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Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na+ channel cell surface expression

2001; Springer Nature; Volume: 20; Issue: 24 Linguagem: Inglês

10.1093/emboj/20.24.7052

1460-2075

Christophe Debonneville,

Genetics and Neurodevelopmental Disorders

Article17 December 2001free access Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na+ channel cell surface expression Christophe Debonneville Christophe Debonneville Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Institute of Anatomy, University of Zurich, CH-8057 Zurich, Switzerland Search for more papers by this author Sandra Y. Flores Sandra Y. Flores Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Institute of Anatomy, University of Zurich, CH-8057 Zurich, Switzerland Search for more papers by this author Elena Kamynina Elena Kamynina Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Search for more papers by this author Pamela J. Plant Pamela J. Plant Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Search for more papers by this author Caroline Tauxe Caroline Tauxe Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Search for more papers by this author Marc A. Thomas Marc A. Thomas Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Search for more papers by this author Carole Münster Carole Münster Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Search for more papers by this author Ahmed Chraïbi Ahmed Chraïbi Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Search for more papers by this author J.Howard Pratt J.Howard Pratt Endocrinology/Hypertension, Department of Medicine, Indiana University, Indianapolis, IN, 46202 USA Search for more papers by this author Jean-Daniel Horisberger Jean-Daniel Horisberger Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Search for more papers by this author David Pearce David Pearce Department of Medicine and Department of Cellular and Molecular Pharmacology, University of San Francisco, San Francisco, CA, 94143 USA Search for more papers by this author Johannes Loffing Johannes Loffing Institute of Anatomy, University of Zurich, CH-8057 Zurich, Switzerland Search for more papers by this author Olivier Staub Corresponding Author Olivier Staub Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Search for more papers by this author Christophe Debonneville Christophe Debonneville Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Institute of Anatomy, University of Zurich, CH-8057 Zurich, Switzerland Search for more papers by this author Sandra Y. Flores Sandra Y. Flores Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Institute of Anatomy, University of Zurich, CH-8057 Zurich, Switzerland Search for more papers by this author Elena Kamynina Elena Kamynina Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Search for more papers by this author Pamela J. Plant Pamela J. Plant Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Search for more papers by this author Caroline Tauxe Caroline Tauxe Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Search for more papers by this author Marc A. Thomas Marc A. Thomas Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Search for more papers by this author Carole Münster Carole Münster Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Search for more papers by this author Ahmed Chraïbi Ahmed Chraïbi Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Search for more papers by this author J.Howard Pratt J.Howard Pratt Endocrinology/Hypertension, Department of Medicine, Indiana University, Indianapolis, IN, 46202 USA Search for more papers by this author Jean-Daniel Horisberger Jean-Daniel Horisberger Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Search for more papers by this author David Pearce David Pearce Department of Medicine and Department of Cellular and Molecular Pharmacology, University of San