Affinity and specificity of interactions between Nedd4 isoforms and the epithelial Na+ channel

The epithelial Na+ channel (alphabetagammaENaC) regulates salt and fluid homeostasis and blood pressure. Each ENaC subunit contains a PY motif (PPXY) that binds to the WW domains of Nedd4, a Hect family ubiquitin ligase containing 3-4 WW domains and usually a C2 domain. It has been proposed that Nedd4-2, but not Nedd4-1, isoforms can bind to and suppress ENaC activity. Here we challenge this notion and show that, instead, the presence of a unique WW domain (WW3*) in either Nedd4-2 or Nedd4-1 determines high affinity interactions and the ability to suppress ENaC. WW3* from either Nedd4-2 or Nedd4-1 binds ENaC-PY motifs equally well (e.g. Kd approximately 10 microm for alpha- or betaENaC, 3-6-fold higher affinity than WW4), as determined by intrinsic tryptophan fluorescence. Moreover, dNedd4-1, which naturally contains a WW3* instead of WW2, is able to suppress ENaC function equally well as Nedd4-2. Homology models of the WW3*.betaENaC-PY complex revealed that a Pro and Ala conserved in all WW3*, but not other Nedd4-WW domains, help form the binding pocket for PY motif prolines. Extensive contacts are formed between the betaENaC-PY motif and the Pro in WW3*, and the small Ala creates a large pocket to accommodate the peptide. Indeed, mutating the conserved Pro and Ala in WW3* reduces binding affinity 2-3-fold. Additionally, we demonstrate that mutations in PY motif residues that form contacts with the WW domain based on our previously solved structure either abolish or severely reduce binding affinity to the WW domain and that the extent of binding correlates with the level of ENaC suppression. Independently, we show that a peptide encompassing the PY motif of sgk1, previously proposed to bind to Nedd4-2 and alter its ability to regulate ENaC, does not bind (or binds poorly) the WW domains of Nedd4-2. Collectively, these results suggest that high affinity of WW domain-PY-motif interactions rather than affiliation with Nedd4-1/Nedd-2 is critical for ENaC suppression by Nedd4 proteins.     

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.     

Liddle综合征触发蛋白ENaC的PY模体突变型的结合肽段的筛选

Liddle综合征是一种常染色体显性、盐敏感型的高血压综合征,其分子发病机制研究认为是上皮钠离子通道(epithelial Na+channel,ENaC)的β亚基或γ亚基的胞质侧羧基端区域低频率的点突变或缺失突变导致肾远曲小管钠离子重吸收增加.本研究提出了一种从分子水平上治疗Liddle综合征的设想,即构建一种可识别Liddle综合征患者中ENaC蛋白突变的PY模体的人工泛素连接酶E3,使其结合并降解突变的ENaC蛋白,从而使肾远曲小管上皮细胞膜上ENaC的表达数量和活性恢复.而识别PY突变体的E3,可通过用患者中的PY突变体筛选随机多肽文库获得与之结合的随机肽段,用其替换PY模体天然配体蛋白Nedd4的WW结构域,从而得到一个新的人工E3.本研究中以一种Liddle综合征突变型Y620H为诱饵蛋白,筛选新型随机多肽文库,获得了1个至少能与2种PY突变体(Y620H和P618L)特异性结合的随机肽段,为进一步构建可降解ENaC突变体的人工E3积累了重要的实验数据.     

All three WW domains of murine Nedd4 are involved in the regulation of epithelial sodium channels by intracellular Na+

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 which is necessary for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddle's syndrome where mutations delete or alter the PY motif of either the beta or gamma subunits, results in increased ENaC activity. We have recently shown using the whole-cell patch clamp technique that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. In this paper, we demonstrate that WW domains 2 and 3 bind alpha-, beta-, and gamma-ENaC with varying degrees of affinity, whereas WW domain 1 does not bind to any of the subunits. We further show using whole-cell patch clamp techniques that Nedd4-mediated down-regulation of ENaC in mouse mandibular duct cells involves binding of the WW domains of Nedd4 to three distinct sites. We propose that Nedd4-mediated down-regulation of Na+ channels involves the binding of WW domains 2 and 3 to the Na+ channel and of WW domain 1 to an unknown associated protein.     

