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Molecular Endocrinology 19(12):3073­3084 Copyright © 2005 by The Endocrine Society doi: 10.1210/me.2005-0193

Serum- and Glucocorticoid-Regulated Kinase 1 Regulates Ubiquitin Ligase Neural Precursor CellExpressed, Developmentally Down-Regulated Protein 4-2 by Inducing Interaction with 14-3-3

Vivek Bhalla, Dorothee Daidie, Hongyan Li, Alan C. Pao, Lila P. LaGrange, Jian Wang, ´ ´ Alain Vandewalle, James D. Stockand, Olivier Staub, and David Pearce Division of Nephrology, Department of Medicine (V.B., H.L., A.C.P., J.W., D.P.), and Department of Cellular and Molecular Pharmacology (D.P.), University of California, San Francisco, California 941430532; Department of Pharmacology and Toxicology, University of Lausanne (D.D., O.S.), CH-1005 Lausanne, Switzerland; Department of Physiology, University of Texas Health Science Center (L.P.L., J.D.S.), San Antonio, Texas 78229-3900; and Institut National de la Sante et de la Recherche ´ Medicale, Unite 478, Faculte de Medecine Xavier Bichat (A.V.), 75870 Paris, France ´ ´ ´ ´

Serum- and glucocorticoid-regulated kinase 1 (SGK1) is an aldosterone-regulated early response gene product that regulates the activity of several ion transport proteins, most notably that of the epithelial sodium channel (ENaC). Recent evidence has established that SGK1 phosphorylates and inhibits Nedd4-2 (neural precursor cell-expressed, developmentally down-regulated protein 4-2), a ubiquitin ligase that decreases cell surface expression of the channel and possibly stimulates its degradation. The mechanistic basis for this SGK1-induced Nedd4-2 inhibition is currently unknown. In this study we show that SGK1-mediated phosphorylation of Nedd4-2 induces its interaction with members of the 14-3-3 family of regulatory proteins. Through functional characterization of Nedd4-2-mutant proteins, we demonstrate that this interaction is required for SGK1-mediated inhibition of Nedd4-2. The concerted action of SGK1 and 14-3-3 appears to disrupt Nedd4-2-mediated ubiquitination of ENaC, thus providing a mechanism by which SGK1 modulates the ENaC-mediated Na current. Finally, the expression pattern of 14-3-3 is also consistent with a functional role in distal nephron Na transport. These results demonstrate a novel, physiologically significant role for 14-3-3 proteins in modulating ubiquitin ligasedependent pathways in the control of epithelial ion transport. (Molecular Endocrinology 19: 3073­3084, 2005)

T

HE EPITHELIAL SODIUM channel (ENaC) constitutes the rate-limiting step in transepithelial Na transport in kidney, colon, lung, and other electrically high resistance epithelia. Disruption of channel function or of its key regulators causes profound disease states and in some cases is incompatible with life (1, 2). ENaC-mediated sodium transport is under physiological control, primarily by the adrenal steroid hormones, which act through the mineralocorticoid and glucocorticoid receptors (MR and GR, respectively) to stimulate localization of functional ENaC to the apical plasma membrane. The ENaC stimulatory effects of

First Published Online August 11, 2005 Abbreviations: ENaC, Epithelial sodium channel; GR, glucocorticoid receptor; GST, glutathione-S-transferase; h, human; HA, hemagglutinin; HEK, human embryonic kidney; MR, mineralocorticoid receptor; Nedd4-2, neural precursor cellexpressed, developmentally down-regulated protein 4-2; PI3-kinase, phosphatidylinositol 3-kinase; PMSF, phenylmethylsulfonylfluoride; SGK1, serum- and glucocorticoid-regulated kinase 1; WT, wild type; x, Xenopus. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.

corticosteroid hormones are mediated in part by the serine-threonine kinase, serum- and glucocorticoidregulated kinase 1 (SGK1), whose gene transcription is stimulated by MR and GR (3, 4). Other hormonal and nonhormonal regulators of ENaC, including insulin and osmotic shock, also appear to act through SGK1 (5, 6) to stimulate Na current (7­9). One of the cellular substrates of SGK1 is Nedd4-2 (neural precursor cellexpressed, developmentally down-regulated protein 4-2), a HECT (homologous to E6-AP carboxyl terminus) domain-containing ubiquitin ligase that regulates ENaC trafficking and degradation through ubiquitination (10, 11). Several recent reports support the idea that SGK1 phosphorylates and thereby inhibits Nedd4-2 (7, 12­14), although there are data to suggest that SGK1 also has effects on ENaC that are independent of Nedd4 or Nedd4-2 (15­20). Nedd4-2 contains a series of tryptophan-rich sequences (WW motifs) that interact with a proline-tyrosine PY motif within the C-terminal tail of ENaC (2, 21­23). Interaction of Nedd4-2 with ENaC is essential for its inhibitory effect; notably, the human disease Liddle's syndrome, a form of pseudohyperaldosteronism characterized by severe hypertension and hypo-