Francisco, San Francisco, CA, 94143 USA Search for more papers by this author Johannes Loffing Johannes Loffing Institute of Anatomy, University of Zurich, CH-8057 Zurich, Switzerland Search for more papers by this author Olivier Staub Corresponding Author Olivier Staub Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland Search for more papers by this author Author Information Christophe Debonneville1,4, Sandra Y. Flores1,4, Elena Kamynina1, Pamela J. Plant1, Caroline Tauxe1, Marc A. Thomas1, Carole Münster1, Ahmed Chraïbi1, J.Howard Pratt2, Jean-Daniel Horisberger1, David Pearce3, Johannes Loffing4 and Olivier Staub 1 1Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland 2Endocrinology/Hypertension, Department of Medicine, Indiana University, Indianapolis, IN, 46202 USA 3Department of Medicine and Department of Cellular and Molecular Pharmacology, University of San Francisco, San Francisco, CA, 94143 USA 4Institute of Anatomy, University of Zurich, CH-8057 Zurich, Switzerland ‡C.Debonneville and S.Y.Flores contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:7052-7059https://doi.org/10.1093/emboj/20.24.7052 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The epithelial Na+ channel (ENaC) plays an essential role in the regulation of whole body Na+ balance and blood pressure. The cell surface expression of this channel, a complex of three subunits (α, β and γENaC), has been shown to be regulated by hormones such as aldosterone and vasopressin and by intracellular signaling, including ubiquitylation and/or phosphorylation. However, the molecular mechanisms involving phosphorylation in the regulation of ENaC are unclear. Here we show by expression studies in Xenopus laevis oocytes that the aldosterone-induced Sgk1 kinase interacts with the ubiquitin protein ligase Nedd4-2 in a PY motif-dependent manner and phosphorylates Nedd4-2 on Ser444 and, to a lesser extent, Ser338. Such phosphorylation reduces the interaction between Nedd4-2 and ENaC, leading to elevated ENaC cell surface expression. These data show that phosphorylation of an enzyme involved in the ubiquitylation cascade (Nedd4-2) controls cell surface density of ENaC and propose a paradigm for the control of ion channels. Moreover, they suggest a novel and complete signaling cascade for aldosterone-dependent regulation of ENaC. Introduction Na+ homeostasis, which is crucial for the maintenance of blood volume and pressure, is primarily regulated in the kidney, particularly in the distal areas of the nephron including the cortical collecting duct (CCD). Specialized epithelial cells (principal cells) express the amiloride-sensitive epithelial Na+ channel (ENaC) at the apical side (Garty and Palmer, 1997), allowing entry of Na+ into the cell and a Na+,K+–ATPase, extruding Na+ in exchange for K+, at the basolateral side. The concerted action of these two membrane proteins results in transepithelial Na+ absorption from the urinary to the sanguine compartment. In this context, ENaC activity, the rate-limiting step of Na+ transport, is under complex hormonal regulation, including aldosterone, vasopressin and insulin. However, the molecular mechanisms of this regulation are poorly understood (Verrey et al., 2000). ENaC comprises three homologous subunits (α, β and γ) that are each composed of two transmembrane domains, an extracellular loop and short N- and C-termini (Canessa et al., 1993, 1994). Significantly, each subunit contains a PY motif (xPPxY) in the C-terminal region, which we and others have shown to interact with the WW domains of the ubiquitin protein ligases Nedd4-1 and Nedd4-2 (Staub et al., 1996; Kanelis et al., 1998, 2001; Abriel et al., 1999; Harvey et al., 1999; Farr et al., 2000; Kamynina et al., 2001a,b; Snyder et al., 2001). Because ubiquitylation (the covalent attachment of ubiquitin polypeptides to target proteins) of plasma membrane proteins is recognized