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.     

Hrs Controls Sorting of the Epithelial Na+ Channel between Endosomal Degradation and Recycling Pathways

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 syndrome, an inherited form of hypertension. Previous work found that Nedd4-2 functions through two distinct effects on trafficking, enhancing both ENaC endocytosis and ENaC degradation in lysosomes. To investigate the mechanism by which Nedd4-2 targets ENaC to lysosomes, we tested the role of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a component of the endosomal sorting complexes required for transport (ESCRT)-0 complex. We found that α-, β-, and γENaC each interact with Hrs. These interactions were enhanced by Nedd4-2 and were dependent on the catalytic function of Nedd4-2 as well as its WW domains. Mutation of ENaC PY motifs, responsible for inherited hypertension (Liddle syndrome), decreased Hrs binding to ENaC. Moreover, binding of ENaC to Hrs was reduced by dexamethasone/serum- and glucocorticoid-inducible kinase and cAMP, which are signaling pathways that inhibit Nedd4-2. Nedd4-2 bound to Hrs and catalyzed Hrs ubiquitination but did not alter Hrs protein levels. Expression of a dominant negative Hrs lacking its ubiquitin-interacting motif (Hrs-ΔUIM) increased ENaC surface expression and current. This occurred through reduced degradation of the cell surface pool of proteolytically activated ENaC, which enhanced its recycling to the cell surface. In contrast, Hrs-ΔUIM had no effect on degradation of uncleaved inactive channels. The data support a model in which Nedd4-2 induces binding of ENaC to Hrs, which mediates the sorting decision between ENaC degradation and recycling.     

The PY motif of ENaC, mutated in Liddle syndrome, regulates channel internalization, sorting and mobilization from subapical pool

The epithelial-Na(+)-channel (alphabetagammaENaC) regulates kidney salt-transport and blood pressure. Each ENaC subunit contains a PY motif (PPxY) and its mutation in beta/gammaENaC causes Liddle syndrome, a hereditary hypertension. These (extended) PY motifs (PP(616)xY(618)xxL(621)) serve as binding sites for the ubiquitin ligase Nedd4-2, which decreases cell-surface expression of ENaC by unknown route(s). Using polarized kidney epithelia [Madin-Darby canine kidney I (MDCK-I)] cells stably expressing extracellularly myc-tagged wild type (WT) or PY-motif mutants of betaENaC (P616A, Y618A or L621A, with WT-alphagammaENaC), and live-imaging plus enzyme-linked immunosorbent assay (ELISA)-type assays to analyze routes/rates of ENaC internalization/recycling, we show here that cell-surface half-life of all PY mutants was fourfold longer than WT-ENaC (approximately 120 versus 30 minutes), reflecting primarily reduced channel internalization but also attenuated replenishment of cell-surface ENaC from a large subapical pool. The Y618A mutant revealed more severe internalization and replenishment defects than the other PY mutants. Internalized WT-ENaC was detected in sorting/recycling and late endosomes/lysosomes, while the Y618A mutant accumulated in the former. Nedd4-2 ubiquitinated ENaC at the apical membrane causing channel internalization and degradation. Cyclic AMP (cAMP) accelerated mobilization of subapical ENaC to the cell surface and long-term ENaC recycling, but only mobilization, not recycling, was inhibited in the PY mutants. These results suggest that the ENaC PY motifs (and Nedd4-2) primarily regulate channel internalization but also affect cAMP-dependent replenishment, providing important insight into the Liddle syndrome defects.     

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 cardiac voltage-gated Na+ channel (H1) by the ubiquitin-protein ligase Nedd4

The cardiac voltage-gated Na+ channel H1, involved in the generation of cardiac action potential, contains a C-terminal PY motif (xPPxY). Since PY motifs are known ligands to WW domains, we investigated their role for H1 regulation and the possible involvement of the WW domain containing ubiquitin-protein ligase Nedd4, taking advantage of the Xenopus oocyte system. Mutation of the PY motif leads to higher peak currents when compared to wild-type channel. Moreover, co-expression of Nedd4 reduced the peak currents, whereas an enzymatically inactive Nedd4 mutant increased them, likely by competing with endogenous Nedd4. The effect of Nedd4 was not observed in the PY motif mutated channel or in the skeletal muscle voltage-gated Na+ channel, which lacks a PY motif. We conclude that H1 may be regulated by Nedd4 depending on WW-PY interaction, and on an active ubiquitination site.     