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kalemia, may be caused by mutations in the C-terminal tail of ENaC that irreversibly disrupt this interaction (21). In contrast, reversible physiological disruption of the Nedd4-2-ENaC interaction is achieved through SGK1-mediated phosphorylation of Nedd4-2 at key substrate residues within the region containing the four WW domains (7, 12). However, the molecular mechanism by which phosphorylation alters Nedd4-2 activity remains unknown. SGK1 is a member of the phosphatidylinositol 3kinase (PI3-kinase)-dependent SGK/Akt family (24), which phosphorylates substrates with the general motif RXRXX(S/T) (25, 26). Such phosphorylation can, in principle, have a variety of effects on target protein function, including altering conformation or inducing interaction with other regulatory proteins. Interestingly, Akt-mediated phosphorylation of the Forkhead transcription factor, FKHRL1, induces its interaction with members of the 14-3-3 family of signaling proteins (27). The 14-3-3 interaction motif (28) within FKHRL1 overlaps an SGK/Akt target sequence, RSX(pS/pT)XP (where pS/pT represents the substrate phosphoserine/phosphothreonine). Importantly, inspection of the functionally critical SGK1 motif within Nedd4-2 revealed that it conforms perfectly to this consensus 14-3-3 interaction motif, whereas two other sites partially match this sequence (14, 29) (Fig. 1). Moreover, these SGK1 sites are conserved in vertebrate species from Xenopus to human, and SGK1mediated phosphorylation of Nedd4-2 (7, 12) has been demonstrated in vivo and in vitro at two of these motifs (7) (Fig. 1). In particular, Ser444 (Xenopus numbering), which is embedded in a perfect 14-3-3 interaction motif, is critical for SGK1-induced inhibition; Nedd4-2 mutants with Ala substitution at this residue inhibit ENaC constitutively and do not respond to SGK1. 14-3-3 proteins were initially described as acidic proteins in brain tissue (30). Since then, they have been identified in virtually all tissues, and a variety of physiological and pathophysiological roles have been

identified in neurodegenerative diseases, carcinogenesis, and congestive heart failure (31­33). There exist seven mammalian isoforms of 14-3-3 ( , , , , , , and ), encoded by distinct genes, which can homodimerize and heterodimerize to increase binding affinity for specific targets (34). Their biology spans a variety of intracellular processes, such as cell cycle regulation, proliferation, metabolism, and ion channel function (31, 35, 36). In general, phosphorylation-dependent interactions of 14-3-3 proteins with targets of cellular kinases alter the activity and/or subcellular location of these target proteins, thereby propagating kinase-dependent signaling events. Together, these observations suggested that SGK1mediated phosphorylation at Ser444 induces an inhibitory interaction of Nedd4-2 with 14-3-3 proteins thereby increasing ENaC-mediated Na transport. To explore this hypothesis, we examined the interaction of 14-3-3 with Nedd4-2 in cultured cells and in vitro and characterized the functional regulation and ubiquitination of ENaC by SGK1 together with 14-3-3.

RESULTS Endogenous Nedd4-2 and 14-3-3 Interact in Kidney Tubule Epithelial Cells To determine whether 14-3-3 proteins physically interact with Nedd4-2 in vivo, we performed coimmunoprecipitation experiments with antibodies directed against endogenous Nedd4-2 and 14-3-3 in cultured mpkCCDc14 cells. This mouse collecting duct cell line forms a monolayer with high electrical resistance when grown on Transwell filters and expresses several key mediators of Na transport regulation, including the corticosteroid receptors (MR and GR), SGK1, Nedd4-2, and ENaC (11, 37­39). In this and other collecting duct cell lines, corticosteroids induce a robust increase in SGK1 expression, and insulin increases SGK1 activity in a PI3-kinase dependent fashion. Hence, corticosteroids and insulin together strongly stimulate, whereas PI3-kinase inhibitors block, ENaC-mediated Na currents (38­41) (Fig. 2). In this study we compared the interaction of endogenous Nedd4-2 and 14-3-3 by coimmunoprecipitation, under basal conditions, SGK1 activation (dexamethasone plus insulin), or PI3-kinase inhibition (by LY294002). As shown in Fig. 2, A and B, the interaction between Nedd4-2 and 14-3-3 was markedly stimulated by dexamethasone plus insulin, whereas it was attenuated by PI3-kinase inhibition with LY294002. Parallel changes in equivalent current were also observed (Fig. 2C). Importantly, total Nedd4-2 and 14-3-3 expression were unchanged, consistent with the idea that the effects are due to changes in interaction per se and not to changes in the level of expression of either component.

Fig. 1. The Major SGK1 Substrate Sequence in Nedd4-2 Overlaps a 14-3-3 Interaction Motif A, Strip diagram depicting the major functional domains of Nedd4-2, including three SGK1 target motifs (Xenopus numbering). B, Sequences surrounding Ser338, Thr363, and Ser444 of xNedd4-2 are shown. The SGK1 site that includes Ser444 is required for SGK1 regulation of Nedd4-2. Amino acids overlapping this SGK1 site constitute a consensus sequence for a high affinity 14-3-3 interaction motif.

