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Originally published In Press as doi:10.1074/jbc.M611329200 on May 14, 2007

J. Biol. Chem., Vol. 282, Issue 28, 20207-20212, July 13, 2007
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Nedd4-2 Catalyzes Ubiquitination and Degradation of Cell Surface ENaC*

Ruifeng Zhou, Saumil V. Patel, and Peter M. Snyder1

From the Departments of Internal Medicine and Molecular Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242

Received for publication, December 11, 2006 , and in revised form, April 27, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {gamma}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 {alpha}-, beta-, and {gamma}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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The epithelial Na+ channel ENaC 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 ({alpha}-, beta-, and {gamma}ENaC) (reviewed in Refs. 1 and 2). Mutations in beta- and {gamma}ENaC cause Liddle 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 {alpha}-, beta-, and {gamma}ENaC to multiple WW domains in Nedd4-2 (5). Importantly, ENaC mutations that disrupt this interaction cause Liddle syndrome by increasing ENaC surface expression (6–8). 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 cAMP-dependent protein kinase, 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 whether Nedd4-2 regulates ENaC directly, by catalyzing ubiquitination of one or more ENaC subunits, or indirectly, by catalyzing ubiquitination of an accessory protein. Staub et al. (13) reported that {alpha}- and {gamma}ENaC are substrates for ubiquitination and that mutation of lysines at the N termini of these subunits increased ENaC surface expression. More recent work suggests that betaENaC might also be a substrate for ubiquitination (14, 15). However, it is not known whether 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 its 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
cDNA Constructs—Human {alpha}-, beta-, and {gamma}ENaC (16, 17), Nedd4-2 (11), and Nedd4-2C821A (18) in pMT3 were cloned as described previously. {alpha}-FLAG, beta-FLAG, and {gamma}-FLAG were generated by the insertion of a FLAG epitope (DYKDDDDK) at the C terminus. PY motif mutations were generated in {alpha}ENaC (Y644A), betaENaC (R566X and Y620A), and {gamma}ENaC (Y627A) as described previously (6). Nedd4-2WW1–4 was generated by mutating two residues in each of the four WW domains (WW1, V210W and H212G; WW2, V367W and H369G; WW3, I440W and H442G; WW4, I492W and H494G). These mutations abolish Nedd4-2 binding to ENaC, similar to previous work with Nedd4 (19). HA2-tagged ubiquitin (in pMT123) was provided by Dirk Bohmann (University of Rochester), and lysine-less ubiquitin cDNA (Ub-0K) was provided by Joan Conaway (Stowers Institute for Medical Research).

Ubiquitination Assay—To detect ENaC ubiquitination, HEK 293T cells were transfected with or without cDNAs encoding ubiquitin-HA and {alpha}-, beta-, and {gamma}ENaC (one subunit contained FLAG epitope) using Lipofectamine 2000 (Invitrogen). In some studies, cells were cotransfected with Nedd4-2 (wild type or C821A) or green fluorescent protein (GFP, control) cDNA, or with siRNA against Nedd4-2 (10) or GFP (control). The cells were maintained in Dulbecco's modified Eagle's medium containing 10 µM amiloride. To inhibit proteasomal degradation, 10 µM N-acetyl-Leu-Leu-norleucinal (ALLN) was added to some cells 2 h before lysis. 24 h after transfection, cells were solubilized in lysis buffer (150 mM NaCl, 50 mM Tris (pH 7.4), 1% Triton X-100, and protease inhibitor mixture (Sigma)), and 800 µg of protein was immunoprecipitated with anti-HA antibody (1:300, Sigma) and immobilized protein A (Pierce). After extensive washing, immunoprecipitated (ubiquitinated) proteins were separated by SDS-PAGE, detected by immunoblot with anti-FLAG M2 monoclonal antibody-peroxidase conjugate (1:5000, Sigma), and quantitated by densitometry.