Characterization of the interactions between Nedd4-2, ENaC,and sgk-1 using surface plasmon resonance

Previous studies have characterized interactions between the ubiquitin ligase Nedd4-1 and the epithelial Na(+) channel (ENaC). Such interactions control the channel cell surface expression and activity. Recently, evidence has been provided that a related protein, termed Nedd4-2, is likely to be the true physiological regulator of the channel. Unlike Nedd4-1, Nedd4-2 also interacts with the aldosterone-induced channel activating kinase sgk-1. The current study uses surface plasmon resonance to quantify the binding of the four WW domains of Nedd4-2 to synthetic peptides corresponding to the PY motifs of ENaC and sgk-1. The measurements demonstrate that WW3 and WW4 are the only Nedd4-2 domains interacting with both ENaC and sgk-1 and that their binding constants are in the 1-6 microM range.     

Structure and dynamics of human Nedd4-1 WW3 in complex with the αENaC PY motif

Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) is an E3 ubiquitin ligase that interacts with and negatively regulates the epithelial Na⁺ channel (ENaC). The WW domains of Nedd4-1 bind to the ENaC subunits via recognition of PY motifs. Human Nedd4-1 (hNedd4-1) contains four WW domains with the third domain (WW3*) showing the strongest affinity to the PY motif. To understand the mechanism underlying this binding affinity, we have carried out NMR structural and dynamics analyses of the hNedd4-1 WW3* domain in complex with a peptide comprising the C-terminal tail of the human ENaC α-subunit. The structure reveals that the peptide interacts in a similar manner to other WW domain–ENaC peptide structures. Crucial interactions that likely provide binding affinity are the broad XP groove facilitating additional contacts between the WW3* domain and the peptide, compared to similar complexes, and the large surface area buried (83 Å²) between R430 (WW3*) and L647′ (αENaC). This corroborates the model-free analysis of the ¹⁵N backbone relaxation data, which showed that R430 is the most rigid residue in the domain (S² = 0.90 ± 0.01). Carr–Purcell–Meiboom–Gill relaxation dispersion analysis identified two different conformational exchange processes on the μs–ms time-scale. One of these processes involves residues located at the peptide binding interface, suggesting conformational exchange may play a role in peptide recognition. Thus, both structural and dynamic features of the complex appear to define the high binding affinity. The results should aid interpretation of biochemical data and modeling interfaces between Nedd4-1 and other interacting proteins.     

Characterization of interactions between Nedd4 and beta and gammaENaC using surface plasmon resonance

Cell surface expression of the epithelial Na(+) channel ENaC is regulated by the ubiquitin ligase Nedd4. Binding of the WW domains of Nedd4 to the PY region in the carboxy tails of beta and gammaENaC, results in channel ubiquitination and degradation. Kinetic analysis of these interactions has been done using surface plasmon resonance. Synthetic peptides corresponding to the PY regions of beta and gammaENaC were immobilized on a sensor chip and "real-time" kinetics of their binding to recombinant WW proteins was determined. Specificity of the interactions was established by competition experiment, as well as by monitoring effects of a point mutation known to impair Nedd4/ENaC binding. These data provides the first determination of association, dissociation and equilibrium constants for the interactions between WW2 and beta or gammaENaC.     

WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome.