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gous expression in human embryonic kidney 293 (HEK293) cells. Wild-type (WT) FLAG epitope-tagged Nedd4-2 was transfected, with or without a constitutively active mutant of SGK1. FLAG-Nedd4-2 was then immunoprecipitated, and immunoblots were stained for endogenous 14-3-3 (Fig. 3). As shown in Fig. 3, A and B, SGK1 markedly increased the amount of 143-3 coimmunoprecipitated with FLAG-Nedd4-2, even at low levels of coexpressed SGK1, consistent with an enzymatic effect. This idea is supported by the observations that omitting phosphatase inhibitors from the interaction buffer markedly attenuated the interaction and that kinase-dead SGK1 failed to stimulate it (Fig. 3, C and D, respectively). Interestingly, it appears that SGK1 itself stably enters the complex with Nedd4-2 and 14-3-3 under these conditions, because it was coimmunoprecipitated with FLAG-Nedd4-2 (Fig. 3A, third panel). We next examined the role of the functionally important SGK1 substrate sequence within Nedd4-2, and the surrounding amino acids that contribute to the 14-3-3 interaction motif (Fig. 1). For these experiments, we used two mutants of Nedd4-2, S444A and P446A, which were predicted to block 14-3-3 interaction. Nedd4-2/S444A was shown previously to abrogate SGK1 phosphorylation and disrupt inhibition, hence blocking ENaC disinhibition (7, 12). In contrast, Nedd4-2/P446A was predicted to be incapable of 143-3 interaction despite being fully phosphorylated by SGK1 (28, 42, 43). As shown in Fig. 3, D and E, in contrast to WT Nedd4-2, neither mutant was able to specifically interact with 14-3-3 in an SGK1-dependent fashion. Taken together, these results strongly suggest that the SGK1-dependent phosphorylation of Nedd4-2 at Ser444 induces its interaction with a 143-3 family member, and that this interaction is dependent on a proline two residues downstream of the substrate serine.

Fig. 2. Endogenous Nedd4-2 and 14-3-3 Interact in Kidney Tubule Epithelial Cells mpkCCDc14 cells were treated with the PI3-kinase inhibitor LY294002, vehicle, or dexamethasone plus insulin for 3 h before harvest. A, Immunoblots of cell lysates immunoprecipitated with anti-Nedd4 antibody and stained with anti-143-3 (top panel) and anti-Nedd4-2 (second panel) antibodies. Right panels, Whole cell lysates (WCL) were stained as shown. A representative blot from three independent experiments is shown. B, Scanning densitometry of 14-3-3 abundance, normalized to the corresponding amount of Nedd4-2, immunoprecipitated from cells treated with LY294002, vehicle, or dexamethasone plus insulin in three independent experiments. Results are expressed relative to vehicle-treated controls. C, Equivalent current from polarized mpkCCDc14 cells at 3 h after treatment. *, Statistically significant (P 0.05) compared with vehicle treatment.

SGK1 Mediates Nedd4-2 Phosphorylation and Stimulates Its Interaction with 14-3-3 in Vitro To explore the dependence of the Nedd4-2 interaction with 14-3-3 on SGK1-induced phosphorylation at Ser444, we examined the phosphorylation of Nedd4-2 and its interaction with 14-3-3 in vitro. We initially tested the hypothesis that the P446A, but not the S444A, mutant, would be phosphorylated by SGK1 at a comparable rate to WT Nedd4-2. Nedd4-2 peptides encompassing the SGK1 and 14-3-3 motifs surrounding Ser444 were expressed in Escherichia coli as glutathione-S-transferase (GST) fusion proteins (Fig. 4A) and incubated with SGK1 and [ -32P]ATP, and SGK1 kinase activity was assayed by radioactive phosphate incorporation. As shown in Fig. 4, B and C, the WT peptide and the P446A mutant were strongly phosphorylated by SGK1, in contrast to phosphorylation of the S444A mutant, which was minimally above background, consistent with previous results (7, 12). Importantly, phosphorylation of the P446A mutant pep-

SGK1 Modulates Interaction of Nedd4-2 with 14-3-3 To examine directly the dependence of the Nedd4-2/ 14-3-3 interaction on SGK1, we turned to heterolo-

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Fig. 3. Nedd4-2 and 14-3-3 Interaction Is Dependent on Phosphorylation of Ser444 by SGK1 Coimmunoprecipitation of transfected WT FLAG-tagged Nedd4-2 and SGK1 with endogenous 14-3-3 in HEK293 cells, with titration of transfected SGK1 (A), titration of transfected Nedd4-2 (B), or removal of phosphatase inhibitors (C). D, Mutation of Nedd4-2 at Pro446 or Ser444 abrogated the SGK1-dependent interaction of Nedd4-2 with 14-3-3. Kinase-dead (K127M) SGK1 was unable to stimulate interaction. All immunoblots displayed are representative of between two and seven experiments for each condition. E, Scanning densitometry of results from experiments with conditions identical with those depicted in D. Results for 14-3-3 abundance are normalized to the amount of Nedd4-2 immunoprecipitated in the corresponding lane. WCL, Whole cell lysate. *, Statistically significant (P 0.05) compared with Nedd4-2 WT without SGK1 (lane 1).

