Impact of Nedd4 Proteins and Serum and Glucocorticoid- ؉ Induced Kinases on Epithelial Na Transport in the Distal Nephron

Olivier Staub* and Franc¸ois Verrey† *Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland; and †Institute of Physiology, University of Zurich, Zurich, Switzerland

(The precise control of BP occurs via Na؉ homeostasis and involves the precise regulation of the epithelial Na؉ channel (ENaC in the aldosterone-sensitive distal nephron. This has been corroborated by the linkage of mutations in the genes encoding ENaC subunits and Liddle’s syndrome, a heritable form of human hypertension. Mapping of these mutations on ENaC indicated that inactivation of PY motifs is responsible and leads to the proposition that the channel interacts via its PY motifs with the WW domains of the Nedd4/Nedd4-like ubiquitin-protein ligase family. It is now well established that the cell surface expression of ENaC is controlled via ubiquitylation by this protein family and that this ubiquitylation is regulated by the aldosterone-induced protein serum and glucocorticoid induced kinase 1. J Am Soc Nephrol 16: 3167–3174, 2005. doi: 10.1681/ASN.2005050454

ϩ he epithelial Na channel (ENaC) localizes to the apical early findings were subsequently confirmed in other patients membrane of high-resistance epithelia of the aldoste- with Liddle’s syndrome, and mutations or deletions were rone-sensitive distal nephron (ASDN) and plays a ma- found in the C-termini of ␤-or␥-ENaC (8–20). To our knowl- T ϩ jor role in the regulation of whole-body Na homeostasis and edge, there is only one exception, in which a mutation was consequently in the control of blood volume and pressure. mapped to the extracellular loop of ␥-ENaC (21). It is interest- ENaC is composed of three homologous subunits, ␣, ␤ and ␥, ing that all of these mutations in the C-termini of ␤-or␥-ENaC each containing two transmembrane domains, an extracellular are mutations that cause frame shifts, stop codons, or single loop and intracellular N- and C-termini (1–3). In the kidney, amino acid changes and invariably delete or mutate a so-called ENaC is highly regulated by various hormonal pathways, such proline-rich PY motif (22). Such PY motifs, which conform to a as those linked to aldosterone, vasopressin, and insulin (4). In consensus sequence of Pro-Pro-Xaa-Tyr (Xaa: any amino acid), recent years, considerable progress was made in understanding are found in many different proteins, often in plasma mem- this regulation, but many of the molecular mechanisms in- brane proteins or ion channels (23), and are known to interact volved remain unknown. Genetic evidence has given insight with WW (protein:protein interaction) domains (22,24). These into the current view that ENaC is regulated via ubiquitylation sequences are present in the C-termini of all three ENaC sub- and phosphorylation. units. When ENaC channels that bear Liddle’s mutations are Liddle’s syndrome is a rare genetic form of human hyper- expressed in heterologous expression systems, such as Xenopus tension that was originally described in 1963 by Dr. Liddle and laevis oocytes or MDCK cells, they display higher ENaC activity his collaborators (5) and presents with early onset of salt- at the plasma membrane (25–27). This increase is due to a rise sensitive hypertension, hypokalemia, metabolic alkalosis, and in cell surface expression, elevated open probability (Po), and ϩ low circulating levels of renin and aldosterone. Patients can be impairment of Na -dependant feedback inhibition of ENaC ϩ treated with ENaC inhibitors (triamterene or amiloride) and (i.e., inhibition of ENaC by intracellular Na ) (25,28,29). A yeast ϩ low-Na diet (6). Linkage analysis linked Liddle’s syndrome to two-hybrid analysis revealed that the ENaC PY motifs interact mutations in the genes encoding ␤- and ␥-ENaC (7,8). These with the ubiquitin-protein ligase Nedd4, implicating ubiquity- lation as a regulatory mechanism of ENaC activity (30).

؉ Published online ahead of print. Publication date available at www.jasn.org. Regulation of Epithelial Na Channel by

Address correspondence to: Dr. Olivier Staub, Department of Pharmacology & Nedd4/Nedd4-Like Ubiquitin Protein Toxicology, University of Lausanne, Rue du Bugnon 27, 1005 Lausanne, Switzerland. Ligases ϩ ϩ Phone: 41-21-692-5407; Fax: 41-21-692-5355; E-mail: [email protected] or Dr. Ubiquitylation (or ubiquitination) is a process that refers to Franc¸ois Verrey, Institute of Physiology, University of Zurich, Winterthurerstrasse 190, 8057 Zu¨rich, Switzerland. Phone: ϩ41-1-635-50-44/46/11; Fax: ϩ41-1-635-68-14; the posttranslational modification of target proteins with ubiq- E-mail: [email protected] uitin, a 76 amino acid–long polypeptide (31). This modification

Copyright © 2005 by the American Society of Nephrology ISSN: 1046-6673/1611-3167 3168 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 3167–3174, 2005 involves a cascade of enzymes, including a single E1 ubiquitin- activating enzyme, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin-protein ligases. The sequential action of these en- zymes leads to the isopeptide formation of ubiquitin via its C-terminal glycine to the amino group of a lysine on the target protein. Internal lysines on ubiquitin are often used to form polyubiquitin chains. Hundreds of E3 enzymes participate in substrate recognition. These enzymes can be divided into two major protein families, namely the RING finger (31) and the HECT (homologous to E6-AP carboxyterminal) domain-con- taining proteins. Nedd4 is a member of the HECT domain ubiquitin-protein ligases (32). These proteins are characterized by a C-terminal, highly conserved 40-kD enzymatic HECT do- main, and a variable, N-terminal region, involved in substrate recognition. The HECT domain contains a cysteine, which is essential for catalytic activity (32). Nedd4 is the founding mem- ber of a