AMP-activated kinase inhibits the epithelial Na+ channel through functional regulation of the ubiquitin ligase Nedd4-2

We recently found that the metabolic sensor AMP-activated kinase (AMPK) inhibits the epithelial Na+ channel (ENaC) through decreased plasma membrane ENaC expression, an effect requiring the presence of a binding motif in the cytoplasmic tail of the beta-ENaC subunit for the ubiquitin ligase Nedd4-2. To further examine the role of Nedd4-2 in the regulation of ENaC by AMPK, we studied the effects of AMPK activation on ENaC currents in Xenopus oocytes co-expressing ENaC and wild-type (WT) or mutant forms of Nedd4-2. ENaC inhibition by AMPK was preserved in oocytes expressing WT Nedd4-2 but blocked in oocytes expressing either a dominant-negative (DN) or constitutively active (CA) Nedd4-2 mutant, suggesting that AMPK-dependent modulation of Nedd4-2 function is involved. Similar experiments utilizing WT or mutant forms of the serum- and glucocorticoid-regulated kinase (SGK1), modulators of protein kinase A (PKA), or extracellular-regulated kinase (ERK) did not affect ENaC inhibition by AMPK, suggesting that these pathways known to modulate the Nedd4-2-ENaC interaction are not responsible. AMPK-dependent phosphorylation of Nedd4-2 expressed in HEK-293 cells occurred both in vitro and in vivo, suggesting a potential mechanism for modulation of Nedd4-2 and thus cellular ENaC activity. Moreover, cellular AMPK activation significantly enhanced the interaction of the beta-ENaC subunit with Nedd4-2, as measured by co-immunoprecipitation assays in HEK-293 cells. In summary, these results suggest a novel mechanism for ENaC regulation in which AMPK promotes ENaC-Nedd4-2 interaction, thereby inhibiting ENaC by increasing Nedd4-2-dependent ENaC retrieval from the plasma membrane. AMPK-dependent ENaC inhibition may limit cellular Na+ loading under conditions of metabolic stress when AMPK becomes activated.     

Intracellular ubiquitylation of the epithelial Na+ channel controls extracellular proteolytic channel activation via conformational change

The epithelial Na(+) channel ENaC is a key player in the maintenance of whole body Na(+) balance, and consequently of blood pressure. It is tightly regulated by numerous signaling pathways including ubiquitylation via the ubiquitin-protein ligase Nedd4-2. This mechanism is itself under the control of several kinases, which phosphorylate Nedd4-2, thereby interfering with ENaC/Nedd4-2 interaction, or by Usp2-45, which binds to and deubiquitylates ENaC. Another, different regulatory mechanism concerns the proteolytic activation of ENaC, during which the channel is cleaved on its luminal side by intracellular convertases such as furin, and further activated by extracellular proteases such as CAP-1. This process is regulated as well but the underlying mechanisms are not understood. Previously, evidence was provided that the ubiquitylation status of ENaC may affect the cleavage of the channel. When ubiquitylation of ENaC was reduced, either by co-expressing Usp2-45, or mutating either the ENaC PY-motifs (i.e. the binding sites for Nedd4-2) or intracellular lysines (i.e. ubiquitylation sites), the level of channel cleavage was increased. Here we demonstrate that lysine-mutated ENaC channels are not ubiquitylated at the cell surface, are preferentially cleaved, and Usp2-45 does not affect their cleavage efficiency. We further show by limited proteolysis that the intracellular ubiquitylation status of ENaC affects the extracellular conformation of αENaC, by demonstrating that non-ubiquitylated channels are more efficiently cleaved when treated with extracellularly added trypsin or chymotrypsin. These results present a new paradigm in which an intracellular, post-translational modification (e.g. ubiquitylation) of a transmembrane protein can affect its extracellular conformation.     