tide was indistinguishable from that of the WT with either varying concentrations of SGK1 or varying periods of incubation (Fig. 4, B and C). We next demonstrated that GST-14-3-3 interacted with Nedd4-2 in an SGK1-dependent fashion (Fig. 4D). WT or mutant Nedd4-2 was translated in vitro in the presence of [35S]methionine, incubated with purified SGK1 in the presence of (cold) ATP, and then interacted with GST-14-3-3 , chosen as a candidate family member because it is highly expressed in epithelial tissues and cell lines (44). Consistent with the results in cultured cells, binding of either Nedd4-2/P446A or S444A to GST-14-3-3 was markedly lower than that of the WT and was unaffected by SGK1. The dependence of the Nedd4-2/14-3-3 interaction on direct SGK1 phosphorylation was confirmed by performing the assay inversely using GST-fused Nedd4-2 pep-

tides incubated with radiolabeled 14-3-3 shown).

(data not

Functional Role of Nedd4-2/14-3-3 Interaction in Stimulating ENaC-Mediated Na Transport To study the functional role of the Nedd4-2/14-3-3 interaction, we next examined the ability of SGK1 to stimulate ENaC-mediated Na currents in the presence of WT Nedd4-2 or either of the two interactiondeficient mutants. Six Xenopus 14-3-3 isoforms have been identified, at least three of which are expressed in oocytes (45, 46). Hence, we coexpressed ENaC subunits ( , , and ), WT or mutant Nedd4-2, and SGK1 in Xenopus oocytes and used a two-electrode voltage clamp to determine ENaC-mediated (phenamil-sensitive) Na currents. As shown previously, WT

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Fig. 4. SGK1 Phosphorylates WT Nedd4-2 and P446A Mutant, But Not the S444A Mutant, in Vitro A, GST fusion proteins were expressed in E. coli and incubated with purified SGK1. B, In vitro kinase assay with titrated amounts of purified SGK1. SGK1 activity is displayed as counts per minute. C, Similar to B, using 25 ng purified SGK1, and aliquots of each reaction were removed at the incubation times indicated. Kinase assay experiments (B and C) were performed in triplicate. D, 14-3-3 interacts with WT, but not mutant, Nedd4-2 in an SGK1-dependent manner in vitro. 35S-Labeled WT or Nedd4-2 mutant proteins were incubated with equimolar GST or GST-14-3-3 . Input, 10% of radiolabeled WT protein, was directly loaded. The Coomassie Blue-stained gel (bottom panel) shows uniform loading. Data are representative of four independent experiments. *, Statistically significant (P 0.05) lower level of phosphorylation of S444A compared with WT or P446A mutant.

Nedd4-2 markedly inhibited the Na current, an effect that was largely reversed by SGK1 (Fig. 5A). The phosphorylation-deficient S444A Nedd4-2 mutant retained the ability to inhibit ENaC-mediated current, similar to WT; however, SGK1 only weakly stimulated Na current in the presence of this mutant (7). Similarly, the P446A mutant of Nedd4-2 strongly inhibited ENaCmediated current, but stimulation of ENaC currents by SGK1 was markedly blunted. The residual stimulation of Na current by SGK1 in the presence of the P446A and S444A mutants may occur through secondary SGK1 phosphorylation sites as previously described (7), through endogenous Nedd4-2 (10, 14), or through a Nedd4-2-independent effect. To directly test the stimulatory effect of 14-3-3 on ENaC-mediated Na current, we also used the Xenopus oocyte coexpression assay. We measured phenamil-sensitive Na current in oocytes coinjected with

ENaC, Nedd4-2, SGK1, and/or 14-3-3 . ENaC-mediated Na current was modestly, but significantly, increased in the presence of 14-3-3 (Fig. 5B, lane 4). We speculate that the only modest increase observed with 14-3-3 may be due to the abundance of endogenous 14-3-3 in oocytes, or perhaps the isoform, which has not been identified in Xenopus laevis, is not optimal for heterologous expression in oocytes. Patch-clamp recordings of amiloride-sensitive current density (pA/pF) in transfected Chinese hamster ovary cells were consistent with these results (data not shown). 14-3-3 Overexpression Suppresses Nedd4-2Dependent Ubiquitination of ENaC Previous reports have suggested that Nedd4 or Nedd4-2 inhibits ENaC through direct ubiquitination of channel subunits (10, 47, 48). To examine the mech-

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Fig. 5. Interaction of Nedd4-2 and 14-3-3 Augments SGK1-Stimulated Na Transport A, Mutation of the 14-3-3 interaction motif in Nedd4-2 reduces SGK1-mediated ENaC activation in a Xenopus oocyte coexpression assay. Stage V­VI oocytes were injected with cRNA for ENaC subunits, Nedd4-2, and SGK1, as indicated. Phenamil-sensitive Na current was recorded 24 h later by two-electrode voltage clamp at a holding potential of 100 mV. B, 14-3-3 stimulates ENaC-mediated Na current in the presence of SGK1. Currents are expressed compared with ENaC alone. Six oocytes were measured per condition within each experiment. Results show a representative experiment performed three or four times with similar results, using different batches of oocytes. Statistical significance between the different conditions was compared as indicated (*, P 0.01).