as a mechanism to target such proteins for internalization (reviewed in Hicke, 1997, 1999; Rotin et al., 2000), we postulated that Nedd4 isoforms may control cell surface expression of ENaC via a ubiquitylation-dependent mechanism (Staub et al., 1996). This model is supported by the findings that the α and γ ENaC subunits are subjected to ubiquitylation (which controls cell surface expression; Staub et al., 1997), and that Nedd4-2 and, to a weaker extent Nedd4-1, regulate ENaC activity (Dinudom et al., 1998; Goulet et al., 1998; Abriel et al., 1999; Harvey et al., 1999, 2001; Farr et al., 2000; Kamynina et al., 2001a,b; Snyder et al., 2001). However, it is not known whether such ubiquitylation is under physiological control. The importance of the ENaC PY motifs is corroborated by findings that Liddle's syndrome, an inherited form of human hypertension (Liddle et al., 1963; Botero-Velez et al., 1994), is linked to mutations in ENaC that invariably cause either the deletion or the alteration of the β or γ PY motif (Shimkets et al., 1994; Hansson et al., 1995a,b; Tamura et al., 1996; Jeunemaitre et al., 1997; J.Inoue et al., 1998; T.Inoue et al., 1998; Gao et al., 2001; Yamashita et al., 2001). When channels containing Liddle mutations are expressed in heterologous cell systems (e.g. Xenopus laevis oocytes), higher amiloride-sensitive Na+ currents (a measure of ENaC activity) are observed (Schild et al., 1995, 1996; Snyder et al., 1995), which are due to increased cell surface expression, open probability (Firsov et al., 1996) and reduced Na+ feedback inhibition (Kellenberger et al., 1998). Recently, it has been reported that the expression of Sgk1 kinase (serum- and glucocorticoid-regulated kinase; Webster et al., 1993), a member of the PKB/Akt family of serine/threonine kinases, is induced by aldosterone in cells of the CCD and, importantly, stimulates ENaC activity when co-expressed in Xenopus oocytes (Chen et al., 1999; Naray-Fejes-Toth et al., 1999). These findings suggested that Sgk1 may represent an important mediator for aldosterone-dependant ENaC regulation. It was further demonstrated in oocytes that Sgk1 increases the cell surface expression of ENaC (Alvarez de la Rosa et al., 1999; Loffing et al., 2001), may be regulated by insulin via PI-3 kinase-dependent phosphorylation and interacts in vitro with the C-termini of α and β ENaC (Wang et al., 2001). However, the target(s) of Sgk1 and the molecular mechanism(s) of its action on ENaC are not known. In this study, we have determined that the Sgk1 sequence contains a PY motif and Nedd4-2 comprises two conserved consensus sites for phosphorylation by Sgk1 [RXRXX(S/T); Kobayashi and Cohen, 1999; Park et al., 1999], suggesting that (i) Sgk1 may interact with Nedd4-2 in a PY motif–WW domain-dependent mode and (ii) Nedd4-2 may be a target of Sgk1. Indeed, we found in Xenopus oocytes that Sgk1 phosphorylates Nedd4-2 in a PY motif-dependent manner, Nedd4-2 phosphorylation is required for Sgk1 action on cell surface density of ENaC and Sgk1 reduces the interaction between Nedd4-2 and ENaC. These data therefore show a novel mechanism of regulated ubiquitylation of an ion channel and suggest a complete signaling pathway between aldosterone and its main downstream target in the distal nephron, ENaC. Results Sgk1 stimulates phosphorylation of Nedd4-2 The recent reports that the aldosterone-inducible Sgk1 kinase controls ENaC activity (Chen et al., 1999; Naray-Fejes-Toth et al., 1999) and the identification of consensus phosphorylation sites for Sgk1 (Kobayashi and Cohen, 1999; Park et al., 1999) prompted us to look for potential phosphorylation sites in Nedd4-2, which is a regulator of ENaC. Indeed, we found two such conserved consensus motifs at Ser338 and Ser444 in Xenopus Nedd4-2 (Figure 1), suggesting that Nedd4-2 may be a target of Sgk1. To test this hypothesis, we expressed Nedd4-2 