Cell Surface Biotinylation and Protein Interactions—HEK 293T cells were transfected with {alpha}-, beta-, and {gamma}ENaC (1 µg each) and Nedd4-2-HA (0.04 µg). 24 h later, the cells were washed with PBS-CM (PBS with 1 mM MgCl2 and CaCl2), cell surface proteins were labeled with 0.5 mg/ml sulfo-NHS-biotin (Pierce) in PBS-CM for 30 min on ice, and then cells were quenched with 100 mM glycine in PBS-CM for 10 min on ice. After washing three times with PBS-CM, cells were lysed in Nonidet P-40 lysis buffer (0.4% sodium deoxycholate, 1% Nonidet P-40, 63 mM EDTA, 50 mM Tris-HCl (pH 8), and protease inhibitor mixture). Biotinylated (cell surface) and interacting proteins were isolated by incubating cell lysate with immobilized NeutrAvidin beads (Pierce) for 12 h at 4 °C. Following separation by SDS-PAGE, biotinylated {alpha}ENaC and coprecipitated Nedd4-2 were detected by immunoblot. In some experiments, unbiotinylated ENaC subunits were immunoprecipitated from either the NeutrAvidin supernatant or the total cellular lysate.

Cell Surface Ubiquitination—HEK 293T cells transfected with or without {alpha}ENaC, beta-FLAG, and {gamma}ENaC, 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, betaENaC was immunoprecipitated (anti-FLAG M2 affinity gel) from 800 µg of cell lysate. betaENaC 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 betaENaC was isolated by incubation with immobilized NeutrAvidin beads and separated by SDS-PAGE. Ubiquitinated betaENaC at the cell surface was detected by immunoblot (anti-HA antibody, 1:2000).


Figure 1
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  		FIGURE 1.
ENaC ubiquitination. HEK 293T cells were transfected with {alpha}-, beta-, and {gamma}ENaC (1 µg each, one subunit contained C-terminal FLAG) with or without ubiquitin-HA (3 µg) and incubated for 24 h prior to lysis (10 µM ALLN was present for the final 2 h). Ubiquitinated (top panel) and total (bottom panel) ENaC subunits were detected by immunoprecipitation (IP) followed by immunoblot (IB), as indicated. Data are representative of at least three experiments.

 
Degradation of Cell Surface ENaC—HEK 293T cells were transfected with {alpha}ENaC, beta-FLAG, and {gamma}ENaC (wild type or {alpha}Y644A, betaY620A, {gamma}Y627A) with Nedd4-2 or GFP. 24 h after transfection, cells were biotinylated on ice for 30 min and then incubated at 37 °C for 0, 40, 80, or 120 min. Cell surface betaENaC was isolated by incubation with immobilized NeutrAvidin beads, detected by immunoblot with anti-FLAG M2 monoclonal antibody-peroxidase conjugate, and quantitated by densitometry.

Short-Circuit Current—Fischer rat thyroid cells on permeable filter supports were cotransfected with {alpha}-, beta-, and {gamma}ENaC (0.17 µg each) with or without Nedd4-2 (0.4 µg) and Ub-0K (0.5 µg) using TFX50 (Promega), as described previously (11); total cDNA was held constant with GFP cDNA. Short-circuit Na+ current was measured using modified Ussing chambers (Warner Instrument Corp.). The apical and basolateral surfaces were bathed in 135 mM NaCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 2.4 mM K2HPO4, 0.6 mM KH2PO4, 10 mM HEPES (pH 7.4) at 37 °C. Amiloride-sensitive short-circuit current was determined as the current difference with and without amiloride (10 µM) in the apical bathing solution.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Nedd4-2 Catalyzes ENaC Ubiquitination—To determine which ENaC subunits are substrates for ubiquitination, we cotransfected HEK 293T cells with {alpha}-, beta-, and {gamma}ENaC (one of the subunits contained a FLAG epitope at its C terminus) with or without ubiquitin containing an HA epitope (Ub-HA). To observe ubiquitinated ENaC subunits, the cells were treated with ALLN to prevent proteasomal degradation. Total {alpha}-, beta-, and {gamma}ENaC were detected by immunoprecipitation followed by immunoblot (Fig. 1, bottom). Ubiquitinated ENaC subunits were isolated by immunoprecipitation of ubiquitin (anti-HA) and detected by immunoblot (anti-FLAG). We detected ubiquitinated {alpha}-, beta-, and {gamma}ENaC 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 poly-monoubiquitinated) ENaC.