anism of inhibition of ENaC-mediated Na current by the Nedd4-2 and 14-3-3 interaction, we assessed the effect of 14-3-3 overexpression on Nedd4-2-mediated ubiquitination of ENaC. 293 cells were cotransfected with the three ENaC subunits, including hemagglutinin (HA)-tagged ENaC, Nedd4-2, SGK1, and 14-3-3 . HA-tagged ENaC was then immunoprecipitated, and immunoblots were stained for endogenous ubiquitin, and ENaC. The antiubiquitin antibody detected a series of bands above 100 kDa, the intensities of which were markedly increased by Nedd4-2 and attenuated by 14-3-3 (Fig. 6A, lanes 5 and 6). Scanning densitometry of blots from three independent experiments showed that 14-3-3 overexpression diminished ubiquitinated ENaC by approximately 70% (Fig. 6B). Increased ubiquitination of ENaC did not appear to alter its total levels, consistent with the idea that Nedd4-2-mediated ubiquitination may modulate channel trafficking without affecting overall expression (10, 47, 48).

14-3-3 Proteins Are Strongly Expressed in Mammalian Cortical Collecting Duct Finally, to determine whether 14-3-3 expression was appropriate for a role in mediating the effects of SGK1 on the collecting duct, we investigated its expression in rat kidney tissue using a pan-14-3-3 antibody that recognizes all seven mammalian isoforms. Interestingly, 14-3-3 immunostaining was not uniform in renal cortex, showing a substantially stronger signal in collecting duct principal cells (identified by costaining with aquaporin 2) than in other nephron segments (Fig. 7). Proximal tubule showed modest staining, whereas glomerular 14-3-3 was nearly undetectable in multiple sections from four different animals. 14-3-3 was also abundantly expressed in medullary collecting duct, but only at modest levels in medullary thick ascending limb of Henle's loop (not shown). These data demonstrate that 14-3-3 is expressed in the appropriate cell type for SGK1- and Nedd4-2-mediated regulation of

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Fig. 6. 14-3-3 Interferes with ENaC Ubiquitination A, HEK293 cells were transfected as indicated. After 24 h, cells were lysed, and immunoprecipitations carried out with anti-HA ( ENaC) antibodies. Immunoprecipitates were analyzed by SDS-PAGE, using antiubiquitin (upper panel) or antiENaC (lower panel) antibodies. Ubiquitinated ENaC is indicated with brackets. *, Heavy and light chains of the immunoprecipitated antibodies. NT, Nontransfected cells. B, Comparison of ubiquitination of ENaC in cells transfected with ENaC, Nedd4-2, and SGK1 with or without 14-3-3. Shown is scanning densitometry from three independent experiments showing decreased ubiquitination of ENaC (normalized to total ENaC) in the presence 14-3-3. *, P 0.04.

docytosis, or both (49), and recent evidence suggests that this effect is due in part to its phosphorylation and inhibition of the ubiquitin ligase, Nedd4-2. There are three SGK1 motifs in Nedd4-2, two of which have been clearly demonstrated to be SGK1 substrates (S338 and S444). Interestingly, one of these (S444), which appears to be the principal mediator of SGK1 inhibition, conforms to the consensus for a 14-3-3 binding motif. Our present data suggest that SGK1mediated phosphorylation of Nedd4-2 at this motif induces its inhibitory interaction with a 14-3-3 protein(s), and moreover, that this inhibition is physiologically important; Nedd4-2 interacts with 14-3-3 proteins in a PI3-kinase/SGK1-dependent fashion; SGK1 stimulation of ENaC currents is markedly blunted in the presence of Nedd4-2 mutants that cannot interact with 14-3-3; overexpression of 14-3-3 augments ENaC-mediated Na current and diminishes Nedd42-mediated ubiquitination of ENaC; and finally, 14-3-3 expression in rat kidney is highest within aldosteronesensitive distal nephron cells. During the preparation of this manuscript, a publication appeared in which Nedd4-2 was identified as a 14-3-3 -interacting partner in a proteomics screen (50) and which made some of the same observations described here. Our present study confirms 14-3-3 interaction and additionally shows that this interaction is physiologically relevant, in that it occurs in a PI3kinase-dependent fashion in cultured distal nephron cells. Moreover, we demonstrate that 14-3-3 proteins modulate Nedd4-2 regulation of Na current and ENaC ubiquitination and show an expression pattern in rat kidney consistent with a role in aldosteroneregulated ion transport. Although both 14-3-3 and 14-3-3 (50) interact with Nedd4-2 in vitro, which one, if either, is important in vivo remains uncertain. The 14-3-3 antibody used in our immunoprecipitation and immunohistochemistry experiments recognizes all seven mammalian isoforms, and it is not possible to conclude decisively at this time which isoform(s) is physiologically the most relevant. With this caveat, it is interesting to note that 14-3-3 is selectively expressed in epithelia and contains a pleckstrin homology domain, which is known to interact with plasma membrane phosphoinositides (51). Mechanism of Nedd4-2 Inhibition by 14-3-3 14-3-3 proteins are highly conserved, are expressed in a variety of eukaryotic cells, and modulate a remarkable variety of cellular processes (52). Most often the target motifs have an essential phosphoserine that, in conjunction with other key residues, including a proline in the 2 position, mediates interaction. The modulation of target protein function is accomplished through several different classes of activities, including displacement of interacting proteins, exposure of nuclear export signals, alteration of active site conformation, and bridging of target proteins to induce interaction (53). Frequently, target proteins have a high

ENaC, interestingly at substantially higher levels than in other regions of the kidney.