Functional regulation of the epithelial Na+ channel by IkappaB kinase-beta occurs via phosphorylation of the ubiquitin ligase Nedd4-2

We have previously shown that IkappaB kinase-beta (IKKbeta) interacts with the epithelial Na+ channel (ENaC) beta-subunit and enhances ENaC activity by increasing its surface expression in Xenopus oocytes. Here, we show that the IKKbeta-ENaC interaction is physiologically relevant in mouse polarized kidney cortical collecting duct (mpkCCDc14) cells, as RNA interference-mediated knockdown of endogenous IKKbeta in these cells by approximately 50% resulted in a similar reduction in transepithelial ENaC-dependent equivalent short circuit current. Although IKKbeta binds to ENaC, there was no detectable phosphorylation of ENaC subunits by IKKbeta in vitro. Because IKKbeta stimulation of ENaC activity occurs through enhanced channel surface expression and the ubiquitin-protein ligase Nedd4-2 has emerged as a central locus for ENaC regulation at the plasma membrane, we tested the role of Nedd4-2 in this regulation. IKKbeta-dependent phosphorylation of Xenopus Nedd4-2 expressed in HEK-293 cells occurred both in vitro and in vivo, suggesting a potential mechanism for regulation of Nedd4-2 and thus ENaC activity. 32P labeling studies utilizing wild-type or mutant forms of Xenopus Nedd4-2 demonstrated that Ser-444, a key SGK1 and protein kinase A-phosphorylated residue, is also an important IKKbeta phosphorylation target. ENaC stimulation by IKKbeta was preserved in oocytes expressing wild-type Nedd4-2 but blocked in oocytes expressing either a dominant-negative (C938S) or phospho-deficient (S444A) Nedd4-2 mutant, suggesting that Nedd4-2 function and phosphorylation by IKKbeta are required for IKKbeta regulation of ENaC. In summary, these results suggest a novel mode of ENaC regulation that occurs through IKKbeta-dependent Nedd4-2 phosphorylation at a recognized SGK1 and protein kinase A target site.     

Participation of the ubiquitin-conjugating enzyme UBE2E3 in Nedd4-2-dependent regulation of the epithelial Na+ channel

The epithelial Na+ channel (ENaC) is a heteromeric protein complex playing a fundamental role in Na+ homeostasis and blood pressure regulation. Specific mutations inactivating PY motifs in ENaC C termini cause Liddle's syndrome, an inherited form of hypertension. Previously we showed that these PY motifs serve as binding sites for the E3 enzyme Nedd4-2, implying ubiquitination as a regulatory mechanism of ENaC. Ubiquitination involves the sequential action of E1, E2, and E3 enzymes. Here we identify the E2 enzyme UBE2E3, which acts in concert with Nedd4-2, and show by coimmunoprecipitation that UBE2E3 and Nedd4-2 interact together. In Xenopus laevis oocytes, UBE2E3 reduces ENaC activity marginally, consistent with Nedd4-2 being the rate-limiting factor in this process, whereas a catalytically inactive mutant of UBE2E3 (UBE2E3-CS) causes elevated ENaC activity by increasing cell surface expression. No additive effect is observed when UBE2E3-CS is coexpressed with an inactive Nedd4-2 mutant, and the stimulatory role of UBE2E3-CS depends on the integrity of the PY motifs (Nedd4-2 binding sites) and the ubiquitination sites on ENaC. In renal mpkCCD(cl4) cells, displaying ENaC-dependent transepithelial Na+ transport, Nedd4-2 and UBE2E3 can be coimmunoprecipitated and overexpression of UBE2E3 affects Na+ transport, corroborating the concept of a concerted action of UBE2E3 and Nedd4-2 in ENaC regulation.     

A novel mouse Nedd4 protein suppresses the activity of the epithelial Na+ channel.

Liddle's syndrome is a form of inherited hypertension linked to mutations in the genes encoding the epithelial Na+ channel (ENaC). These mutations alter or delete PY motifs involved in protein-protein interactions with a ubiquitin-protein ligase, Nedd4. Here we show that Na+ transporting cells, derived from mouse cortical collecting duct, express two Nedd4 proteins with different structural organization and characteristics of ENaC regulation: 1) the classical Nedd4 (herein referred to as Nedd4-1) containing one amino-terminal C2, three WW, and one HECT-ubiquitin protein ligase domain and 2) a novel Nedd4 protein (Nedd4-2), homologous to Xenopus Nedd4 and comprising four WW, one HECT, yet lacking a C2 domain. Nedd4-2, but not Nedd4-1, inhibits ENaC activity when coexpressed in Xenopus oocytes and this property correlates with the ability to bind to ENaC, as only Nedd4-2 coimmunoprecipitates with ENaC. Furthermore, this interaction depends on the presence of at least one PY motif in the ENaC complex and on WW domains 3 and 4 in Nedd4-2. Thus, these results suggest that the novel suppressor protein Nedd4-2 is the regulator of ENaC and hence a potential susceptibility gene for arterial hypertension.     