subfamily of HECT enzymes, which contains nine members (33). These enzymes are composed of an N-terminal Figure 1. Model of regulation of the epithelial sodium channel 2ϩ (ENaC) by serum and glucocorticoid induced kinase 1 (Sgk1) (Ca -dependent lipid binding) C2 domain (34), two to four and Nedd4-2. Induction of aldosterone leads to the expression WW domains (24), and the C-terminal HECT domain. They are of Sgk1. Sgk1 interacts via PY motif/WW domains with involved in several different cellular functions (33,35,36). The Nedd4-2 and phosphorylates Nedd4-2 on Ser444. Phosphory- isolation of the WW domain containing Nedd4–1 suggested lated Ser444 serves as binding site for 14-3-3, which hinders that ENaC may interact with members of this family via its PY interaction of Nedd4-2 with ENaC. This interferes with ENaC motifs. Upon interaction, Nedd4/Nedd4-like proteins would ubiquitylation, internalization, and degradation in the lysoso- ubiquitylate one or several ENaC subunits, in analogy to many mal pathways, consequently leading to accumulation of ENaC plasma membrane receptors (37), and cause their internaliza- at the cell surface. Illustration by Josh Gramling—Gramling tion and targeting to the endosomal multivesicular body path- Medical Illustration. way and ultimately degradation in the lysosome. In Liddle’s syndrome, in which one of the ENaC PY motifs is missing, interaction with the Nedd4/Nedd4-like proteins would be im- in the case of ENaC, this rapid turnover depends on both paired, leading to reduced ubiquitylation and internalization of proteasomal and lysosomal degradation (60,61,63). It has been ENaC (Figure 1). Many predictions of such a model have been shown using ENaC with cytoplasmic lysines mutated to argi- confirmed since then. nine, hence with mutated putative ubiquitylation sites, that ubiquitylation of ENaC controls its cell surface expression but ENaC Interacts with Nedd4/Nedd4-Like Proteins via PY not its intrinsic activity (i.e., Po or single channel conductance Motif/WW Domain Interaction [60]). A direct demonstration that Nedd4/Nedd4-like proteins Many investigators have shown by various in vitro and in ubiquitylate ENaC is so far missing. vivo approaches that ENaC PY motifs and Nedd4/Nedd4-like WW domains interact. In particular, in vitro interaction of ENaC ENaC Is Regulated by Nedd4/Nedd4-Like Proteins PY motif was shown with WW domains of Nedd4-1 (30,38–47), It has been demonstrated by different approaches that Nedd4-2 (47–49), WWP1 (50), and WWP2 (51). These interac- Nedd4/Nedd4-like proteins, in particular Nedd4, Nedd4-2, or tions with ENaC were confirmed in intact cells by co-immuno- WWP2, can regulate ENaC. In mouse mandibular cells, it is ϩ precipitation only with Nedd4-1 (30,52) and Nedd4-2 (52–55). possible to interfere with Na feedback regulation by adding Subsequently, evidence has accumulated that the PY motifs of Nedd4 antibodies or purified WW domains from Nedd4-1 or ␤-or␥-ENaC interact with the WW domains 3 or 4 of human Nedd4-2 into the pipette of a whole-cell patch-clamp set-up and Nedd4-1 or Nedd4-2 (41,46,47,52,56). letting them diffuse into the cell (38,40,56,64). Such data have been interpreted as evidence that either Nedd4 or Nedd4-2 is ϩ ENaC Is a Protein Complex That Has a Rapid Turnover involved in Na feedback regulation in mandibular cells. and Becomes Ubiquitylated Strong support for an involvement of Nedd4/Nedd4-like pro- Several groups have demonstrated that ENaC is a protein teins in ENaC regulation comes from experiments done in the with a rapid turnover of both the total and the cell surface pool Xenopus laevis oocytes system, in which regulation of ENaC by ϭ (T1/2 approximately 1 h) (57–61), although one report men- co-expressed Nedd4-1 (41,45,65), WWP2 (51), and Nedd4-2 tioned a half-life of the ␣-ENaC at the plasma membrane of 24 (52,53,66) has been demonstrated. The strongest effects are seen to 30 h (62). However, in this report, ␣-ENaC has an apparent with Nedd4-2. Overexpression of Nedd4-2 leads to the nearly ϩ molecular weight of 180 kD instead of the usual 90 kD; hence, complete suppression of amiloride-sensitive Na channels, an the specificity of the used anti–␣-ENaC antibody remains to be effect that is dependant on the PY motifs of ENaC (52,53,66). proved. Rapid turnover is a hallmark of ubiquitylated proteins; Conversely, catalytically inactive Nedd4-2 mutants increase ϩ J Am Soc Nephrol 16: 3167–3174, 2005 Nedd4, Sgk, and Epithelial Na Transport in the Distal Nephron 3169 these currents, most likely by competing against endogenous by Nedd4-2. Garty et al. (83) reported that Thr613 on ␤-ENaC Nedd4-2. Moreover, Nedd4-2 has been shown to control cell and Thr623 on ␥-ENaC just upstream of the PY motifs can be surface expression of ENaC (66). phosphorylated by extracellular signal-regulated kinase and The effect of Nedd4/Nedd4-like proteins was also investi- that this phosphorylation stimulates interaction with Nedd4-2. gated in ENaC transfected thyroid cells (FRT cells) that were Indeed, mutation of these phosphorylation sites leads to in- grown on permeable supports; co-transfection of either Nedd4 creased channel activity. Grk2 is another kinase that seems to or Nedd4-2 was found to diminish transepithelial amiloride- phosphorylate the C-terminus of ␤-ENaC and renders the chan- ϩ ϩ sensitive Na currents (45,49,67). In the same system, it was nel insensitive to Na feedback regulation and inhibition by demonstrated by RNA interference that Nedd4-2 controls Nedd4-2 (84). The physiologic significance of the regulation by ENaC, as interference with endogenous Nedd4-2 but not these kinases remains to be determined. Another kinase, serum ϩ Nedd4-1 increased transepithelial Na transport (68). and glucocorticoid induced kinase 1 (Sgk1), has received con- siderable attention in recent years, as its expression is rapidly Genetic Evidence Linking Nedd4-2 to BP