DISCUSSION Role of 14-3-3 in SGK1 Inhibition of Nedd4-2 SGK1 mediates the effects of a variety of hormones and cellular signals on ion transport proteins, most notably the stimulatory effect of aldosterone and insulin on ENaC. A large body of evidence supports the idea that SGK1 increases ENaC plasma membrane localization by increasing its exocytosis, inhibiting en-

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Fig. 7. 14-3-3 Proteins Are Strongly Expressed in Mammalian Cortical Collecting Duct A, Kidney sections from rats fed a standard chow diet were harvested and stained with antibodies against 14-3-3 and aquaporin-2 (AQP2). 14-3-3 expression overlaps closely with AQP2 expression in principal cells of the cortical collecting duct (CCD). CCD was also identifiable by tubular epithelial cell morphology and the presence of adjacent glomeruli (G). B, An adjacent section was stained with antibodies incubated with blocking peptide to demonstrate the specificity of the 14-3-3 expression pattern. The red signal indicates 14-3-3 expression, and the green signal indicates AQP2 expression. The scale bar is indicated in micrometers.

affinity gatekeeper site that initiates interaction with a 14-3-3 dimer, followed by interaction with a lower affinity motif (54). With regard to Nedd4-2 regulation, it is notable that the critical SGK1 phosphorylation site (Ser444), which has the characteristics of a 14-3-3 gatekeeper motif, lies between two of the tryptophan-rich WW domains implicated in PY motif interactions. One of these, WW3, is immediately downstream of the gatekeeper motif and has been suggested to be the key site for ENaC interaction and inhibition (55, 56). A second WW domain, WW2, is flanked by the gatekeeper site and two of the potential secondary SGK1-regulated motifs (Ser444 and Thr363). In conjunction with previous observations (7, 12), this raises the possibility that 14-3-3 binds as a dimer to the gatekeeper site surrounding Ser444 and one of the other secondary sites, thereby interfering with Nedd4-2-ENaC interaction by steric hindrance. It is also possible that the Nedd4-2/14-3-3 interaction induces a conformational change that alters WW domain conformation or affects ligase activity by altering HECT domain conformation. It is interesting to speculate that 14-3-3 might act as a bridge to induce interaction with other proteins involved in modulating trafficking behavior. In this regard, recent evidence suggests that SGK1 induces its own Nedd4-2-driven, proteasome-mediated, degradation (57), and that in a different context, SGK1 has been shown to interact with 14-3-3 (58). We speculate that SGK1, after recruitment to the Nedd4-2/143-3 complex, as demonstrated here, phosphorylates Nedd4-2 and induces its interaction with a 14-3-3 homo- and/or heterodimer. This interaction may then block the interaction of Nedd4-2 with ENaC (hence reducing channel ubiquitination) and stabilize its interaction with SGK1 itself (12), possibly through bridging,

hence promoting the ubiquitination of SGK1 and its subsequent proteasome-mediated degradation. We cannot rule out that 14-3-3 proteins may stimulate ENaC-mediated Na transport through alternative pathways as well. 14-3-3 proteins have been reported to interact with PI3-kinase, phosphoinositide-dependent kinase 1, MAPK, and, most recently, Na ,K -adenosine triphosphatase (33, 59­61), which all have been implicated in modulation of the ENaCmediated Na current. Role of Ubiquitination in ENaC Regulation Numerous integral membrane proteins, including ion channels, are regulated through modulation of trafficking, and a growing body of evidence supports the idea that ubiquitin ligases such as Nedd4-2 act at key points in trafficking pathways to alter recycling vs. degradative decisions (62­66). The Nedd4 family of E3 ligases appears to act by ubiquitinating their targets as well as components of the endocytic and sorting machinery, such as endophillin and Hrs (63). Our present data strongly support the idea that Nedd4-2 mediates ubiquitination of ENaC, and that 14-3-3 inhibits Nedd4-2 in an SGK1-dependent fashion; however, the present experiments do not establish the cellular compartment(s) in which Nedd4-2-mediated ENaC ubiquitination occurs. We favor the idea that a substantial portion is occurring at the plasma membrane or early endosome, impacting on endocytosis, sorting, and recycling to the plasma membrane. In immunoblots, some of the ubiquitinated adducts were detected below the size of the major ENaC band. Additional studies will be needed to determine whether the lower molecular weight bands represent ubiquitinated, endopeptidase-modified ENaC (67), unglycosylated