Regulation of the epithelial sodium channel by N4WBP5A,a novel Nedd4/Nedd4-2-interacting protein

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of alpha, beta, and gamma subunits. The carboxyl terminus of each ENaC subunit contains a PPXY motif that is believed to be important for interaction with the WW domains of the ubiquitin-protein ligases, Nedd4 and Nedd4-2. Disruption of this interaction, as in Liddle's syndrome where mutations delete or alter the PPXY motif of either the beta or gamma subunits, has been shown to result in increased ENaC activity and arterial hypertension. Here we present evidence that N4WBP5A, a novel Nedd4/Nedd4-2-binding protein, is a potential regulator of ENaC. In Xenopus laevis oocytes N4WBP5A increases surface expression of ENaC by reducing the rate of ENaC retrieval. We further demonstrate that N4WBP5A prevents sodium feedback inhibition of ENaC possibly by interfering with the xNedd4-2-mediated regulation of ENaC. As N4WBP5A binds Nedd4/Nedd4-2 via PPXY motif/WW domain interactions and appears to be associated with specific intracellular vesicles, we propose that N4WBP5A functions by regulating Nedd4/Nedd4-2 availability and trafficking. Because N4WBP5A is highly expressed in native renal collecting duct and other tissues that express ENaC, it is a likely candidate to modulate ENaC function in vivo.     

Multiple WW domains, but not the C2 domain, are required for inhibition of the epithelial Na+ channel by human Nedd4

The epithelial Na+ channel (ENaC) absorbs Na+ across the apical membrane of epithelia. The activity of ENaC is controlled by its interaction with Nedd4; mutations that disrupt this interaction increase Na+ absorption, causing an inherited form of hypertension (Liddle's syndrome). Nedd4 contains an N-terminal C2 domain, a C-terminal ubiquitin ligase domain, and multiple WW domains. The C2 domain is thought to be involved in the Ca2+-dependent localization of Nedd4 at the cell surface. However, we found that the C2 domain was not required for human Nedd4 (hNedd4) to inhibit ENaC in both Xenopus oocytes and Fischer rat thyroid epithelia. Rather, hNedd4 lacking the C2 domain inhibited ENaC more potently than wild-type hNedd4. Earlier work indicated that the WW domains bind to PY motifs in the C terminus of ENaC. However, it is not known which WW domains mediate this interaction. Glutathione S-transferase-fusion proteins of WW domains 2-4 each bound to alpha, beta, and gammaENaC in vitro. The interactions were abolished by mutation of two residues. WW domain 3 (but not the other WW domains) was both necessary and sufficient for the binding of hNedd4 to alphaENaC. WW domain 3 was also required for the inhibition of ENaC by hNedd4; inhibition was nearly abolished when WW domain 3 was mutated. However, the interaction between ENaC and WW domain 3 alone was not sufficient for inhibition. Moreover, inhibition was decreased by mutation of WW domain 2 or WW domain 4. Thus, WW domains 2-4 each participate in the functional interaction between hNedd4 and ENaC in intact cells.     

14-3-3 proteins modulate the expression of epithelial Na+ channels by phosphorylation-dependent interaction with Nedd4-2 ubiquitin ligase

The ubiquitin E3 protein ligase Nedd4-2 is a physiological regulator of the epithelial sodium channel ENaC, which is essential for transepithelial Na+ transport and is linked to Liddle's syndrome, an autosomal dominant disorder of human salt-sensitive hypertension. Nedd4-2 function is negatively regulated by phosphorylation via a serum- and glucocorticoid-inducible protein kinase (Sgk1), which serves as a mechanism to inhibit the ubiquitination-dependent degradation of ENaC. We report here that 14-3-3 proteins participate in this regulatory process through a direct interaction with a phosphorylated form of human Nedd4-2 (a human gene product of KIAA0439, termed hNedd4-2). The interaction is dependent on Sgk1-catalyzed phosphorylation of hNedd4-2 at Ser-468. We found that this interaction preserved the activity of the Sgk1-stimulated ENaC-dependent Na+ current while disrupting the interaction decreased ENaC density on the Xenopus laevis oocytes surface possibly by enhancing Nedd4-2-mediated ubiquitination that leads to ENaC degradation. Our findings suggest that 14-3-3 proteins modulate the cell surface density of ENaC cooperatively with Sgk1 kinase by maintaining hNedd4-2 in an inactive phosphorylated state.     