Variation induced by aldosterone and appears to regulate ENaC. There are also some genetic features that seem to be compat- ible with the hypothesis that Nedd4-2 plays a role in the control Aldosterone-Induced Sgk1 Regulates ENaC ϩ of Na reabsorption and BP. The Nedd4-2 gene localizes to Activity and Cell Surface Expression chromosome 18q21-22, extends over 400 kb, and is composed of Sgk1 was originally identified as a kinase that is inducible by at least 40 exons. Numerous splice variants are expressed in a serum and glucocorticoids (85). It is a member of the AGC tissue- or cell-specific manner and encode proteins that do or family of Ser/Thr kinases and shares approximately 50% iden- do not include the C2 domain, have various numbers of WW tity in its catalytic domain with other AGC kinases such as domains, or lack certain phosphorylation sites (69–71). The protein kinase A, Akt, or protein kinase C (85). There are two functional role of these alternatively spliced isoforms is not yet close homologues, Sgk2 and Sgk3 (or CISK), that share approx- entirely clear. The C2 domain of Nedd4-2 has been shown to be imately 80% sequence identity with Sgk1 (86,87). They also required for the translocation of this enzyme to the plasma share a high degree of similarity with Akt/protein kinase B. ϩ membrane upon elevation of intracellular Ca2 (72), similar to Sgk1 was one of the earliest aldosterone-induced gene products the C2 domain of Nedd4-1 (73,74). In Xenopus oocytes, the C2 in renal epithelial cells (88–94). Interest in Sgk1 rose consider- domain is not required for the downregulation of ENaC (52,72), ably when it was found that Sgk1 stimulates ENaC activity in whereas a splice variant that lacks both the C2 domain and the oocytes (49,54,88–91,95–100). This increase of ENaC activity is WW domains 2 and 3 is inactive (72). mostly due to elevated cell surface expression (54,91,95,98), Genetic studies have indicated a link between markers in the although one report also observed an effect on Po (97). Further ϩ chromosomal region that contains the Nedd4-2 gene and dif- evidence for a role in epithelial Na transport came from ferent BP phenotypes (75–79). Although a number of polymor- transduction experiments in renal epithelial cells, which dem- phisms have been identified in the Nedd4-2 gene (55,69), no onstrated that expression of Sgk1 in various cell lines increased ϩ linkage between specific Nedd4-2 variants and BP phenotypes transepithelial Na currents (101–104). Thereby, Alvarez De La have been reported so far. Rosa et al. (104) observed in A6 cells that were transduced with constitutive active Sgk1 a 4-fold effect on ENaC cell surface Nedd4-2 Binding or Modifying Proteins expression as well as an increase of 43% in Po. It is not clear Not surprising, there are a number of proteins that interact how Sgk1 stimulates the Po of ENaC, as the Sgk1-dependent with or modify Nedd4-2 action on ENaC. As outlined above, phosphorylation site on ␣-ENaC, described by Diakov et al. ubiquitylation involves a cascade of enzymes, including E1, E2, (105) below, is not conserved in Xenopus laevis ␣-ENaC. Sgk1 is and an E3 enzyme (Nedd4-2). An E2 enzyme that acts in known to be regulated by the phosphatidylinositol-3 kinase concert with Nedd4-2 has been identified by a two-hybrid pathway and needs to be phosphorylated by PDK1, demon- screen using the HECT domain as bait. Further investigations strating that its function depends on the activity of this path- have shown that this enzyme, referred to as UBE2E3, co-immu- way. Indeed, evidence was provided from several groups that ϩ noprecipitates with Nedd4-2, that its inactive mutant interferes stimulation of Na transport by aldosterone and/or Sgk1 is with Nedd4-2–dependent regulation of ENaC, and that when it inhibited by phosphatidylinositol-3 kinase inhibitors (99,106,107). is overexpressed in mpkCCDcl4 cells (a mouse cortical collect- Antisense (103), RNA interference (94), or overexpression of cat- ϩ ing duct [CCD] cell line [80]), it inhibits transepithelial Na alytically inactive Sgk1 (101–103) further corroborates the role of ϩ current (81). Another protein that interacts with Nedd4-1 and Sgk1 in positive regulation of transepithelial Na transport. The Nedd4-2 is N4WBP5a, a transmembrane protein expressed in strongest evidence for such a function comes from the Sgk1 knock- the CCD (82). This protein interacts via PY motif/WW domain out mice, which present a pseudohypoaldosteronism type 1 phe- ϩ interaction with Nedd4-1 and Nedd4-2, and when co-expressed notype when kept under low-Na diet (108). ϩ in Xenopus oocytes, it prevents Na feedback regulation. It was proposed that this protein localizes to intracellular organelles Mechanisms of Sgk1 Action and binds Nedd4-2, thereby segregating it away from ENaC Several mechanisms have been proposed for Sgk1-dependent and thus preventing ENaC downregulation (82). Several ki- regulation of ENaC. Wang et al. (99) showed that Sgk1 directly nases also have been suggested to influence regulation of ENaC interacts with the C-terminus of ␤-ENaC. Diakov et al. (103) 3170 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 3167–3174, 2005 identified a consensus phosphorylation site on the C-terminus units related to ubiquitylation, and the control of cell surface of ␣-ENaC (S621) and found by the outside-out patch-clamp expression of ENaC by members of this E3 family. Neverthe- technique and inclusion of recombinant Sgk1 in the pipette that less, many questions remain open, in particular that of the this site may be important for ENaC stimulation. However, identity of the physiologically relevant ubiquitin-protein li- mutation of this site to alanine (S321A) does not impair the gase(s). Functional, biochemical, and genetic evidence point at stimulatory effect of Sgk1 (as well as of Sgk2 and Sgk3) on Nedd4-2 as the primary regulator, but to date, we cannot