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forms, or degradation products. Importantly for the present context, it is clear that Nedd4-2 increases ENaC ubiquitination, and that this effect is blunted by 14-3-3 overexpression. Physiological Implications of Nedd4-2/14-3-3 Interactions The association of 14-3-3 proteins and the regulation of ENaC and Nedd4-2 impacts on aldosterone-regulated fluid and electrolyte homeostasis and may also impact on other cellular functions. In addition to the distal nephron, ENaC is expressed in a variety of tight epithelia in the lung, salivary ducts, distal colon, eye, and skin. Moreover, two new targets of Nedd4-2-mediated ubiquitination have been identified, Nav 1.5 and N4WBP5A (66, 68), and 14-3-3 proteins may play a role in their regulation. It appears that 14-3-3 modulation of Nedd4-2 and other ubiquitin ligases will be an important general mechanism in the control of ion transporters, and the role of 14-3-3 proteins in Na transport will guide future study of their role in saltsensitive hypertension and edema-forming states.

antibody conjugated to resin (Sigma-Aldrich Corp.), and samples were separated by SDS-PAGE and immunoblotted with anti-FLAG, anti-14-3-3, or anti-SGK1 antibodies. When indicated, phosphatase inhibitors were omitted from the lysis buffer. Scanning densitometry was measured as described above. In Vitro Kinase Assay GST fusion peptides encompassing Ser444 of Xenopus (x) Nedd4-2 were expressed in E. coli and isolated by glutathione-Sepharose 4B beads according to the manufacturer's protocol (Amersham Biosciences). Uniform expression was confirmed by SDS-PAGE and Coomassie Blue staining and was quantified by Bradford assay. Ten micrograms of peptide was used as substrate for a kinase reaction with purified, active SGK1 (Upstate Biotechnology, Inc.) and [ -32P]ATP according to the manufacturer's protocol. The reaction product was then blotted onto P81 phosphocellulose squares (Whatman, Inc., Clifton, NJ), washed, and counted in a scintillation counter. GST Pull-Down Assay Full-length cDNA encoding human (h) 14-3-3 (MGC-8516, IMAGE clone ID 2822397) was obtained from American Type Culture Collection (Manassas, VA) and subcloned into the pGEX-4T1 (Amersham Biosciences) vector using standard techniques. GST fusion proteins were prepared as described above. WT and mutant xNedd4-2 were synthesized in vitro in the presence of [35S]methionine and incubated as described above with 25 ng SGK1 for 60 min. Phosphorylated WT or mutant xNedd4-2 was incubated with 15 mg GST-h14-3-3 (or equimolar amounts of GST alone) and glutathione-Sepharose beads in binding buffer [PBS (pH 7.4), 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, and 1 protease inhibitor mixture] at room temperature for 30 min. Samples were washed in binding buffer and then incubated with 50 l of the kinase reaction described above and 150 l interaction buffer (71) at 4 C overnight. Pelleted beads were then washed three times in interaction buffer, boiled in Laemmli's sample buffer, and separated by SDS-PAGE. The gel was stained with Coomassie Blue as a loading control and dried, and [35S]-labeled proteins were visualized by autoradiography. Construction of Expression Vectors and X. laevis Oocyte Coexpression Assay All cDNAs were subcloned into the in vitro expression vector pSDEasy or pMO. Capped cRNAs were synthesized as described previously (3). Xenopus -, -, and ENaC subunit cRNAs were coinjected (3 ng each) into stage V­VI oocytes together with xNedd4-2 or mutant cRNA (0.6­1.5 ng), xSGK1 cRNA (5 ng), h14-3-3 cRNA (20 ng), or diethylpyrocarbonate-treated water as a control. After injection, the oocytes were incubated in a low-Na Barth's solution, and current measurements were performed as described previously (3). The macroscopic phenamil-sensitive current was defined as the difference between currents obtained in the presence (2 M) and the absence of phenamil. In Vivo Ubiquitination Assay HEK293 cells, grown on 75-cm2 dishes, were transfected by the calcium phosphate technique with 4.5 g of the plasmids encoding differentially tagged rat ENaC subunits (HA-tagged ENaC and ENaC, and FLAG-tagged ENaC) (47, 72), 2 g hNedd4-2 (68), 1 g mSGK1 S422D, and 13.5 g h14-3-3 . Twenty-four hours after transfection, cells were dissociated