Figure 2
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  		FIGURE 2.
Nedd4-2 catalyzes ENaC ubiquitination. HEK 293T cells were transfected with {alpha}-, beta-, and {gamma}ENaC (the indicated subunits contained FLAG) (1 µg each). ENaC subunits were wild type (wt) or contained the following mutations: betaR566X (betaL), betaY620A (betaY-A), {alpha}Y644A ({alpha}Y-A), and {gamma}Y627A ({gamma}Y-A). The cells were cotransfected with or without Nedd4-2 (wild type or C821A (H), 2 µg) and ubiquitin-HA (3 µg), as indicated. Total transfected DNA was held constant using GFP cDNA. Ubiquitinated ENaC was detected by immunoprecipitation of ubiquitin (anti-HA) followed by immunoblot for {alpha}-, beta-, or {gamma}ENaC (anti-FLAG). A–C, representative immunoblots; D–F, quantitation of multi- (Multi-ubiq.) and mono/oligoubiquitinated (Mono/oligo.-ubiq.) ENaC subunits by densitometry relative (Rel.) to wild-type ENaC + Nedd4-2 groups in lane 2 (mean ± S.E., n = 3; *, p < 0.05 versus ENaC + Nedd4-2 by Student's t test).

 


Figure 3
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  		FIGURE 3.
Endogenous Nedd4-2 ubiquitinates ENaC. HEK 293T cells were transfected with ENaC ({alpha}-FLAG, betaENaC, and {gamma}ENaC, 0.8 µg each), ubiquitin-HA (2.4 µg), and siRNA targeting Nedd4-2 or GFP (C) (1.6 µg). Ubiquitinated {alpha}ENaC was detected by immunoprecipitation (IP) (anti-HA) followed by immunoblot (anti-FLAG). A, representative immunoblot. B, quantitation by densitometry (mean ± S.E., n = 3; * p < 0.005 by Student's t test). Multi-ubiq., multiubiquitinated; Mono/oligo.-ubiq., mono/oligoubiquitinated; Rel., relative.

 
To test whether Nedd4-2 catalyzes ubiquitination of one or more ENaC subunits, we used two strategies. First, we overexpressed Nedd4-2. In these experiments, ALLN was omitted to reduce basal levels of ubiquitination. Fig. 2, A–C, shows representative immunoblots and Fig. 2, D–F, shows quantitation of multiubiquitinated (left panel) and mono/oligoubiquitinated (right panel) ENaC subunits. Nedd4-2 significantly increased ubiquitination of {alpha}-, beta-, and {gamma}ENaC (Fig. 2, A–C, compare lanes 1 and 2). Moreover, Nedd4-2 increased both the faster and the slower migrating forms. As controls for specificity, we did not detect ubiquitinated proteins in cells lacking ENaC (Fig. 2, A and C, lanes 5 and 7, and Fig. 2B, lane 7) or in cells expressing ENaC without Ub-HA and Nedd4-2 (lane 6).

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 {alpha}ENaC (Fig. 3, A and B).

Nedd4-2 HECT Domain and ENaC PY Motifs Are Required for ENaC 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 {alpha}- and betaENaC, as well as the slower migrating form of {gamma}ENaC (Fig. 2, A–C, compare lanes 2 and 3, quantified in Fig. 2, D–F). Interestingly, the mutant Nedd4-2 increased the faster migrating mono- or oligoubiquitinated form of {gamma}ENaC (when compared with the group without Nedd4-2), although much less than wild-type Nedd4-2 (Fig. 2, C and F). Thus, the catalytic activity of the HECT domain is required for Nedd4-2 to induce ubiquitination of {alpha}ENaC, betaENaC, and the slower migrating form of {gamma}ENaC but not mono/oligoubiquitinated {gamma}ENaC.

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 whether binding is required for ubiquitination, we disrupted the PY motifs. Deletion of this motif in betaENaC by a Liddle syndrome mutation (R566X, indicated in Fig. 2 by betaL) reduced Nedd4-2-induced ubiquitination of {alpha}- and {gamma}ENaC (Fig. 2, A and C, compare lanes 2 and 4, quantitated in Fig. 2, D and F). Mutation of the PY motif (Y620A, indicated in Fig. 2 by betaY-A) produced a minimal decrease in the faster migrating form of ubiquitinated betaENaC (which 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 {alpha}-, beta-, and {gamma}ENaC abolished ubiquitination of betaENaC (Fig. 2, B and E). Taken together, these data suggest that Nedd4-2 binds to the PY motifs of ENaC subunits and then catalyzes ubiquitination via the HECT domain.