ϩ amiloride-sensitive Na currents in Xenopus laevis oocytes exclude the involvement of other family members of the (109). Demonstrating that ␣-ENaC indeed becomes phosphor- Nedd4/Nedd4-like proteins. It is possible that several of these ylated by Sgk1 will be required to shed some light on this proteins are implicated, as has been shown recently for the mechanism. An alternative model proposes that Nedd4-2 is a ubiquitylation and degradation of a membrane protein in me- substrate of Sgk1, as Nedd4-2 contains three consensus phos- lanocytes, Melan-A, which seems to be ubiquitylated by phorylation sites for Sgk1 (49,54), and Sgk1 itself contains a PY Nedd4–1, whereas its degradation seems to be controlled by motif, rendering it possible that Sgk1 and Nedd4-2 interact via Itch (a Nedd4/Nedd4-like protein) (114). Direct genetic linkage PY motif/WW domain (Figure 1). In support, it was demon- of BP variation with mutations in the Nedd4-2 gene or knock- strated that Sgk1 does interact with Nedd4-2 (48,49) and that out mice will be required to answer these questions defini- the PY motif on Sgk1 is partly required for Sgk1-dependent tively. That Nedd4-2 can be phosphorylated by Sgk1, an aldo- stimulation of ENaC in Xenopus laevis oocytes (54). In oocytes, sterone-induced protein in the CCD, suggests its potential it was also shown that Sgk1 induces phosphorylation of physiologic importance. Recently, it was shown that aldoste- Nedd4-2 on two sites, primarily on Ser444 but also on Ser338. rone stimulates phosphorylation of Nedd4-2, one of the predic- Moreover, mutation of these sites interfered with Sgk1-depen- tions of such a mechanism. This phosphorylation of Nedd4-2 dent phosphorylation of Nedd4-2 and stimulation of ENaC on Ser444 induces the binding of the 14-3-3 protein, likely (54). It was further demonstrated that Sgk1 reduced the inter- causing steric hindrance of the interaction of Nedd4-2 with action between Nedd4-2 and ENaC, indicating that Sgk1 im- ENaC. Although this mechanism is attractive, it probably does pairs ENaC ubiquitylation and subsequent internalization. not explain entirely the effect of Sgk1 on ENaC and certainly Taken together, these data suggest that one of the mechanisms not the complexity of aldosterone action on ENaC function. It is by which aldosterone regulates ENaC function is by transcrip- more than likely that other molecular events play a role. Fur- tionally inducing Sgk1 that in turn phosphorylates Nedd4-2. As thermore, ubiquitylation of ENaC may be reversible and in- expected from such a mechanism, the phosphorylation of volve de-ubiquitylating enzymes that are under the control of Nedd4-2 on Ser444 is increased upon aldosterone treatment in hormones such as aldosterone, vasopressin, or insulin. It is mpkCCDcl4 cells and in adrenalectomized rats (107). When clear that the regulatory pathways involving members of the phosphorylated, Nedd4-2–Ser444 is part of a consensus binding Nedd4/Nedd4-like family and Sgk1 play a central role in ENaC site for 14-3-3 proteins, suggesting that it is the binding of regulation. 14-3-3 proteins to Nedd4-2 that sterically interferes with the Note added in proof: Since submission of this article, Bhalla interaction of Nedd4-2 with ENaC and prevents ENaC ubiqui- et al. showed that 14-3-3 proteins bind to phosphorylated tylation (96). Nedd4-2 and interfere with Nedd4-2/ENaC interaction and ENaC ubiquitylation (Mol Endocrinol 2005, in press). Regulation of ENaC by Sgk2 and Sgk3 As outlined above, there are two Sgk paralogues, referred to as Sgk2 and Sgk3 (110). These two kinases share the same Acknowledgments consensus phosphorylation sites as Sgk1 [RXRXX(S/T)], and We thank Michael Harris for critically reading this review. they are both expressed in renal epithelial cells. In contrast to Sgk1, their expression is not regulated by glucocorticoids or by aldosterone (86,111,112). A possible role in ENaC regulation References has been studied in the Xenopus laevis system, and it has been 1. Canessa CM, Me´rillat A-M, Rossier BC: Membrane topol- found that Sgk2 and Sgk3 are as efficient in ENaC regulation as ogy of the epithelial sodium channel in intact cells. Am J Sgk1 (109). However, an Sgk3 knockout does not show any Physiol 267: C1682–C1690, 1994 ϩ discernable phenotype with respect to Na handling, suggest- 2. Canessa CM, Horisberger J-D, Rossier BC: Epithelial so- ing that Sgk3 does not play a major role in ENaC regulation dium channel related to proteins involved in neurodegen- (113). eration. Nature 361: 467–470, 1993 3. Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger J-D, Rossier BC: Amiloride-sensitive epithelial Perspectives Naϩ channel is made of three homologous subunits. Na- The past decade has provided us with a novel and at the time ture 367: 463–467, 1994 unexpected mode of regulation of ENaC, involving regulated 4. Kellenberger S, Schild L: Epithelial sodium channel/de- ubiquitylation of this channel complex by Nedd4/Nedd4-like generin family of ion channels: A variety of functions for a proteins (see Figure 1). Many of the aspects of such a model shared structure. Physiol Rev 82: 735–767, 2002 have been demonstrated experimentally, such as the PY mo- 5. Liddle GW, Bledsoe T, Coppage WS Jr: A familial renal tif/WW domain interaction, the rapid turnover of ENaC sub- disorder simulating primary aldosteronism but with neg- ϩ J Am Soc Nephrol 16: 3167–3174, 2005 Nedd4, Sgk, and Epithelial Na Transport in the Distal Nephron 3171