The amiloride-sensitive epithelial sodium channel (ENaC) plays a major role in sodium transport in kidney and other epithelia, and in regulating blood pressure. The channel is composed of three subunits (alphabetagamma) each containing two proline-rich sequences (P1 and P2) at its C-terminus. The P2 regions in human beta and gammaENaC, identical to the rat betagammarENaC, were recently shown to be deleted in patients with Liddle's syndrome (a hereditary form of hypertension), leading to hyperactivation of the channel. Using a yeast two-hybrid screen, we have now identified the rat homologue of Nedd4 (rNedd4) as the binding partner for the P2 regions of beta and gammarENaC. rNedd4 contains a Ca2+ lipid binding (CaLB or C2) domain, three WW domains and a ubiquitin ligase (Hect) domain. Our yeast two-hybrid and in vitro binding studies revealed that the rNedd4-WW domains mediate this association by binding to the P2 regions, which include the PY motifs (XPPXY) of either betarENaC (PPPNY) or gammarENaC (PPPRY). SH3 domains were unable to bind these sequences. Moreover, mutations to Ala of Pro616 or Tyr618 within the betarENaC P2 sequence (to PPANY or PPPNA, respectively), recently described in Liddle's patients, led to abrogation of rNedd4-WW binding. Nedd4-WW domains also bound to the proline-rich C-terminus (containing the sequence PPPAY) of alpharENaC, and endogenous Nedd4 co-immunoprecipitated with alpharENaC expressed in MDCK cells. These results demonstrate that the WW domains of rNedd4 bind to the PY motifs deleted from beta or gammaENaC in Liddle's syndrome patients, and suggest that Nedd4 may be a regulator (suppressor) of the epithelial Na+ channel.     

Characterization of the interactions between Nedd4-2, ENaC, and sgk-1 using surface plasmon resonance

Previous studies have characterized interactions between the ubiquitin ligase Nedd4-1 and the epithelial Na⁺ channel (ENaC). Such interactions control the channel cell surface expression and activity. Recently, evidence has been provided that a related protein, termed Nedd4-2, is likely to be the true physiological regulator of the channel. Unlike Nedd4-1, Nedd4-2 also interacts with the aldosterone-induced channel activating kinase sgk-1. The current study uses surface plasmon resonance to quantify the binding of the four WW domains of Nedd4-2 to synthetic peptides corresponding to the PY motifs of ENaC and sgk-1. The measurements demonstrate that WW3 and WW4 are the only Nedd4-2 domains interacting with both ENaC and sgk-1 and that their binding constants are in the 1-6 μM range.     

Structural determinants for high-affinity binding in a Nedd4 WW3* domain-Comm PY motif complex

Interactions between the WW domains of Drosophila Nedd4 (dNedd4) and Commissureless (Comm) PY motifs promote axon crossing at the CNS midline and muscle synaptogenesis. Here we report the solution structure of the dNedd4 WW3* domain complexed to the second PY motif (227'TGLPSYDEALH237') of Comm. Unexpectedly, there are interactions between WW3* and ligand residues both N- and C-terminal to the PY motif. Residues Y232'-L236' form a helical turn, following the PPII helical PY motif. Mutagenesis and binding studies confirm the importance of these extensive contacts, not simultaneously observed in other WW domain complexes, and identify a variable loop in WW3* responsible for its high-affinity interaction. These studies expand our general understanding of the molecular determinants involved in WW domain-ligand recognition. In addition, they provide insights into the specific regulation of dNedd4-mediated ubiquitination of Comm and subsequent internalization of Comm or the Comm/Roundabout complex, critical for CNS and muscle development.     

The Nedd4-like protein KIAA0439 is a potential regulator of the epithelial sodium channel

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 which is believed to be important for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. 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 proposed to result in increased ENaC activity. Here we present evidence that KIAA0439 protein, a close relative of Nedd4, is also a potential regulator of ENaC. We demonstrate that KIAA0439 WW domains bind all three ENaC subunits. We show that a recombinant KIAA0439 WW domain protein acts as a dominant negative mutant that can interfere with the Na(+)-dependent feedback inhibition of ENaC in whole-cell patch clamp experiments. We propose that KIAA0439 and Nedd4 proteins either play a redundant role in ENaC regulation or function in a tissue- and/or signal-specific manner to down-regulate ENaC.     