Figure 4
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  		FIGURE 4.
Nedd4-2 binds to and ubiquitinates ENaC at the cell surface. A, HEK 293T cells were transfected with or without{alpha}-FLAG, betaENaC, and{gamma}ENaC (1 µg each) and Nedd4-2-HA (0.04 µg). Cell surface proteins were biotinylated, isolated with NeutrAvidin beads, and then immunoblotted (IB) with anti-HA to detect Nedd4-2 (top panel) or anti-FLAG to detect {alpha}ENaC (bottom panel). B, HEK 293T cells were transfected with or without{alpha}ENaC, beta-FLAG, and {gamma}ENaC (1 µg each), Nedd4-2 (2 µg), and ubiquitin-HA (3 µg). Cell surface proteins were biotinylated or not biotinylated at 4 °C, as indicated. Ubiquitinated betaENaC at the cell surface was isolated by immunoprecipitation (IP) (anti-FLAG) followed by binding to NeutrAvidin beads and then detected by immunoblot (anti-HA). Data are representative of at least three experiments.

 
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 {alpha}ENaC (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 whether 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) and 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 betaENaC 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—If 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 ({alpha}ENaC, beta-FLAG, {gamma}ENaC) and Nedd4-2 (0–0.5 µg). We separated biotinylated (surface) proteins from non-biotinylated (intracellular) proteins by binding to NeutrAvidin beads and then detected betaENaC 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 betaENaC (Fig. 5, A and B, top panels). In contrast, Nedd4-2 produced a dose-dependent decrease in betaENaC at the cell surface (Fig. 5, A and B, bottom panels). Thus, Nedd4-2 selectively regulates ENaC at the cell surface.


Figure 5
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  		FIGURE 5.
Nedd4-2 selectively decreases ENaC surface expression. HEK 293T cells were transfected with {alpha}ENaC, beta-FLAG (wild type or Y620A), and {gamma}ENaC (1 µg each) and Nedd4-2 (0–0.5 µg). Nedd4-2 was wild type (A) or contained mutations in the four WW domains (C, Nedd4-2WW) or the HECT domain (D, Nedd4-2C821A). Control was transfected with GFP. Cell surface proteins were biotinylated and then isolated by binding to NeutrAvidin beads. Unbiotinylated proteins in the supernatant were subjected to immunoprecipitation (anti-FLAG). betaENaC in the biotinylated (surface) and unbiotinylated (intracellular) fractions were detected by immunoblot (anti-FLAG). A, C, and D, representative immunoblots. B, quantitation of betaENaC in the intracellular (top panel) and cell surface (bottom panel) fractions by densitometry (relative (Rel.) to 0 Nedd4-2 groups, n = 4). In C and D, total betaENaC was detected by immunoprecipitation of cell lysates followed by immunoblot (anti-FLAG). Data are representative of at least three experiments.

 
To test whether binding is required for Nedd4-2 to reduce ENaC surface expression, we used two strategies. First, we mutated the PY motif in betaENaC (betaY620A). 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. 5, A and B, bottom panels). In contrast, this mutation had little effect on levels of intracellular ENaC (Fig. 5, A and B, 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).


Figure 6
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  		FIGURE 6.
Nedd4-2 induces degradation of the cell surface pool of ENaC. HEK 293T cells were transfected with {alpha}ENaC, beta-FLAG, and {gamma}ENaC (wild type (WT) or Y-A mutants, 1 µg each) with Nedd4-2 or GFP (0.04 µg), as indicated. Cell surface proteins were labeled by biotinylation at 4 °C and then incubated at 37 °C for 0–120 min. Biotinylated betaENaC from 500 µg(upper two panels) and 50µg(lower panel) of cell lysates was isolated with neutravidin beads and detected by immunoblot (A). B and C, quantitation of the time-dependent loss of biotinylated betaENaC relative to 0 min (n = 3).

 
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), and 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 time-dependent loss of biotinylated betaENaC (Fig. 6, A and B); the half-life of degradation was approximately 1 h. Nedd4-2 dramatically increased the rate of degradation of cell surface ENaC, shortening the half-life to <20 min (Fig. 6, A and B). Mutation of the PY motifs in {alpha}-, beta-, and {gamma}ENaC abolished the effect of Nedd4-2 (Fig. 6, A and C). Thus, once ENaC reaches the cell surface, Nedd4-2 induces its degradation. This negative regulation is disrupted in Liddle syndrome.

ENaC Monoubiquitination Is Sufficient for Nedd4-2-induced Degradation—The data in Fig. 2 suggest that Nedd4-2 catalyzes both monoubiquitination and polyubiquitination of {alpha}-, beta-, and {gamma}ENaC. To assess the relative functional importance of these two species, we used a mutant ubiquitin; mutation of each lysine (Ub-0K) prevents the formation of polyubiquitin chains (21, 22). Expression of Ub-0K increased levels of betaENaC 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. 7, A and B). In Fischer rat thyroid epithelia transfected with {alpha}-, beta-, and {gamma}ENaC, 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.