ligible aldosterone secretion. Trans Assoc Am Physicians 76: mura K, Tomita K, Sasaki S, Shimamoto K: A family with 199–213, 1963 Liddle’s syndrome caused by a mutation in the beta sub- 6. Botero-Velez M, Curtis JJ, Warnock DG: Brief report: Lid- unit of the epithelial sodium channel. Clin Exp Hypertens dle’s syndrome revisited. N Engl J Med 330: 178–181, 1994 23: 471–478, 2001 7. Shimkets RA, Warnock DG, Bositis CM, Nelson-Williams 20. Furuhashi M, Kitamura K, Adachi M, Miyoshi T, Wakida C, Hansson JH, Schambelan M, Gill JR, Ulick S, Milora RV, N, Ura N, Shikano Y, Shinshi Y, Sakamoto K, Hayashi M, Findling JW, Canessa CM, Rossier BC, Lifton RP: Liddle’s Satoh N, Nishitani T, Tomita K, Shimamoto K: Liddle’s syndrome: Heritable human hypertension caused by mu- syndrome caused by a novel mutation in the proline-rich tations in the beta subunit of the epithelial sodium channel. PY motif of the epithelial sodium channel beta-subunit. Cell 79: 407–414, 1994 J Clin Endocrinol Metab 90: 340–344, 2005 8. Hansson JH, Nelson-Williams C, Suzuki H, Schild L, 21. Hiltunen TP, Hannila-Handelberg T, Petajaniemi N, Kan- Shimkets RA, Lu Y, Canessa CM, Iwasaki T, Rossier BC, tola I, Tikkanen I, Virtamo J, Gautschi I, Schild L, Kontula Lifton RP: Hypertension caused by a truncated epithelial K: Liddle’s syndrome associated with a point mutation in sodium channel gamma subunit: Genetic heterogeneity of the extracellular domain of the epithelial sodium channel Liddle syndrome. Nat Genet 11: 76–82, 1995 gamma subunit. J Hypertens 20: 2383–2390, 2002 9. Freundlich M, Ludwig M: A novel epithelial sodium chan- 22. Chen HI, Sudol M: The WW domain of Yes-associated nel beta-subunit mutation associated with hypertensive protein binds a novel proline-rich ligand that differs from Liddle syndrome. Pediatr Nephrol 20: 512–551, 2005 the consensus established for SH3-binding modules. Proc 10. Yamashita Y, Koga M, Takeda Y, Enomoto N, Uchida S, Natl Acad Sci U S A 92: 7819–7823, 1995 Hashimoto K, Yamano S, Dohi K, Marumo F, Sasaki S: Two 23. Einbond A, Sudol M: Towards prediction of cognate com- sporadic cases of Liddle’s syndrome caused by de novo plexes between the WW domain and proline-rich ligands. ENaC mutations. Am J Kidney Dis 37: 499–504, 2001 FEBS Lett 384: 1–8, 1996 11. Gao PJ, Zhang KX, Zhu DL, He X, Han ZY, Zhan YM, Yang 24. Staub O, Rotin D: WW domains. Structure 4: 495–499, 1996 LW: Diagnosis of Liddle syndrome by genetic analysis of 25. Snyder PM, Price MP, McDonald FJ, Adams CM, Volk KA, beta and gamma subunits of epithelial sodium channel—A Zeiher BG, Stokes JB, Welsh MJ: Mechanism by which report of five affected family members. J Hypertens 19: Liddle’s syndrome mutations increase activity of a human 885–889, 2001 epithelial Naϩ channel. Cell 83: 969–978, 1995 12. Uehara Y, Sasaguri M, Kinoshita A, Tsuji E, Kiyose H, 26. Schild L, Canessa CM, Shimkets RA, Warnock DG, Lifton Taniguchi H, Noda K, Ideishi M, Inoue J, Tomita K, Ara- RP, Rossier BC: A mutation in the epithelial sodium chan- kawa K: Genetic analysis of the epithelial sodium channel nel causing Liddle’s disease increases channel activity in in Liddle’s syndrome. J Hypertens 16: 1131–1135, 1998 the Xenopus laevis oocyte expression system. Proc Natl Acad 13. Nakano Y, Ishida T, Ozono R, Matsuura H, Yamamoto Y, Sci U S A 92: 5699–5703, 1995 Kambe M, Chayama K, Oshima T: A frameshift mutation 27. Schild L, Lu Y, Gautschi I, Schneeberger E, Lifton RP, of beta subunit of epithelial sodium channel in a case of Rossier BC: Identification of a PY motif in the epithelial isolated Liddle syndrome. J Hypertens 20: 2379–2382, 2002 Naϩ channel subunits as a target sequence for mutations 14. Jackson SNJ, Williams B, Houtman P, Trembath RC: The causing channel activation found in Liddle syndrome. diagnosis of Liddle syndrome by identification of a muta- EMBO J 15: 2381–2387, 1996 tion in the beta subunit of the epithelial sodium channel. 28. Firsov D, Schild L, Gautschi I, Me´rillat A-M, Schneeberger J Med Genet 35: 510–512, 1998 E, Rossier BC: Cell surface expression of the epithelial Naϩ 15. Hansson JH, Schild L, Lu Y, Wilson TA, Gautschi I, channel and a mutant causing Liddle syndrome: A quan- Shimkets RA, Nelson-Williams C, Rossier BC, Lifton RP: A titative approach. Proc Natl Acad Sci U S A 93: 15370–15375, de novo missense mutation of the beta subunit of the 1996 epithelial sodium channel causes hypertension and Liddle 29. Kellenberger S, Gautschi I, Rossier BC, Schild L: Mutations syndrome, identifying a proline-rich segment critical for causing Liddle syndrome reduce sodium-dependent regulation of channel activity. Proc Natl Acad Sci U S A 25: downregulation of the epithelial sodium channel in the 11495–11499, 1995 Xenopus oocyte expression system. J Clin Invest 101: 2741– 16. Tamura H, Schild L, Enomoto N, Matsui N, Marumo F, 2750, 1998 Rossier BC, Sasaki S: Liddle disease caused by a missense 30. Staub O, Dho S, Henry PC, Correa J, Ishikawa T, McGlade mutation of beta subunit of the epithelial sodium channel J, Rotin D: WW domains of Nedd4 bind to the proline-rich gene. J Clin Invest 97: 1780–1784, 1996 PY motifs in the epithelial Naϩ channel deleted in Liddle’s 17. Inoue J, Iwaoka T, Tokunaga H, Takamune K, Naomi S, syndrome. EMBO J 15: 2371–2380, 1996 Araki M, Takahama K, Yamaguchi K, Tomita K: A family 31. Glickman MH, Ciechanover A: The ubiquitin-proteasome with Liddle’s syndrome caused by a new missense muta- proteolytic pathway: Destruction for the sake of construc- tion in the beta subunit of the epithelial sodium channel. tion. Physiol Rev 82: 373–428, 2002 J Clin Endocrinol Metab 83: 2210–2213, 1998 32. Huibregtse JM, Scheffner M, Beaudenon S, Howley PM: A 18. Inoue T, Okauchi Y, Matsuzaki Y, Kuwajima K, Kondo H, family of proteins structurally and functionally related to Horiuchi N, Nakao K, Iwata M, Yokogoshi Y, Shintani Y, the E6-AP ubiquitin-protein ligase. Proc Natl Acad Sci U S A Bando H, Saito S: Identification of a single cytosine base 92: 2563–2567, 1995 insertion mutation at Arg-597 of the beta subunit of the 33. Ingham RJ, Gish G, Pawson T: The Nedd4 family of E3 human epithelial sodium channel in a family with Liddle’s ubiquitin ligases: Functional diversity within a common disease. Eur J Endocrinol 138: 691–697, 1998 modular architecture. Oncogene 23: 1972–1984, 2004 19. Kyuma M, Ura N, Torii T, Takeuchi H, Takizawa H, Kita- 34. Nalefski EA, Falke JJ: The C2 domain calcium-binding 3172 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 3167–3174, 2005