in Xenopus oocytes with or without c-myc-tagged Sgk1, incubated the oocytes with [32P]ortho–phosphate and immunoprecipitated Nedd4-2 from a cellular lysate. We found that Nedd4-2 was strongly phosphorylated when Sgk1 was co-expressed (Figure 2A, lanes 2 and 6, top). Weak phosphorylation, possibly due to endogenous Sgk1 activity, was observed when Sgk1 was omitted (lane 5) or when a catalytically inactive Sgk1 was used (lane 3; Sgk1-KA, K130A). We then mutated, either together or separately, the two putative phosphorylation sites on Nedd4-2. This led to complete inhibition of Nedd4-2 phosphorylation when both sites were mutated [Figure 2A, lane 8; N4-2(S-A)2, S338AS444A], weak reduction when Ser338 was mutated (lane 10; Nedd4-2-S338A) and a much stronger reduction when Ser444 was changed to Ala (lane 12; Nedd4-2-S444A). As mentioned, Sgk1 also contains a PY motif, which may serve as an interaction site with the Nedd4-2 WW domains. Indeed, mutation of the PY motif abrogated Nedd4-2 phosphorylation almost completely (Figure 2A, lane 4; Sgk1ΔPY), although the same mutant was still able to phosphorylate a synthetic peptide substrate (Sgktide; Park et al., 1999), demonstrating that mutation of the PY motif does not impair catalytic activity (Figure 2C). As shown in Figure 2B, Sgk1 did not efficiently phosphorylate the paralog Nedd4-1, which contains no consensus sequences for Sgk1-dependent phosphorylation and does not seem to be primarily involved in ENaC regulation (Kamynina et al., 2001a). Hence, Sgk1 induces phosphorylation of Nedd4-2 (but not Nedd4-1) via a PY motif-dependent mechanism, essentially on Ser444 and to a lesser extent on Ser338. Figure 1.Schematic view of Nedd4-2 and Sgk1. (A) Scheme of Xenopus Nedd4-2 with the consensus phosphorylation sites and Xenopus Sgk1 with the indication of the catalytic domain, the catalytically essential Lys130 and the PY motif. (B) Conserved consensus phosphorylation sites in mouse, human and Xenopus Nedd4-2. Download figure Download PowerPoint Figure 2.Sgk1 phosphorylates Nedd4-2, but not Nedd4-1. (A) Oocytes expressing either wild-type Nedd4-2 (N4-2) or the phosphorylation mutants Nedd4-2-S338A-S444A [N4-2(S-A)2], Nedd4-2-S338A, Nedd4-2-S444A and myc-Sgk1 [wild-type, catalytically inactive (KA, K130A) or PY motif-mutated (ΔPY, Y301A)] were incubated with [32P]ortho–phosphate and treated as follows: top, immunoprecipitation from lysates with anti-Nedd4-2 antibodies and autoradiography; middle, western blot on lysates with anti-myc antibody (recognizing Sgk1); bottom, western blot on lysates with anti-Nedd4-2 antibodies. (B) Mouse Nedd4-1 (mNedd4-1/T7) or Nedd4-2 (mNedd4-2/T7), both epitope-tagged with a T7 epitope (Novagen), were expressed with or without Sgk1 (as indicated) and phosphorylation was followed as described in (A), except that the Nedd4 proteins were immunoprecipitated with anti-T7 antibody (top). Expression of mNedd4-1, mNedd4-2 and Sgk1 was followed by western blot analysis on lysates, using both anti-T7 (Nedd4-1 or Nedd4-2) and anti-myc (Sgk1) antibodies. (C) Phosphorylation of a synthetic peptide substrate (Sgktide) by Sgk1. Wild-type and mutant myc-Sgk1 (lacking a functional PY motif) were expressed in Xenopus oocytes and immunoprecipitated with anti-myc antibodies. The immunoprecipitated kinases were assayed in a kinase assay with Sgktide or a mutant peptide lacking the phosphorylation site as described in Materials and methods. Phosphorylated peptides were then analyzed by separation on a tricine acrylamide gel followed by autoradiography. Both wild-type and mutant Sgk1 were able to phosphorylate Sgktide, but not its mutant (top). Bottom, Coomassie Blue staining. Download figure Download PowerPoint Sgk1 regulates ENaC via Nedd4-2 phosphorylation If phosphorylation of Nedd4-2 is the mechanism by which Sgk1 stimulates ENaC activity, mutation of the phosphorylation sites on Nedd4-2 