Regulation of the epithelial Na+ channel by the protein kinase CK2

CK2 is a ubiquitous, pleiotropic, and constitutively active Ser/Thr protein kinase that controls protein expression, cell signaling, and ion channel activity. Phosphorylation sites for CK2 are located in the C terminus of both beta- and gamma-subunits of the epithelial Na(+) channel (ENaC). We examined the role of CK2 on the regulation of both endogenous ENaC in native murine epithelia and in Xenopus oocytes expressing rENaC. In Ussing chamber experiments with mouse airways, colon, and cultured M1-collecting duct cells, amiloride-sensitive Na(+) transport was inhibited dose-dependently by the selective CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB). In oocytes, ENaC currents were also inhibited by TBB and by the structurally unrelated inhibitors heparin and poly(E:Y). Expression of a trimeric channel lacking both CK2 sites (alphabeta(S631A)gamma(T599A)) produced a largely attenuated amiloride-sensitive whole cell conductance and rendered the mutant channel insensitive to CK2. In Xenopus oocytes, CK2 was translocated to the cell membrane upon expression of wt-ENaC but not of alphabeta(S631A)gamma(T599A)-ENaC. Phosphorylation by CK2 is essential for ENaC activation, and to a lesser degree, it also controls membrane expression of alphabetagamma-ENaC. Channels lacking the Nedd4-2 binding motif in beta-ENaC (R561X, Y618A) no longer required the CK2 site for channel activity and siRNA-knockdown of Nedd4-2 eliminated the effects of TBB. This implies a role for CK2 in inhibiting the Nedd4-2 pathway. We propose that the C terminus of beta-ENaC is targeted by this essential, conserved pleiotropic kinase that directs its constitutive activity toward many cellular protein complexes.     

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.     

Progesterone down-regulates the open probability of the amiloride-sensitive epithelial sodium channel via a Nedd4-2-dependent mechanism

Activation of the mitogen-activated protein (MAP) kinase cascade by progesterone in Xenopus oocytes leads to a marked down-regulation of activity of the amiloride-sensitive epithelial sodium channel (ENaC). Here we have studied the signaling pathways involved in progesterone effect on ENaC activity. We demonstrate that: (i) the truncation of the C termini of the alphabetagammaENaC subunits results in the loss of the progesterone effect on ENaC; (ii) the effect of progesterone was also suppressed by mutating conserved tyrosine residues in the Pro-X-X-Tyr (PY) motif of the C termini of the beta and gamma ENaC subunits (beta(Y618A) and gamma(Y628A)); (iii) the down-regulation of ENaC activity by progesterone was also suppressed by co-expression ENaC subunits with a catalytically inactive mutant of Nedd4-2, a ubiquitin ligase that has been previously demonstrated to decrease ENaC cell-surface expression via a ubiquitin-dependent internalization/degradation mechanism; (iv) the effect of progesterone was significantly reduced by suppression of consensus sites (beta(T613A) and gamma(T623A)) for ENaC phosphorylation by the extracellular-regulated kinase (ERK), a MAP kinase previously shown to facilitate the binding of Nedd4 ubiquitin ligases to ENaC; (v) the quantification of cell-surface-expressed ENaC subunits revealed that progesterone decreases ENaC open probability (whole cell P(o), wcP(o)) and not its cell-surface expression. Collectively, these results demonstrate that the binding of active Nedd4-2 to ENaC is a crucial step in the mechanism of ENaC inhibition by progesterone. Upon activation of ERK, the effect of Nedd4-2 on ENaC open probability can become more important than its effect on ENaC cell-surface expression.     