motif: Structural and functional diversity. Protein Sci 5: Dhermy D: Identification of new partners of the epithelial 2375–2390, 1996 sodium channel alpha subunit. C R Biol 326: 615–624, 2003 35. Rotin D, Staub O, Haguenauer-Tsapis R: Ubiquitination 51. McDonald FJ, Western AH, McNeil JD, Thomas BC, Olson and endocytosis of plasma membrane proteins; role of DR, Snyder PM: Ubiquitin-protein ligase WWP2 binds to Nedd4/Rsp5 family of ubiquitin-protein ligases. J Membr and downregulates the epithelial Na(ϩ) channel. Am J Biol 176: 1–17, 2000 Physiol 283: F431–F436, 2002 36. Harvey KF, Kumar S: Nedd4-like proteins: An emerging 52. Kamynina E, Tauxe C, Staub O: Differential characteristics family of ubiquitin-protein ligases implicated in diverse of two human Nedd4 proteins with respect to epithelial cellular functions. Trends Cell Biol 9: 166–169, 1999 Naϩ channel regulation. Am J Physiol 281: F469–F477, 2001 37. Hicke L, Dunn R: Regulation of membrane protein trans- 53. Kamynina E, Debonneville C, Bens M, Vandewalle A, port by ubiquitin and ubiquitin-binding proteins. Annu Staub O: A novel mouse Nedd4 protein suppresses the ϩ Rev Cell Dev Biol 19: 141–172, 2003 activity of the epithelial Na channel. FASEB J 15: 204– 38. Dinudom A, Harvey BJ, Komwatana P, Young JA, Kumar 214, 2001 S, Cook DI: Nedd4 mediates control of an epithelial Naϩ 54. Debonneville C, Flores SY, Kamynina E, Plant PJ, Tauxe C, channel in salivary duct cells by cytosolic Naϩ. Proc Natl Thomas MA, Munster C, Chraibi A, Pratt JH, Horisberger Acad Sci U S A 95: 7169–7173, 1998 JD, Pearce D, Loffing J, Staub O: Phosphorylation of ϩ 39. Kanelis V, Farrow NA, Kay LE, Rotin D, Forman-Kay JD: Nedd4-2 by Sgk1 regulates epithelial Na( ) channel cell NMR studies of tandem WW domains of Nedd4 in com- surface expression. EMBO J 20: 7052–7059, 2001 plex with a PY motif-containing region of the epithelial 55. Fouladkou F, Alikhani-Koopaei R, Vogt B, Flores SY, sodium channel. Biochem Cell Biol 76: 341–350, 1998 Malbert-Colas L, Lecomte MC, Loffing J, Frey FJ, Frey BM, 40. Harvey KF, Dinudom A, Komwatana P, Jolliffe CN, Day Staub O: A naturally occurring human Nedd4-2 variant ML, Parasivam G, Cook DI, Kumar S: All three WW do- displays impaired ENaC regulation in Xenopus laevis oo- mains of murine Nedd4 are involved in the regulation of cytes. Am J Physiol Renal Physiol 287: F550–F561, 2004 epithelial sodium channels by intracellular Naϩ. J Biol 56. Fotia AB, Dinudom A, Shearwin KE, Koch JP, Korbmacher Chem 274: 12525–12530, 1999 C, Cook DI, Kumar S: The role of individual Nedd4-2 (KIAA0439) WW domains in binding and regulating epi- 41. Farr TJ, Coddington-Lawson SJ, Snyder PM, McDonald FJ: thelial sodium channels. FASEB J 17: 70–72, 2003 Human Nedd4 interacts with the human epithelial Naϩ 57. May A, Puoti A, Gaeggeler, H-P, Horisberger J-D, Rossier channel: WW3 but not WW1 binds to Naϩ-channel sub- BC: Early effect of aldosterone on the rate of synthesis of units. Biochem J 345: 503–509, 2000 the epithelial sodium channel alpha subunit in A6 renal 42. Kanelis V, Donaldson L, Muhandiram DR, Rotin D, cells. J Am Soc Nephrol 8: 1813–1822, 1997 Forman-Kay JD, Kay LE: Sequential assignment of proline- 58. Alvarez de la Rosa D, Li H, Canessa CM: Effects of aldo- rich regions in proteins: Application to modular binding sterone on biosynthesis, traffic, and functional expression domain complexes. J Biomol NMR 16: 253–259, 2000 of epithelial sodium channels in A6 cells. J Gen Physiol 119: 43. Asher C, Chigaev A, Garty H: Characterization of Interac- 427–442, 2002 tions between Nedd4 and beta and gammaENaC using 59. Hanwell D, Ishikawa T, Saleki R, Rotin D: Trafficking and surface plasmon resonance. Biochem Biophys Res Commun cell surface stability of the epithelial Naϩ channel ex- 286: 1228–1231, 2001 pressed in epithelial Madin-Darby canine kidney cells. 44. Kanelis V, Rotin D, Forman-Kay JD: Solution structure of a J Biol Chem 277: 9772–9779, 2002 Nedd4 WW domain-ENaC peptide complex. Nat Struct 60. Staub O, Gautschi I, Ishikawa T, Breitschopf K, Ciech- Biol 8: 1–6, 2001 anover A, Schild L, Rotin D: Regulation of stability and 45. Snyder PM, Olson DR, McDonald FJ, Bucher DB: Multiple function of the epithelial Naϩ channel (ENaC) by ubiquiti- WW domains, but not the C2 domain, are required for the nation. EMBO J 16: 6325–6336, 1997 inhibition of ENaC by human Nedd4. J Biol Chem 276: 61. Malik B, Schlanger L, Al-Khalili O, Bao HF, Yue G, Price 28321–28326, 2001 SR, Mitch WE, Eaton DC: Enac degradation in A6 cells by 46. Lott JS, Coddington-Lawson SJ, Teesdale-Spittle PH, the ubiquitin-proteosome proteolytic pathway. J Biol Chem McDonald FJ: A single WW domain is the predominant 276: 12903–12910, 2001 mediator of the interaction between the human ubiquitin- 62. Kleyman TR, Zuckerman JB, Middleton P, McNulty KA, protein ligase Nedd4 and the human epithelial sodium Hu B, Su X, An B, Eaton DC, Smith PR: Cell surface channel. Biochem J 361: 481–488, 2002 expression and turnover of the alpha-subunit of the epi- 47. Henry PC, Kanelis V, O’Brien MC, Kim B, Gautschi I, thelial sodium channel. Am J Physiol Renal Physiol 281: Forman-Kay J, Schild L, Rotin D: Affinity and specificity of F213–F221, 2001 interactions between Nedd4 isoforms and the epithelial 63. Malik B, Yue Q, Yue G, Chen XJ, Price SR, Mitch WE, Eaton Naϩ channel. J Biol Chem 278: 20019–20028, 2003 DC: Role of Nedd4-2 and polyubiquitination in epithelial 48. Asher C, Sinha I, Garty H: Characterization of the interac- sodium channel degradation in untransfected renal A6 tions between Nedd4-2, ENaC, and sgk-1 using surface cells expressing endogenous ENaC subunits. Am J Physiol plasmon resonance. Biochim Biophys Acta 1612: 59–64, 2003 Renal Physiol 289: F107–F116, 2005 49. Snyder PM, Olson DR, Thomas BC: Serum and glucocor- 64. Komwatana P, Dinudom A, Young JA, Cook DI: Cytosolic ticoid-regulated kinase modulates Nedd4-2-mediated inhi- Naϩ controls an epithelial Naϩ channel via the Go gua- bition of the epithelial Naϩ channel. J Biol Chem 277: 5–8, nine nucleotide-binding regulatory protein. Proc Natl Acad 2002 Sci U S A 93: 8107–8111, 1996 50. Malbert-Colas L, Nicolas G, Galand C, Lecomte MC, 65. Goulet CC, Volk KA, Adams CM, Prince LS, Stokes JB, ϩ J Am Soc Nephrol 16: 3167–3174, 2005 Nedd4, Sgk, and Epithelial Na Transport in the Distal Nephron 3173