would be expected to abrogate the stimulatory effect of Sgk1. To test this hypothesis, we expressed ENaC together with various Sgk1 and Nedd4-2 mutants in Xenopus oocytes (Figure 3). As reported previously, wild-type Nedd4-2 strongly reduced ENaC currents, whereas Sgk1 stimulated them (Figure 3A). When Sgk1 was expressed in excess over Nedd4-2, the inhibitory effect of Nedd4-2 on ENaC was reduced, leading to currents that were sometimes even higher than in the control oocytes (Figure 3A; N4-2 + Sgk1). However, Sgk1 did not increase ENaC activity when co-expressed with Nedd4-2 lacking both phosphorylation sites [Figure 3A, compare N4-2(S-A)2 with N4-2(S-A)2 + Sgk1]. In a separate experiment, we determined the importance of the individual phosphorylation sites for ENaC regulation. We found that when Ser338 alone was mutated (Figure 3B; Nedd4-2 S338A + Sgk1), Sgk1's effect was markedly reduced, whereas when Ser444 was mutated, Sgk1's effect was blunted (Nedd4-2 S444A + Sgk1). Thus, phosphorylation of Nedd4-2 on Ser444 is essential for the regulation of ENaC by Sgk1, while phosphorylation of Ser338 may also play a role. Finally, mutation of the Sgk1 PY motif reduced the ability of Sgk1 to interfere with Nedd4-2-dependent inhibition of ENaC (Figure 3B), consistent with an Sgk1–Nedd4-2 interaction involving the Sgk1 PY motif. Figure 3.Sgk1-dependent regulation of ENaC requires Nedd4-2 phosphorylation sites and the PY motifs on Sgk1. (A) Oocytes were injected with cRNA encoding Nedd4-2 or the Nedd4-2 phosphorylation mutant, Sgk1 cRNA and ENaC (as indicated). Amiloride-sensitive Na+ currents were measured and normalized to control oocytes (expressing only ENaC). n = 15 oocytes from three animals; **p <0.01 level of significance versus N4-2 + Sgk1. (B) Same as (A), but cRNA encoding either wild-type or mutant Sgk1 (Sgk1ΔPY), wild-type or phosphorylation site mutant Nedd4-2 and ENaC (as indicated) were injected, ** p 0.01 versus ENaC. Download figure Download PowerPoint Sgk1-dependent phosphorylation of Nedd4-2 controls cell surface expression of ENaC We then investigated whether Sgk1-dependent phosphorylation of Nedd4-2 affects cell surface density of ENaC. We expressed ENaC, which was FLAG-tagged at the extracellular loops of the β and γ subunits, together with Sgk1 and various forms of Nedd4-2. This allowed quantification of ENaC surface expression by binding of 125I-labeled FLAG antibodies (Firsov et al., 1996). As shown previously, both Nedd4-2 (negatively; Abriel et al., 1999) and Sgk1 (positively; Alvarez de la Rosa et al., 1999; Loffing et al., 2001) influenced amiloride-sensitive Na+ currents (Figure 5, filled bars) and antibody binding proportionally (non-filled bars), confirming that they mostly affect the expression of channels at the cell surface and not the intrinsic properties of ENaC. When Nedd4-2 and Sgk1 were co-expressed, channel density was high, which is compatible with the idea that Sgk1 interferes with Nedd4-2-dependent suppression of ENaC. In contrast, mutations of the phosphorylation sites on Nedd4-2 lead to a low number of ENaC channels even in the presence of Sgk1 [Nedd4-2(S-A)2 + Sgk1], corroborating that phosphorylation of Nedd4-2 is required for Sgk1-dependent control of ENaC levels at the plasma membrane. We further confirmed this effect by immunocytochemical detection of FLAG-tagged ENaC on cryosections of Xenopus oocytes (Figure 6). Consistent with the binding experiments, co-expression of ENaC with Nedd4-2 and Sgk1 (+ Nedd4-2 + Sgk1) increased ENaC cell surface abundance, whereas co-expression of ENaC with Sgk1 and the phosphorylation mutant Nedd4-2 [+Nedd4-2(S-A)2 + Sgk1] decreased it. Figure 5.Sgk1-dependent phosphorylation of Nedd4-2 controls ENaC cell surface expression. Oocytes were co-injected with cRNA encoding FLAG-tagged ENaC together with either H2O, wild-type or mutant Nedd4-2 lacking both phosphorylation