Multiple interactions drive adaptor-mediated recruitment of the ubiquitin ligase rsp5 to membrane proteins in vivo and in vitro

Recognition of membrane proteins by the Nedd4/Rsp5 ubiquitin ligase family is a critical step in their targeting to the multivesicular body pathway. Some substrates contain "PY" motifs (PPxY), which bind to WW domains in the ligase. Others lack PY motifs and instead rely on adaptors that recruit the ligase to them. To investigate the mechanism of adaptor-mediated ubiquitination, we have characterized the interactions between the adaptor Bsd2, the ubiquitin ligase Rsp5, and the membrane proteins Cps1, Tre1, and Smf1 from Saccharomyces cerevisiae. We have reconstituted adaptor-mediated modification of Cps1 and Tre1 in vitro, and we show that two PY motifs in Bsd2 and two WW domains (WW2 and WW3) in Rsp5 are crucial for this. The binding of a weak noncanonical DMAPSY motif in Bsd2 to WW3 is an absolute requirement for Bsd2 adaptor function. We show that sorting of the manganese transporter Smf1, which requires both Bsd2 and Tre1, depends upon two PY motifs in Bsd2 and one motif in Tre1 but only two WW domains in Rsp5. We suggest that sequential assembly of first a Bsd2/Rsp5 complex, then a Tre1/Bsd2/Rsp5 complex followed by a rearrangement of PY-WW interactions is required for the ubiquitination of Smf1.     

Rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction

The matrix (M) proteins of vesicular stomatitis virus (VSV) and rabies virus (RV) play a key role in both assembly and budding of progeny virions. A PPPY motif (PY motif or late-budding domain) is conserved in the M proteins of VSV and RV. These PY motifs are important for virus budding and for mediating interactions with specific cellular proteins containing WW domains. The PY motif and flanking sequences of the M protein of VSV were used as bait to screen a mouse embryo cDNA library for cellular interactors. The mouse Nedd4 protein, a membrane-localized ubiquitin ligase containing multiple WW domains, was identified from this screen. Ubiquitin ligase Rsp5, the yeast homolog of Nedd4, was able to interact both physically and functionally with full-length VSV M protein in a PY-dependent manner. Indeed, the VSV M protein was multiubiquitinated by Rsp5 in an in vitro ubiquitination assay. To demonstrate further that ubiquitin may be involved in the budding process of rhabdoviruses, proteasome inhibitors (e.g., MG132) were used to decrease the level of free ubiquitin in VSV- and RV-infected cells. Viral titers measured from MG132-treated cells were reproducibly 10- to 20-fold lower than those measured from untreated control cells, suggesting that free ubiquitin is important for efficient virus budding. Last, release of a VSV PY mutant was not inhibited in the presence of MG132, signifying that the functional L domain of VSV is required for the inhibitory effect exhibited by MG132. These data suggest that the cellular ubiquitin-proteasome machinery is involved in the budding process of VSV and RV.     

Interactions of beta and gamma ENaC with Nedd4 can be facilitated by an ERK-mediated phosphorylation.

Phosphorylation of the epithelial Na(+) channel (ENaC) has been suggested to play a role in its regulation. Here we demonstrate that phosphorylating the carboxyl termini of the beta and gamma subunits facilitates their interactions with the ubiquitin ligase Nedd4 and inhibits channel activity. Three protein kinases, which phosphorylate the carboxyl termini of beta and gammaENaC, have been identified by an in vitro assay. One of these phosphorylates betaThr-613 and gammaThr-623, well-conserved C-tail threonines in the immediate vicinity of the PY motifs. Phosphorylation of gammaThr-623 has also been demonstrated in vivo in channels expressed in Xenopus oocytes, and mutating betaThr-613 and gammaThr-623 into alanine increased the channel activity by 3.5-fold. Effects of the above phosphorylations on interactions between ENaC and Nedd4 have been studied using surface plasmon resonance. Peptides having phospho-threonine at positions beta613 or gamma623 bind the WW domains of Nedd4 two to three times better than the non-phosphorylated analogues, due to higher association rate constants. Using a number of different approaches it was demonstrated that the protein kinase acting on betaThr-613 and gammaThr-623 is the extracellular regulated kinase (ERK). It is suggested that an ERK-mediated phosphorylation of betaThr-613 and gammaThr-623 down-regulates the channel by facilitating its interaction with Nedd4.     

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.