Regulation of amino acid transporter ATA2 by ubiquitin ligase Nedd4-2

We report here that ubiquitin ligase Nedd4-2 regulates amino acid transporter ATA2 activity on the cell surface. We first found that a proteasome inhibitor MG132 increased the uptake of alpha-(methylamino)isobutyric acid, a model substrate for amino acid transport system A, in 3T3-L1 adipocytes as well as the preadipocytes. Transient expression of Nedd4-2 in Xenopus oocytes and Chinese hamster ovary cells down-regulated the ATA2 transport activity induced by injected cRNA and transfected cDNA, respectively. Neither the Nedd4-2 mutant with defective catalytic domain nor c-Cbl affected the ATA2 activity significantly. RNA-mediated interference of Nedd4-2 increased the ATA2 activity in the cells, and this was associated with decreased polyubiquitination of ATA2 on the cell surface membrane. Immunofluorescent analysis of Nedd4-2 in the adipocytes stably transfected with the enhanced green fluorescent protein (EGFP)-tagged ATA2 showed the co-localization of Nedd4-2 and EGFP-ATA2 in the plasma membrane but not in the perinuclear ATA2 storage site, supporting the idea that the primary site for the ubiquitination of ATA2 is the plasma membrane. These data suggest that ATA2 on the plasma membrane is subject to polyubiquitination by Nedd4-2 with consequent endocytotic sequestration and proteasomal degradation and that this process is an important determinant of the density of ATA2 functioning on the cell surface.     

Interaction of Serum- and Glucocorticoid Regulated Kinase 1 (SGK1) with the WW-Domains of Nedd4-2 Is Required for Epithelial Sodium Channel Regulation

Background

The epithelial sodium channel (ENaC) is an integral component of the pathway for Na⁺ absorption in epithelial cells. The ubiquitin ligases Nedd4 and Nedd4-2 bind to ENaC and decrease its activity. Conversely, Serum- and Glucocorticoid regulated Kinase-1 (SGK1), a downstream mediator of aldosterone, increases ENaC activity. This effect is at least partly mediated by direct interaction between SGK and Nedd4-2. SGK binds both Nedd4 and Nedd4-2, but it is only able to phosphorylate Nedd4-2. Phosphorylation of Nedd4-2 reduces its ability to bind to ENaC, due to the interaction of phosphorylated Nedd4-2 with 14-3-3 proteins, and hence increases ENaC activity. WW-domains in Nedd4-like proteins bind PY-motifs (PPXY) present in ENaC subunits, and SGK also has a PY-motif.

Principal Finding

Here we show that single or tandem WW-domains of Nedd4 and Nedd4-2 mediate binding to SGK and that different WW-domains of Nedd4 and Nedd4-2 are involved. Our data also show that WW-domains 2 and 3 of Nedd4-2 mediate the interaction with SGK in a cooperative manner, that activated SGK has increased affinity for the WW-domains of Nedd4-2 in vitro, and a greater stimulatory effect on ENaC Na⁺ transport compared to wildtype SGK. Further, SGK lacking a PY motif failed to stimulate ENaC activity in the presence of Nedd4-2.

Conclusions

Binding of Nedd4-2 WW-domains to SGK is necessary for SGK-induced ENaC activity.     

Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain

Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 domain, four WW domains (protein-protein interaction domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) domain. In the present paper we show that the WW domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT domain and inhibit auto-ubiquitination. Pulse-chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-domain-HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.     

GRK2 interacts with and phosphorylates Nedd4 and Nedd4-2

Epithelial Na(+) channels (ENaC) mediate the transport of sodium (Na) across epithelia in the kidney, gut, and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4 and Nedd4-2, which bind to proline-rich motifs (PY motifs) present in the C-termini of ENaC subunits. Loss of inhibition leads to hypertension. ENaC channels are maintained in the active state by G-protein-coupled receptor kinase 2 (GRK2), an enzyme implicated in the development of essential hypertension. Here, we report that GRK2 interacts not only with ENaC, but also with both Nedd4 and Nedd4-2. Additionally, GRK2 is capable of phosphorylating both Nedd4 and Nedd4-2 at multiple sites. Of possible significance is the phosphorylation of the threonine at position 466 in Nedd4, which is located in the area of the ww3 domain that binds ENaC. These results support and extend the role of GRK2 in sodium transport regulation.     

Nedd4-2 induces endocytosis and degradation of proteolytically cleaved epithelial Na+ channels

As a pathway for Na(+) reabsorption, the epithelial Na(+) channel ENaC is critical for Na(+) homeostasis and blood pressure control. Na(+) transport is regulated by Nedd4-2, an E3 ubiquitin ligase that decreases ENaC expression at the cell surface. To investigate the underlying mechanisms, we proteolytically cleaved/activated ENaC at the cell surface and then quantitated the rate of disappearance of cleaved channels using electrophysiological and biochemical assays. We found that cleaved ENaC channels were rapidly removed from the cell surface. Deletion or mutation of the Nedd4-2 binding motifs in alpha, beta, and gammaENaC dramatically reduced endocytosis, whereas a mutation that disrupts a YXX? endocytosis motif had no effect. ENaC endocytosis was also decreased by silencing of Nedd4-2 and by expression of a dominant negative Nedd4-2 construct. Conversely, Nedd4-2 overexpression increased ENaC endocytosis in human embryonic kidney 293 cells but had no effect in Fischer rat thyroid epithelia. In addition to its effect on endocytosis, Nedd4-2 also increased the rate of degradation of the cell surface pool of cleaved alphaENaC. Together the data indicate that Nedd4-2 reduces ENaC surface expression by altering its trafficking at two distinct sites in the endocytic pathway, inducing endocytosis of cleaved channels and targeting them for degradation.     

Neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) regulation by 14-3-3 protein binding at canonical serum and glucocorticoid kinase 1 (SGK1) phosphorylation sites

Regulation of epithelial Na(+) channel (ENaC)-mediated transport in the distal nephron is a critical determinant of blood pressure in humans. Aldosterone via serum and glucocorticoid kinase 1 (SGK1) stimulates ENaC by phosphorylation of the E3 ubiquitin ligase Nedd4-2, which induces interaction with 14-3-3 proteins. However, the mechanisms of SGK1- and 14-3-3-mediated regulation of Nedd4-2 are unclear. There are three canonical SGK1 target sites on Nedd4-2 that overlap phosphorylation-dependent 14-3-3 interaction motifs. Two of these are termed "minor," and one is termed "major," based on weak or strong binding to 14-3-3 proteins, respectively. By mass spectrometry, we found that aldosterone significantly stimulates phosphorylation of a minor, relative to the major, 14-3-3 binding site on Nedd4-2. Phosphorylation-deficient minor site Nedd4-2 mutants bound less 14-3-3 than did wild-type (WT) Nedd4-2, and minor site Nedd4-2 mutations were sufficient to inhibit SGK1 stimulation of ENaC cell surface expression. As measured by pulse-chase and cycloheximide chase assays, a major binding site Nedd4-2 mutant had a shorter cellular half-life than WT Nedd4-2, but this property was not dependent on binding to 14-3-3. Additionally, a dimerization-deficient 14-3-3ε mutant failed to bind Nedd4-2. We conclude that whereas phosphorylation at the Nedd4-2 major site is important for interaction with 14-3-3 dimers, minor site phosphorylation by SGK1 may be the relevant molecular switch that stabilizes Nedd4-2 interaction with 14-3-3 and thus promotes ENaC cell surface expression. We also propose that major site phosphorylation promotes cellular Nedd4-2 protein stability, which potentially represents a novel form of regulation for turnover of E3 ubiquitin ligases.     

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

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.     

cAMP and serum and glucocorticoid-inducible kinase (SGK) regulate the epithelial Na(+) channel through convergent phosphorylation of Nedd4-2

The epithelial Na(+) channel (ENaC) functions as a pathway for epithelial Na(+) transport, contributing to Na(+) homeostasis and blood pressure control. Vasopressin increases ENaC expression at the cell surface through a pathway that includes cAMP and cAMP-dependent protein kinase (PKA), but the mechanisms that link PKA to ENaC are unknown. Here we found that cAMP regulates Na(+) transport in part by inhibiting the function of Nedd4-2, an E3 ubiquitin-protein ligase that targets ENaC for degradation. Consistent with this model, we found that cAMP inhibited Nedd4-2 by decreasing its binding to ENaC. Moreover, decreased Nedd4-2 expression (RNA interference) or overexpression of a dominant negative Nedd4-2 construct disrupted ENaC regulation by cAMP. Nedd4-2 was a substrate for phosphorylation by PKA in vitro and in cells; three Nedd4-2 residues were phosphorylated by PKA and were required for cAMP to inhibit Nedd4-2 (relative functional importance Ser-327 > Ser-221 > Thr-246). Previous work found that these residues are also phosphorylated by serum and glucocorticoid-inducible kinase (SGK), a downstream mediator by which aldosterone regulates epithelial Na(+) transport. Consistent with a functional interaction between these pathways, overexpression of SGK blunted ENaC stimulation by cAMP, whereas inhibition of SGK increased stimulation. Conversely, cAMP agonists decreased ENaC stimulation by SGK. The data suggest that cAMP regulates ENaC in part by phosphorylation and inhibition of Nedd4-2. Moreover, Nedd4-2 is a central convergence point for kinase regulation of Na(+) transport.