sites [Nedd4(S-A)2] and Sgk1, as indicated. Amiloride-sensitive Na+ currents (filled bars) and binding of iodinated anti-FLAG antibodies (non-filled bars) to quantitate the number of channels at the cell surface were measured in the same oocytes, as described previously (Firsov et al., 1996; Abriel et al., 1999). Current and binding values were normalized to control values (ENaC + H2O). n = 18 oocytes from three animals; *p <0.05 versus control, **p <0.01 versus Nedd4-2 + Sgk1. Download figure Download PowerPoint Figure 6.Immunostaining of ENaC in oocytes expressing either wild-type or phosphorylation mutant Nedd4-2 and Sgk1. FLAG-tagged ENaC was followed by immunofluorescence with anti-FLAG antibodies on cryosections in either uninjected oocytes or oocytes expressing ENaC alone, ENaC plus wt-Nedd4-2 plus Sgk1 (+ Nedd4-2 + Sgk1) or ENaC plus Nedd4-2 S338A-S444A plus Sgk1 [+ Nedd4(S-A)2 + Sgk1]. Download figure Download PowerPoint Sgk1 interferes with the interaction between Nedd4-2 and ENaC We wished to know whether Sgk1-dependent phosphorylation of Nedd4-2 influenced ENaC cell surface expression. Sgk1 may interfere either with Nedd4-2–ENaC interaction or with other parameters, such as enzymatic Nedd4-2 activity. To determine whether the Nedd4-2–ENaC interaction was regulated by Sgk1, we expressed FLAG-tagged ENaC subunits together with mutant Nedd4-2 (Nedd4-2-CS) and various Sgk1 mutants. We chose to use the catalytically inactive Nedd4-2-CS (Abriel et al., 1999) in order to avoid interference of ENaC ubiquitylation with the Nedd4-2–ENaC interaction. Cells were labeled overnight with [35S]methionine and immunoprecipitations with anti-FLAG antibodies (recognizing FLAG-tagged ENaC) were performed. The immunoprecipitated material was analyzed by SDS–PAGE and autoradiography or western blotting. As can be seen, all three ENaC subunits were immunoprecipitated (Figure 7A, arrows). When Nedd4-2-CS was co-expressed, an additional band was observed (Figure 7A, arrowhead), which was identified as the Nedd4-2 mutant protein using anti-Nedd4-2 antibodies (Figure 7B, arrowhead). When Sgk1 was added, the intensity of the Nedd4-2 band was reduced (Figure 7A, lane 3) and dropped below the detection limit by the anti-Nedd4-2 antibody (Figure 7B, lane 3). When either catalytically inactive Sgk1 (Sgk1-KA, lane 4) or Sgk1 lacking the PY motif (Sgk1-ΔPY, lane 5) were expressed, the quantity of co-immunoprecipitated Nedd4-2-CS was not affected. Therefore, these data suggest that Sgk1 interferes with the interaction of Nedd4-2 and ENaC in a phosphorylation- and PY motif-dependent manner. This reduced interaction is likely to be the cause of the observed stimulation of ENaC surface expression by Sgk1. Figure 7.Sgk1 interferes with ENaC–Nedd4-2 interaction. Oocytes were injected with cRNA encoding FLAG-tagged ENaC, Nedd4-2-CS and Sgk1, as indicated. After overnight incubation with [35S]methionine, membrane fractions were lysed and immunoprecipitations performed with anti-FLAG antibodies. (A) Immunoprecipitated material analyzed by SDS–PAGE autoradiography and (B) western blotting using an anti-Nedd4-2 antiserum. Lysates were analyzed by western blotting using (C) anti-c-myc (i.e. Sgk1) and (D) anti-Nedd4-2 antibodies. Download figure Download PowerPoint Discussion In this study, we demonstrate that Sgk1-dependent phosphorylation of Nedd4-2 regulates ENaC cell surface expression and suggest a novel mechanism for the hormonal regulation of ENaC by aldosterone. Phosphorylation events have long been recognized to play a role in the regulation of ENaC (Sariban-Sohraby et al., 1988; Ling and Eaton, 1989; Matsumoto et al., 1993; Shimkets et al., 1998; Blazer-Yost et al., 1999; Chigaev et al., 2001), but the molecular mechanisms (identity of the substrates, phosphorylation sites and involved kinases) remained vague. The recent discoveries that aldosterone-induced