Snyder PM: Inhibition of the epithelial Naϩ channel by Rafestin-Oblin ME, Rossier BC, Vandewalle A: Corticoste- interaction of Nedd4 with a PY motif deleted in Liddle’s roid-dependent sodium transport in a novel immortalized syndrome. J Biol Chem 273: 30012–30017, 1998 mouse collecting duct principal cell line. J Am Soc Nephrol 66. Abriel H, Loffing J, Rebhun JF, Pratt JH, Horisberger J-D, 10: 923–934, 1999 Rotin D, Staub O: Defective regulation of the epithelial 81. Debonneville C, Staub O: Participation of the ubiquitin- Naϩ channel (ENaC) by Nedd4 in Liddle’s syndrome. conjugating enzyme UBE2E3 in Nedd4-2-dependent regu- J Clin Invest 103: 667–673, 1999 lation of the epithelial Naϩ channel. Mol Cell Biol 24: 67. Snyder PM, Olson DR, Kabra R, Zhou R, Steines JC: cAMP 2397–2409, 2004 and serum and glucocorticoid-inducible kinase (SGK) reg- 82. Konstas AA, Shearwin-Whyatt LM, Fotia AB, Degger B, ulate the epithelial Na(ϩ) channel through convergent Riccardi D, Cook DI, Korbmacher C, Kumar S: Regulation phosphorylation of Nedd4-2. J Biol Chem 279: 45753–45758, of the epithelial sodium channel by N4WBP5A, a novel 2004 Nedd4/Nedd4-2-interacting protein. J Biol Chem 277: 68. Snyder PM, Steines JC, Olson DR: Relative contribution of 29406–29416, 2002 Nedd4 and Nedd4-2 to ENaC regulation in epithelia de- 83. Shi H, Asher C, Chigaev A, Yung Y, Reuveny E, Seger R, termined by RNA interference. J Biol Chem 279: 5042–5046, Garty H: Interactions of beta and gamma ENaC with 2004 Nedd4 can be facilitated by an ERK-mediated phosphory- 69. Dunn DM, Ishigami T, Pankow J, Von Niederhausern A, lation. J Biol Chem 277: 13539–13547, 2002 Alder J, Hunt SC, Leppert MF, Lalouel JM, Weiss RB: 84. Dinudom A, Fotia AB, Lefkowitz RJ, Young JA, Kumar S, Common variant of human NEDD4L activates a cryptic Cook DI: The kinase Grk2 regulates Nedd4/Nedd4-2-de- splice site to form a frameshifted transcript. J Hum Genet pendent control of epithelial Naϩ channels. Proc Natl Acad 47: 665–676, 2002 Sci U S A 101: 11886–11890, 2004 70. Itani OA, Campbell JR, Herrero J, Snyder PM, Thomas CP: 85. Webster MK, Goya L, Ge Y, Maiyar AC, Firestone GL: Alternative promoter usage and differential splicing leads Characterization of sgk, a novel member of the serine/ to human Nedd4-2 isoforms with a C2 domain and with threonine protein kinase gene family which is transcrip- variable number of WW domains. Am J Physiol 285: F916– tionally induced by glucocorticoids and serum. Mol Cell F929, 2003 Biol 13: 2031–2040, 1993 71. Chen H, Ross CA, Wang N, Huo Y, MacKinnon DF, Potash 86. Kobayashi T, Deak M, Morrice N, Cohen P: Characteriza- JB, Simpson SG, McMahon FJ, DePaulo JR Jr, McInnis MG: tion of the structure and regulation of two novel isoforms NEDD4L on human chromosome 18q21 has multiple of serum- and glucocorticoid-induced protein kinase. Bio- forms of transcripts and is a homologue of the mouse chem J 344: 189–197, 1999 Nedd4-2 gene. Eur J Hum Genet 9: 922–930, 2001 87. Liu D, Yang X, Songyang Z: Identification of CISK, a new 72. Itani OA, Stokes JB, Thomas CP: Nedd4-2 isoforms differ- member of the SGK kinase family that promotes IL-3- entially associate with ENaC and regulate its activity. Am J dependent survival. Curr Biol 10: 1233–1236, 2000 Physiol Renal Physiol 289: F334–F346, 2005 88. Naray-Fejes-Toth A, Canessa CM, Cleaveland ES, Aldrich 73. Plant P, Yeger H, Staub O, Howard P, Rotin D: The C2 G, Fejes-Toth G: Sgk is an aldosterone-induced kinase in domain of the ubiquitin protein ligase Nedd4 mediates the renal collecting duct. J Biol Chem 274: 16973–16978, 1999 Ca2ϩ-dependent plasma membrane localization. J Biol 89. Chen S, Bhargava A, Mastroberardino L, Meijer OC, Wang Chem 272: 32329–32336, 1997 J, Buse P, Firestone GL, Verrey F, Pearce D: Epithelial 74. Plant P, Lafont F, Lecat S, Verkade P, Simons K, Rotin D: sodium channel regulated by aldosterone-induced protein Apical membrane localization of Nedd4 is mediated by an sgk. Proc Natl Acad Sci U S A 96: 2514–2519, 1999 association of its C2 domain with annexin XIIIb. J Cell Biol 90. Shigaev A, Asher C, Latter H, Garty H, Reuveny E: Regu- 149: 1473–1483, 2000 lation of sgk by aldosterone and its effects on the epithelial 75. Kristjansson K, Manolescu A, Kristinsson A, Hardarson T, Na(ϩ) channel. Am J Physiol 278: F613–F619, 2000 Knudsen H, Ingason S, Thorleifsson G, Frigge ML, Kong A, 91. Loffing J, Zecevic M, Feraille E, Kaissling B, Asher C, Gulcher JR, Stefansson K: Linkage of essential hyperten- Rossier BC, Firestone GL, Pearce D, Verrey F: Aldosterone sion to chromosome 18q. Hypertension 39: 1044–1049, 2002 induces rapid apical translocation of ENaC in early portion 76. Atwood LD, Samollow PB, Hixson JE, Stern MP, MacCluer of renal collecting system: Possible role of SGK. Am J JW: Genome-wide linkage analysis of blood pressure in Physiol 280: F675–F682, 2001 Mexican Americans. Genet Epidemiol 20: 373–382, 2001 92. Brennan FE, Fuller PJ: Rapid upregulation of serum and 77. Pankow JS, Rose KM, Oberman A, Hunt SC, Atwood LD, glucocorticoid-regulated kinase (sgk) gene expression by Djousse L, Province MA, Rao DC: Possible locus on chro- corticosteroids in vivo. Mol Cell Endocrinol 166: 129–136, mosome 18q influencing postural systolic blood pressure 2000 changes. Hypertension 36: 471–476, 