Figure 7
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  		FIGURE 7.
ENaC monoubiquitination is sufficient for Nedd4-2-induced degradation. A and B, HEK 293T cells were transfected with ENaC ({alpha}ENaC, beta-FLAG, {gamma}ENaC, 1 µg each), Nedd4-2 (0–0.5 µg) with or without lysine-less ubiquitin (Ub-0K, 2.5 µg). Biotinylated betaENaC was detected by immunoblot (A) and quantitated (B) by densitometry relative (Rel.) to 0 Nedd4-2 groups (n = 4). C, amiloride (Amil.)-sensitive short-circuit current in Fischer rat thyroid epithelia expressing ENaC ({alpha}ENaC, betaENaC, and {gamma}ENaC) (0.17 µg each) with or without Nedd4-2 (0.4 µg) and Ub-0K (0.5 µg) (n = 19–24).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
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 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 be either 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 {alpha}-, beta-, and {gamma}ENaC. 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 serum- and glucocorticoid-induced kinase (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).

Does Nedd4-2 catalyze monoubiquitination or polyubiquitination of ENaC? Our data suggest that Nedd4-2 does both. Nedd4-2 increased the quantity of faster migrating ubiquitinated forms of {alpha}-, beta-, and {gamma}ENaC. 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 {alpha}-, beta-, and {gamma}ENaC in cells not transfected with Nedd4-2. This is consistent with previous work from other laboratories (although there was disagreement about whether betaENaC 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 monoubiquitination 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 Ca2+-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, in either the biosynthetic or the 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 despite 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 whether the same mechanisms are operative in native epithelia.

We found that Liddle syndrome mutations disrupted Nedd4-2-mediated ubiquitination of ENaC. Because these mutations delete or alter PY motifs that mediate ENaC binding to Nedd4-2, this suggests that binding facilitates ubiquitination. Moreover, it provides a mechanistic link between the disease-causing mutations and the excessive Na+ reabsorption that leads to hypertension in patients with Liddle syndrome. By disrupting ubiquitination, Liddle syndrome mutations increase ENaC surface expression, causing excessive renal Na+ absorption. Nedd4-2 is regulated by the renin-angiotensin-aldosterone pathway. Moreover, defects in this pathway are responsible for nearly all of the known inherited forms of hypertension. Thus, understanding the mechanisms that mediate ENaC ubiquitination may help unravel the pathogenesis of more common forms of hypertension.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by the National Institutes of Health. To whom correspondence should be addressed: University of Iowa College of Medicine, 371 EMRB, Iowa City, IA 52242. E-mail: peter-snyder{at}uiowa.edu.

2 The abbreviations used are: siRNA, small interfering RNA; GFP, green fluorescent protein; ALLN, N-acetyl-Leu-Leu-norleucinal; HA, hemagglutinin; PBS, phosphate-buffered saline; Ub, ubiquitin; Ub-0K, lysine-less ubiquitin cDNA. Back


