubiquitinated βENaC (that did not reach statistical significance), but not the slower migrating form (Fig. 2B lane 4 and Fig. 2E). However, simultaneous mutation of the PY motifs in α, β, and γENaC abolished ubiquitination of βENaC ( Fig. 2B and 2E). Taken together, these data suggest that Nedd4-2 binds to the PY motifs of ENaC subunits, then catalyzes ubiquitination via the HECT domain.
Nedd4-2 and ENaC Interact at the Cell Surface -Although it is clear from previous work that Nedd4-2 binds to ENaC, the cellular location where this interaction occurs has not been identified. We hypothesized that Nedd4-2 interacts with ENaC at the cell surface. To test this hypothesis, we transfected HEK 293T cells with ENaC and Nedd4-2 (containing an HA epitope). Cell surface proteins were then biotinylated and isolated with NeutrAvidin beads. In this fraction of cell surface biotinylated proteins, we detected (by immunoblot) full length (90 kDa) and proteolytically cleaved (65 kDa) forms of αENaC (Fig. 4A, bottom panel). Nedd4-2 was also present in this cell surface fraction in cells expressing ENaC, but not in cells lacking ENaC (Fig. 4A, top panel). These results indicate that Nedd4-2 binds to ENaC at the cell surface. Moreover, they show that Nedd4-2 is not itself a substrate for biotinylation, consistent with its intracellular location.
To test if Nedd4-2 ubiquitinates ENaC at the cell surface, we subjected cells to cell surface biotinylation, followed by sequential precipitation with anti-FLAG beads (to isolate ENaC), then NeutrAvidin beads (to isolate cell surface ENaC). We then analyzed the ubiquitination state of cell surface ENaC by immunoblot for Ub-HA. Fig. 4B shows that βENaC was highly ubiquitinated in the presence but not in the absence of Nedd4-2. Together the data suggest that Nedd4-2 binds to and ubiquitinates ENaC at the cell surface.
Nedd4-2 Selectively Decreases Steady-State Levels of ENaC at the Cell Surface-I f Nedd4-2 regulates ENaC at the cell surface, we predict that it should decrease steady state levels of ENaC at the cell surface, but have little effect on intracellular ENaC (which primarily reflects immature ENaC in the biosynthetic pathway). To test this prediction, we biotinylated cell surface proteins in cells transfected with ENaC (α, β-FLAG, γ) and Nedd4-2 (0-0.5 µg). We separated biotinylated (surface) proteins from nonbiotinylated (intracellular) proteins by binding to NeutrAvidin beads, then detected βENaC in each fraction by immunoblotting; Fig. 5A shows representative immunoblots and Fig. 5B shows quantitation of the Nedd4-2 dose-response relationship. Nedd4-2 did not alter levels of intracellular βENaC (Fig. 5A and 5B, top panels). In contrast, Nedd4-2 produced a dose-dependent decrease in βENaC at the cell surface ( Fig. 5A and 5B, bottom panels). Thus, Nedd4-2 selectively regulates ENaC at the cell surface.
To test if binding is required for Nedd4-2 to reduce ENaC surface expression, we used two strategies. First, we mutated the PY motif in βENaC (β Y620A ). In the absence of Nedd4-2, this mutation increased ENaC surface expression (Fig.  5A, bottom panel), consistent with previous work (6,8). Moreover, it reduced the effect of Nedd4-2 on ENaC surface expression ( Fig. 5A and 5B, bottom panels). In contrast, this mutation had little effect on levels of intracellular ENaC ( Fig.  5A and 5B, top panels). As a second strategy, we mutated the four Nedd4-2 WW domains (WW domains 2-4 mediate binding to ENaC). The mutant Nedd4-2 failed to decrease ENaC surface expression (Fig. 5C). The ubiquitin ligase activity of Nedd4-2 was also required; mutation of the HECT domain (C821A) prevented Nedd4-2 from decreasing ENaC surface expression (Fig. 5D).
Nedd4-2 Induces Degradation of the Cell Surface Pool of ENaC-To further test the hypothesis that Nedd4-2 selectively regulates ENaC at the cell surface, we asked whether Nedd4-2 induces degradation of channels that have reached the cell surface. We selectively measured the rate of degradation of the cell surface pool of ENaC. This pool was labeled by biotinylation (at 4 ˚C to prevent trafficking), then cells were warmed to 37 ˚C for 0-120 min to allow endocytosis and degradation of biotinylated ENaC. In the absence of Nedd4-2, there was a timedependent loss of biotinylated βENaC (Fig. 6A and 6B); the half-life of degradation was approximately one hour. Nedd4-2 dramatically increased the rate of degradation of cell surface ENaC, shortening the half-life to < 20 min ( Fig.  6A and 6B). Mutation of the PY motifs in α, β, and γENaC abolished the effect of Nedd4-2 ( Fig.  6A and 6C). Thus, once ENaC reaches the cell surface, Nedd4-2 induces its degradation. This negative regulation is disrupted in Liddle's syndrome.