2000 93. Muller OG, Parnova RG, Centeno G, Rossier BC, Firsov D, 78. DeStefano AL, Baldwin CT, Burzstyn M, Gavras I, Handy Horisberger JD: Mineralocorticoid effects in the kidney: DE, Joost O, Martel T, Nicolaou M, Schwartz F, Streeten Correlation between alphaENaC, GILZ, and Sgk-1 mRNA DH, Farrer LA, Gavras H: Autosomal dominant orthostatic expression and urinary excretion of Na(ϩ) and K(ϩ). JAm hypotensive disorder maps to chromosome 18q. Am J Hum Soc Nephrol 14: 1107–1115, 2003 Genet 63: 1425–1430, 1998 94. Bhargava A, Wang J, Pearce D: Regulation of epithelial ion 79. Atwood LD, Samollow PB, Hixson JE, Stern MP, MacCluer transport by aldosterone through changes in gene expres- JW: Genome-wide linkage analysis of pulse pressure in sion. Mol Cell Endocrinol 217: 189–196, 2004 Mexican Americans. Hypertension 37: 425–428, 2001 95. Alvarez de la Rosa D, Zhang P, Naray-Fejes-Toth A, Fejes- 80. Bens M, Vallet V, Cluzeaud F, Pascual-Letallec L, Kahn A, Toth G, Canessa CM: The serum and glucocorticoid kinase 3174 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 3167–3174, 2005

sgk increases the abundance of epithelial sodium channels ticoid-inducible kinase consensus motif in the C terminus in the plasma membrane of Xenopus oocytes. J Biol Chem of the channel’s alpha-subunit. J Biol Chem 279: 38134– 274: 37834–37839, 1999 38142, 2004 96. Ichimura T, Yamamura H, Sasamoto K, Tominaga Y, Taoka 106. Blazer-Yost BL, Paunescu TG, Helman SI, Lee KD, Vlahos M, Kakiuchi K, Shinkawa T, Takahashi N, Shimada S, CJ: Phosphoinositide 3-kinase is required for aldosterone- Isobe T: 14-3-3 proteins modulate the expression of epithe- regulated sodium reabsorption. Am J Physiol 46: C531– lial Naϩ channels by phosphorylation-dependent interac- C536, 1999 tion with Nedd4-2 ubiquitin ligase. J Biol Chem 280: 13187– 107. Flores SY, Loffing-Cueni D, Kamynina E, Daidie D, Gerbex 13194, 2005 C, Chabanel S, Dudler J, Loffing J, Staub O: Aldosterone 97. Vuagniaux G, Vallet V, Jaeger NF, Hummler E, Rossier BC: induced Sgk1 expression is accompanied by Nedd4-2 Synergistic activation of ENaC by three membrane-bound phosphorylation and increased Naϩ transport in cortical channel- activating serine proteases (mCAP1, mCAP2, and collecting duct cells. J Am Soc Nephrol 16: 2279–2287, 2005 mCAP3) and serum- and glucocorticoid-regulated kinase 108. Wulff P, Vallon V, Huang DY, Volkl H, Yu F, Richter K, (Sgk1) in Xenopus oocytes. J Gen Physiol 120: 191–201, 2002 Jansen M, Schlunz M, Klingel K, Loffing J, Kauselmann G, 98. Wagner CA, Ott M, Klingel K, Beck S, Melzig J, Friedrich B, Bosl MR, Lang F, Kuhl D: Impaired renal Na(ϩ) retention Wild KN, Broer S, Moschen I, Albers A, Waldegger S, in the sgk1-knockout mouse. J Clin Invest 110: 1263–1268, Tummler B, Egan ME, Geibel JP, Kandolf R, Lang F: Effects 2002 of the serine/threonine kinase SGK1 on the epithelial 109. Friedrich B, Feng Y, Cohen P, Risler T, Vandewalle A, Na(ϩ) channel (ENaC) and CFTR: Implications for cystic Broer S, Wang J, Pearce D, Lang F: The serine/threonine fibrosis. Cell Physiol Biochem 11: 209–218, 2001 kinases SGK2 and SGK3 are potent stimulators of the 99. Wang J, Barbry P, Maiyar AC, Rozansky DJ, Bhargava A, epithelial Na(ϩ) channel alpha, beta, gamma-ENaC. Leong M, Firestone GL, Pearce D: SGK integrates insulin Pflugers Arch 445: 693–696, 2003 and mineralocorticoid regulation of epithelial sodium 110. Gamper N, Fillon S, Feng Y, Friedrich B, Lang PA, Henke transport. Am J Physiol 280: F303–F313, 2001 G, Huber SM, Kobayashi T, Cohen P, Lang F: Kϩ channel 100. Lang F, Klingel K, Wagner CA, Stegen C, Warntges S, activation by all three isoforms of serum- and glucocorti- Friedrich B, Lanzendorfer M, Melzig J, Moschen I, Steuer S, coid-dependent protein kinase SGK. Pflugers Arch 445: 60– Waldegger S, Sauter M, Paulmichl M, Gerke V, Risler T, 66, 2002 Gamba G, Capasso G, Kandolf R, Hebert SC, Massry SG, 111. Naray-Fejes-Toth A, Helms MN, Stokes JB, Fejes-Toth G: Broer S: Deranged transcriptional regulation of cell-vol- Regulation of sodium transport in mammalian collecting ume-sensitive kinase hSGK in diabetic nephropathy. Proc duct cells by aldosterone-induced kinase, SGK1: Struc- Natl Acad Sci U S A 97: 8157–8162, 2000 ture/function studies. Mol Cell Endocrinol 217: 197–202, 101. Faletti CJ, Perrotti N, Taylor SI, Blazer-Yost BL: Sgk: An 2004 essential convergence point for peptide and steroid hor- 112. Robert-Nicoud M, Flahaut M, Elalouf JM, Nicod M, Salinas mone regulation of ENaC-mediated Naϩ transport. Am J M, Bens M, Doucet A, Wincker P, Artiguenave F, Horis- Physiol 282: C494–C500, 2002 berger JD, Vandewalle A, Rossier BC, Firsov D: Transcrip- 102. Alvarez De La Rosa D, Canessa CM: Role of SGK in hor- tome of a mouse kidney cortical collecting duct cell line: monal regulation of epithelial sodium channel in A6 cells. Effects of aldosterone and vasopressin. Proc Natl Acad Sci Am J Physiol 284: C404–C414, 2003 USA98: 2712–2716, 2001 103. Helms MN, Fejes-Toth G, Naray-Fejes-Toth A: Hormone- 113. McCormick JA, Feng Y, Dawson K, Behne MJ, Yu B, Wang regulated transepithelial Naϩ transport in mammalian J, Wyatt AW, Henke G, Grahammer F, Mauro TM, Lang F, CCD cells requires SGK1 expression. Am J Physiol 284: Pearce D: Targeted disruption of the protein kinase SGK3/ F480–487, 2003 CISK impairs postnatal hair follicle development. Mol Biol 104. Alvarez de la Rosa D, Paunescu TG, Els WJ, Helman SI, Cell 15: 4278–4288, 2004 Canessa CM: Mechanisms of regulation of epithelial so- 114. Levy F, Muehlethaler K, Salvi S, Peitrequin AL, Lindholm dium channel by SGK1 in A6 cells. J Gen Physiol 124: CK, Cerottini JC, Rimoldi D: Ubiquitylation of a melano- 395–407, 2004 somal protein by HECT-E3 ligases serves as sorting signal 105. Diakov A, Korbmacher C: A novel pathway of epithelial for lysosomal degradation. Mol Biol Cell 16: 1777–1787, sodium channel activation involves a serum- and glucocor- 2005