expression of Sgk1 kinase correlates with induction of transepithelial Na+ transport and that this kinase stimulates the ENaC activity at the cell membrane (Alvarez de la Rosa et al., 1999; Chen et al., 1999; Naray-Fejes-Toth et al., 1999; Loffing et al., 2001) pointed to Sgk1 as an important player in ENaC regulation, but the molecular mechanisms remained elusive. The present data reveal the mechanism of Sgk1-dependent ENaC regulation. As indicated in Figure 1, Nedd4-2 contains two conserved consensus sites for phosphorylation by Sgk1 kinase [RXRXX(S/T); Kobayashi and Cohen, 1999; Park et al., 1999]. Indeed, we demonstrate by co-expression of Sgk1 and Nedd4-2 that primarily Ser444 and, to a lesser extent, Ser338 are phosphorylated by Sgk1 kinase and that this phosphorylation depends on intrinsic Sgk1 kinase activity (Figure 2A). In Xenopus oocytes, when both phosphorylation sites are mutated, no phosphorylation of Nedd4-2 is observed. Phosphorylation of Nedd4-2 by the overexpressed Sgk1 kinase in the oocytes appears to be specific, as the paralog Nedd4-1, which does not contain a consensus for Sgk1 phosphorylation, is not primarily involved in ENaC regulation and is not phosphorylated by Sgk1. In addition, co-expression of Sgk1 together with ENaC in Xenopus oocytes does not induce phosphorylation of ENaC subunits (P.J.Plant and O.Staub, unpublished observations), which is in accordance with other reports finding no evidence for ENaC phosphorylation (Alvarez de la Rosa et al., 1999; Chigaev et al., 2001). Several lines of evidence suggest that Sgk1 and Nedd4-2 interact via the PY motif on Sgk1 and, consequently, on the WW domains on Nedd4-2: (i) Nedd4-2 phosphorylation is largely reduced when Sgk1 is mutated on the tyrosine of the PY motif, (ii) mutation of the Sgk1 PY motif leads to a reduced effect of Sgk1 on Nedd4-2-dependent inhibition of ENaC activity, (iii) the PY mutant of Sgk1 does not interfere with ENaC–Nedd4-2 interaction, (iv) the PY mutant retains catalytic activity toward a synthetic peptide substrate and (v) in a two-hybrid screen using Sgk1 as a bait, Nedd4-1, which contains highly similar WW domains to Nedd4-2, was pulled out as a weak interacting protein with Sgk1 (E.Kamynina and O.Staub, unpublished observations). The fact that we are unable to co-immunoprecipitate the two proteins when co-expressed in oocytes suggests that the interaction between Sgk1 and Nedd4-2 is of a transient nature or, alternatively, this interaction may be indirect. We found that the PY motifs of ENaC are required for Sgk1 to stimulate ENaC activity, which would be expected if the effect of Sgk1 is via Nedd4-2. These results apparently differ from earlier published data in which additional effects of Liddle mutations and Sgk1 were observed (Alvarez de la Rosa et al., 1999; Shigaev et al., 2000). However, the differences may be explained by the fact that only one subunit (β) was mutated (Shigaev et al., 2000) and that both studies employed heterologous (mouse) Sgk1 in Xenopus oocytes (Alvarez de la Rosa et al., 1999; Shigaev et al., 2000). Alternatively, Sgk1 may act through other signaling pathways, in addition to phosphorylation of Nedd4-2. Sgk1-dependent phosphorylation is likely to interfere with the ENaC–Nedd4-2 interaction, as suggested by the co-immunoprecipitation experiments, which show that less Nedd4-2 is co-immunoprecipitated with ENaC when Sgk1 is present. These effects depend on the catalytic activity of Sgk1 (the inactive SgkK130A does not interfere with ENaC–Nedd4-2 binding), which excludes the idea that the observed reduction of co-immunoprecipitated Nedd4-2 protein is simply due to a displacement of Nedd4-2 by Sgk1 via WW domain–PY motif interaction. Intriguingly, the phosphorylation sites are not localized in the vicinity of the WW domains (the interacting domains with ENaC). In fact, the predominantly used Ser444 is situat