ENaC Monoubiquitination is Sufficient for Nedd4-2-induced Degradation-Data in Fig. 2 suggest that Nedd4-2 catalyzes both monoubiquitination and polyubiquitination of α, β, and γENaC. To assess the relative functional importance of these two species, we used a mutant ubiquitin; mutation of each lysine (Ub-0K) prevents formation of polyubiquitin chains (21,22). Expression of Ub-0K increased levels of βENaC at the cell surface (Fig. 7A, compare lanes  1 and 5). This suggests that ENaC surface expression is in part controlled by polyubiquitination. However, Ub-0K did not prevent Nedd4-2 from decreasing ENaC surface expression; the Nedd4-2 dose-response relationship was identical in the presence or absence of Ub-0K (when each group was normalized to surface expression in the absence of Nedd4-2) (Fig. 7A and 7B). In Fischer rat thyroid epithelia transfected with α, β, and γENaC, Ub-0K did not prevent Nedd4-2 from decreasing ENaC current (Fig. 7C). Together the data suggest that monoubiquitination is sufficient for Nedd4-2 to regulate ENaC.

DISCUSSION
From previous work, it is clear that Nedd4-2 plays a key role in regulating epithelial Na + transport. By decreasing the expression of ENaC at the cell surface, Nedd4-2 reduces renal Na + absorption, which is critical in the maintenance of Na + homeostasis. Defects in this regulation cause Liddle's syndrome and may contribute to more common forms of hypertension.
Does Nedd4-2 regulate ENaC surface expression by ubiquitinating one or more ENaC subunit(s)? Nedd4-2 is an E3 ubiquitin-protein ligase. Moreover, its catalytic HECT domain is required for ENaC regulation. Thus, it seems likely that Nedd4-2 regulates ENaC by catalyzing ubiquitination of one or more substrates. These substrates could either be ENaC subunit(s) themselves, or a trans-acting protein that modulates ENaC surface expression. In support of the first possibility, we found that Nedd4-2 catalyzes ubiquitination of α, β, and γENaC. We cannot exclude the possibility that ubiquitination of a trans-acting protein by Nedd4-2 also contributes to regulation of ENaC surface expression. For example, we previously found that Nedd4-2 ubiquitinates and induces degradation of SGK, a Ser/Thr kinase that stimulates ENaC (18). In this regard, it is also intriguing that a yeast homologue of Nedd4-2 (Rsp5) induces endocytosis of Ste2p by catalyzing ubiquitination of a component of the endocytosis machinery (23).
D o e s N e d d 4 -2 c a t a l y z e monoubiquitination or polyubiquitination of ENaC? Our data suggest that Nedd4-2 does both. Nedd4-2 increased the quantity of faster migrating ubiquitinated forms of α, β, and γENaC. Based on the relative molecular mass, this form is consistent with subunits containing one or a small number of ubiquitins (mono-or oligoubiquitinated). Nedd4-2 also increased a high molecular mass smear, consistent with polyubiquitin chains. However, because the N-terminus of each ENaC subunit contains multiple lysines, attachment of a single ubiquitin to multiple residues (polymonoubiquitination) could also contribute to the higher molecular mass forms. In addition to ENaC ubiquitination induced by overexpression of Nedd4-2, we also observed ubiquitinated α, β, and γENaC in cells not transfected with Nedd4-2. This is consistent with previous work from other labs (although there was disagreement about whether βENaC was a substrate for ubiquitination) (13-15). Our data suggest that endogenous Nedd4-2 contributes to this basal level of ubiquitination; silencing of Nedd4-2 reduced ENaC ubiquitination. Importantly, ENaC regulation by Nedd4-2 was intact under conditions that prevented polyubiquitination (Ub-0K). Thus, although Nedd4-2 catalyzes both mono-and polyubiquitination of ENaC, monoubiquitination is sufficient for Nedd4-2 to induce ENaC degradation (conversely, polyubiquitination is not necessary).
E3 ligases function at a variety of cellular locations.
For example, gp78 and CHIP participate in quality control in the biosynthetic pathway, targeting misfolded proteins in the endoplasmic reticulum for degradation in the proteasome (24,25). Other E3 ligases (e.g. Rsp5, Mdm2) target membrane proteins for endocytosis and degradation (23,26). Previous work localized Nedd4-2 both in the cytoplasm and at the cell surface (27). Moreover, the C2 domain (required for Ca 2+ -induced localization to the cell surface (27)) is not required for Nedd4-2 or the related E3 ligase Nedd4 to reduce ENaC surface expression (11,19,28).
These observations raised the possibility that Nedd4-2 and ENaC interact at an intracellular location (27). Our current data suggest an alternative model in which Nedd4-2 binds to ENaC and catalyzes its ubiquitination at the cell surface. First, we found that Nedd4-2 bound to ENaC at the cell surface. Second, Nedd4-2 catalyzed ubiquitination of cell surface ENaC.
Third, Nedd4-2 decreased ENaC expression at the cell surface, but had no effect on intracellular ENaC, which principally reflects immature channels in the biosynthetic pathway. Finally, following ENaC trafficking to the cell surface, Nedd4-2 induced its degradation. However, we cannot exclude an additional role for Nedd4-2 at a cytoplasmic location, either in the biosynthetic or endosomal sorting pathways. Moreover, it seems likely that additional E3 ligases might also regulate ENaC in these locations. Consistent with this notion, silencing of Nedd4-2 did not abolish ENaC ubiquitination. Moreover, disruption of polyubiquitination (Ub-0K) increased ENaC surface expression in spite of is lack of effect on ENaC regulation by Nedd4-2. One caveat is that our studies were carried out in heterologous cell systems; additional work will be required to determine if the same mechanisms are operative in native epithelia.