MATERIALS AND METHODS

Coimmunoprecipitation in mpkCCDc14 Cells Murine cortical collecting duct (mpkCCDc14) cells were grown in defined medium, and electrical properties were measured as described previously (37, 40). Cells were pretreated for 30 min with 50 M LY294002 (Calbiochem, La Jolla, CA) or vehicle (dimethylsulfoxide) before addition of 10 7 M dexamethasone (Sigma-Aldrich Corp., St. Louis, MO) and 100 nM insulin (Sigma-Aldrich Corp.) or vehicle (ethanol). After 3 h, cells were lysed in 900 l lysis buffer (69) containing protease inhibitors [1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM, benzamidine, and 1 Complete Protease Inhibitor Cocktail (Roche, Indianapolis, IN)] and phosphatase inhibitors [25 mM sodium fluoride, 2 M microcystin-LR, and 1 Phosphatase-Inhibitor Cocktail Sets I and II (Calbiochem)]. Immunoprecipitation was performed overnight with anti-Nedd4 antibody (Upstate Biotechnology, Inc., Lake Placid, NY) and protein A-agarose. Samples were separated by SDS-PAGE and immunoblotted with anti-Nedd4-2 antibody (69) or anti-14-3-3 (K-19) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), which recognizes all seven mammalian isoforms (70). Densitometry was measured using ImageQuant TL software (Amersham Biosciences, Arlington Heights, IL), and values for immunoprecipitated 14-3-3 were normalized to Nedd4-2 within the same lane. Coimmunoprecipitation in HEK293 Cells HEK293 cell cultures were maintained at 37 C in culture medium containing DMEM, 10% fetal bovine serum, and 1% penicillin/streptomycin. Cells were transfected with FLAGtagged Xenopus Nedd4-2, mouse SGK1, and/or empty vector using Lipofectamine 2000 (Invitrogen Life Technologies, Inc., Gaithersburg, MD). Twenty-four hours after transfection, cells were harvested in 500 l lysis buffer [50 mM Tris-HCl (pH 7.5), 120 mM NaCl, and 1% Nonidet P-40] containing protease inhibitors and phosphatase inhibitors as described above. Immunoprecipitation was performed with anti-FLAG

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Bhalla et al. · SGK1 Inhibits Nedd4-2 through 14-3-3

in 1 ml dissociation buffer [5% glycerol, 1 mM EDTA, and 1 mM EGTA (pH 8.0)]. Cells were recovered with 10 ml PBS and centrifuged at 4 C, 1500 g, for 2 min. Cell pellets were frozen at 70 C. Pellets were then resuspended in 1 ml lysis buffer [50 mM HEPES (pH 8.0), 150 mM NaCl, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1 mM dithiothreitol, 100 mM sodium fluoride, 10 mM disodium pyrophosphate, 1 mM PMSF, and 1 protease mixture], and lysates were centrifuged for 15 min at 21,500 g (4 C). Supernatants were recovered, and immunoprecipitations were carried out with monoclonal anti-HA antibodies (Santa Cruz Biotechnology, Inc.) and protein G-Sepharose. Samples were analyzed by 5­15% (ubiquitin-blot) or 8% (HA-blot) SDS-PAGE and immunoblotted with monoclonal antiubiquitin antibodies (FK2, Affinity, Nottingham, UK) or polyclonal anti-rat ENaC antibodies (73). Quantification of the signals on fluorograms was performed using a molecular imager FX (Bio-Rad Laboratories, Hercules, CA), and the results were normalized to the control values. Immunofluorescence Fifty- to 75-gm Sprague-Dawley male rats (Harlan Laboratories, Indianapolis, IN) were maintained on a regular chow diet with normal Na content. Rats were anesthetized by ip injection with pentobarbital (40­60 mg/kg) according to the standard protocol and perfused with chilled 0.9% NaCl, followed by chilled PBS (pH 7.4). Kidneys were removed, bisected sagittally, and incubated in 4% paraformaldehyde at 4 C overnight, then rinsed several times with PBS, embedded in paraffin, sliced into 4- to 5- m sections, and mounted on glass slides. Slides were washed in PBS for 5 min to remove their coverslips and incubated in blocking solution [10% donkey serum (Chemicon International, Temecula, CA) and 0.1% BSA in PBS] for 1 h at room temperature. Sections were then incubated with primary antibody [rabbit anti-14-3-3 and goat anti-aquaporin 2 (Santa Cruz Biotechnology, Inc.)] or antibody incubated with 5 (wt/vol) 14-3-3 blocking peptide (Santa Cruz Biotechnology, Inc.) overnight at 4 C. Sections were then rinsed three times with PBS and overlaid with the appropriate secondary antibodies (Texas Red-conjugated donkey anti-rabbit and fluorescein isothiocyanate-conjugated donkey anti-goat IgG, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Slides were rinsed in PBS and mounted with VectaMount (Vector Laboratories, Inc., Burlingame, CA). Images were obtained at 400 magnification using an Axioscope epifluorescence microscope (Carl Zeiss, Inc., New York, NY) and MetaMorph imaging software (Molecualr Devices, Sunnyvale, CA). Statistical Analysis

SEM. Unless otherwise Data are presented as the mean specified, all statistical comparisons were performed using Student's unpaired t test, and significance was defined as P 0.05.

Received May 13, 2005. Accepted August 2, 2005. Address all correspondence and requests for reprints to: Dr. David Pearce, 600 16th Street, N272 Genentech Hall, Mission Bay, Box 2140, University of California, San Francisco, California 94107-214. E-mail: [email protected] ucsf.edu. This work was supported by grants from the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (F32-DK-066968 and R01-DK5­ 6695), and the Swiss National Science Foundation (3100A0-103779).

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Experimental Animal Safety All animal experimentation described in this manuscript was conducted in accord with accepted standards of humane animal care and was approved by the committee on animal research at University of California-San Francisco.

Acknowledgments

We thank Martin McMahon, Anthony Lau, Jeffrey Lau, and Fred Cohen for helpful discussions, and Bernard Rossier and Laurent Schild for ENaC plasmids.

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