   ACKNOWLEDGMENTS
 
We thank Diane Olson, Kristin Knight, Kaela Kramer, Dan Collier, and Danielle Wentzlaff for assistance, and acknowledge the University of Iowa DNA Core Facility for their services.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Schild, L. (2004) Rev. Physiol. Biochem. Pharmacol. 151, 93–107[CrossRef][Medline] [Order article via Infotrieve]
  2. Snyder, P. M. (2005) Endocrinology 146, 5079–5085[Abstract/Free Full Text]
  3. Lifton, R. P. (1996) Science 272, 676–680[Abstract]
  4. Boucher, R. C., Stutts, M. J., Knowles, M. R., Cantley, L., and Gatzy, J. T. (1986) J. Clin. Investig. 78, 1245–1252[Medline] [Order article via Infotrieve]
  5. Kamynina, E., Debonneville, C., Bens, M., Vandewalle, A., and Staub, O. (2001) FASEB J. 15, 204–214[Abstract/Free Full Text]
  6. Snyder, P. M., Price, M. P., McDonald, F. J., Adams, C. M., Volk, K. A., Zeiher, B. G., Stokes, J. B., and Welsh, M. J. (1995) Cell 83, 969–978[CrossRef][Medline] [Order article via Infotrieve]
  7. Firsov, D., Schild, L., Gautschi, I., Merillat, A. M., Schneeberger, E., and Rossier, B. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 15370–15375[Abstract/Free Full Text]
  8. Knight, K. K., Olson, D. R., Zhou, R., and Snyder, P. M. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 2805–2808[Abstract/Free Full Text]
  9. Debonneville, C., Flores, S. Y., Kamynina, E., Plant, P. J., Tauxe, C., Thomas, M. A., Munster, C., Chraibi, A., Pratt, J. H., Horisberger, J. D., Pearce, D., Loffing, J., and Staub, O. (2001) EMBO J. 20, 7052–7059[CrossRef][Medline] [Order article via Infotrieve]
  10. Snyder, P. M., Steines, J. C., and Olson, D. R. (2004) J. Biol. Chem. 279, 5042–5046[Abstract/Free Full Text]
  11. Snyder, P. M., Olson, D. R., and Thomas, B. C. (2002) J. Biol. Chem. 277, 5–8[Abstract/Free Full Text]
  12. Snyder, P. M., Olson, D. R., Kabra, R., Zhou, R., and Steines, J. C. (2004) J. Biol. Chem. 279, 45753–45758[Abstract/Free Full Text]
  13. Staub, O., Gautschi, I., Ishikawa, T., Breitschopf, K., Ciechanover, A., Schild, L., and Rotin, D. (1997) EMBO J. 16, 6325–6336[CrossRef][Medline] [Order article via Infotrieve]
  14. Malik, B., Yue, Q., Yue, G., Chen, X., Price, S. R., Mitch, W. E., and Eaton, D. C. (2005) Am. J. Physiol. 289, F107–F116
  15. Wang, H., Traub, L. M., Weixel, K. M., Hawryluk, M. J., Shah, N., Edinger, R. S., Perry, C. J., Kester, L., Butterworth, M. B., Peters, K. W., Kleyman, T. R., Frizzell, R. A., and Johnson, J. P. (2006) J. Biol. Chem. 281, 14129–14135[Abstract/Free Full Text]
  16. McDonald, F. J., Snyder, P. M., McCray, P. B., Jr., and Welsh, M. J. (1994) Am. J. Physiol. 266, L728–L734[Medline] [Order article via Infotrieve]
  17. McDonald, F. J., Price, M. P., Snyder, P. M., and Welsh, M. J. (1995) Am. J. Physiol. 268, C1157–C1163[Medline] [Order article via Infotrieve]
  18. Zhou, R., and Snyder, P. M. (2005) J. Biol. Chem. 280, 4518–4523[Abstract/Free Full Text]
  19. Snyder, P. M., Olson, D. R., McDonald, F. J., and Bucher, D. B. (2001) J. Biol. Chem. 276, 28321–28326[Abstract/Free Full Text]
  20. Goulet, C. C., Volk, K. A., Adams, C. M., Prince, L. S., Stokes, J. B., and Snyder, P. M. (1998) J. Biol. Chem. 273, 30012–30017[Abstract/Free Full Text]
  21. Petroski, M. D., and Deshaies, R. J. (2005) Cell 123, 1107–1120[CrossRef][Medline] [Order article via Infotrieve]
  22. Hampe, C., Ardila-Osorio, H., Fournier, M., Brice, A., and Corti, O. (2006) Hum. Mol. Genet. 15, 2059–2075[Abstract/Free Full Text]
  23. Dunn, R., and Hicke, L. (2001) J. Biol. Chem. 276, 25974–25981[Abstract/Free Full Text]
  24. Fang, S., Ferrone, M., Yang, C., Jensen, J. P., Tiwari, S., and Weissman, A. M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 14422–14427[Abstract/Free Full Text]
  25. Jiang, J., Ballinger, C. A., Wu, Y., Dai, Q., Cyr, D. M., Hohfeld, J., and Patterson, C. (2001) J. Biol. Chem. 276, 42938–42944[Abstract/Free Full Text]
  26. Shenoy, S. K., McDonald, P. H., Kohout, T. A., and Lefkowitz, R. J. (2001) Science 294, 1307–1313[Abstract/Free Full Text]
  27. Itani, O. A., Stokes, J. B., and Thomas, C. P. (2005) Am. J. Physiol. 289, F334–F346
  28. Kamynina, E., Tauxe, C., and Staub, O. (2001) Am. J. Physiol. 281, F469–F477

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