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【摘要】 The epithelial Na + channel (ENaC) is regulated by the ubiquitin-protein ligase Nedd4-2 via interaction with ENaC PY-motifs. These PY-motifs are mutated/deleted in Liddle's syndrome, resulting in elevated Na + reabsorption and hypertension explained partly by impaired ENaC-Nedd4-2 interaction. We hypothesized that Nedd4-2 is a susceptibility gene for hypertension and screened 856 renal patients and healthy controls for mutations in a subset of exons of the human Nedd4-2 gene that are relevant for ENaC regulation by PCR/single-strand conformational polymorphism. Several variants were identified, and one nonsynonymous mutation (Nedd4-2-P355L) was further characterized. This mutation next to the 3' donor site of exon 15 does not affect in vitro splicing of Nedd4-2 mRNA. However, in the Xenopus oocyte expression system, Nedd4-2-P355L-dependent ENaC inhibition was weaker compared with the wild type (Nedd4-2-WT), and this difference depended on the presence of intact PY-motifs on ENaC. This could not be explained by the amount of wild type or mutant Nedd4-2 coimmunoprecipitating with ENaC. When the phosphorylation level of human Nedd4-2 Ser 448 (known to be phosphorylated by the Sgk1 kinase) was determined with a specific anti-pSer 448 antibody, we observed stronger basal phosphorylation of Nedd4-2-P355L. Both the phosphorylation level and the accompanying amiloride-sensitive Na + currents could be further enhanced to approximately the same levels by coexpressing Sgk1. In addition, the role of the two other putative Sgk1 phosphorylation sites (S342 and T367) appears also to be affected by the P355L mutation. The differential phosphorylation status between wild-type and mutant Nedd4-2 provides an explanation for the different potential to inhibit ENaC activity.
【关键词】 genetic variation sodium ion homeostasis ubiquitination phosphorylation
THE EPITHELIAL NA + CHANNEL (ENaC), composed of three homologous subunits (,, and ), plays a fundamental role in the control of the whole body Na + balance and consequently regulation of blood volume and pressure ( 26, 52 ). It is expressed in the apical membrane of segment-specific cells of the aldosterone-sensitive distal nephron ( 30 ), and facilitates the entry of Na + in the cell. The regulation of ENaC is complex and involves the action of various hormones, including aldosterone, vasopressin, or insulin. The cell surface expression of ENaC appears to be tightly regulated, both by clathrin-dependant endocytosis ( 43, 48 ) and by ubiquitination via the ubiquitin-protein ligase Nedd4 (neuronal precursor cell expressed developmentally downregulated; also referred to as Nedd4-1; see Refs. 1 and 15 ), its closely related isoform Nedd4-2 ( 12, 18, 23, 25, 49 ), and/or WWP2 (WW-domain containing protein; see Ref. 35 ). Ubiquitination (also referred to as ubiquitylation) is a posttranslational modification in which ubiquitin, a polypeptide of 76 amino acids, is covalently linked to the -NH 2 group of lysines in a target protein. This process involves an enzymatic cascade including the ubiquitin-activating enzyme E1, ubiquitin-conjugating (or ubiquitin-carrier) enzyme E2, and ubiquitin-protein ligase E3, the latter being involved in substrate recognition ( 14 ). In the case of ENaC, Nedd4-1, Nedd4-2, or WWP2 bind via WW-domains to the COOH-terminal PY-motifs on the ENaC subunits and reduce in a ubiquitination-dependent manner the cell surface expression of ENaC ( 1, 9, 15, 35, 50 ). Indeed ENaC is known to be ubiquitinated on its - and -subunit and has been shown to be regulated via ubiquitination ( 51 ). Interestingly, the PY-motif of either - or -ENaC is deleted/mutated in most forms of Liddle's syndrome, an inherited form of human hypertension ( 16, 17, 44 ). This disease is characterized by the early onset of salt-sensitive hypertension, hypokalemia, metabolic alkalosis, and low circulating aldosterone and renin levels. It is thought that this is the result of elevated ENaC activity, most likely because of reduced Nedd4-2/ENaC interaction, ENaC ubiquitination, and internalization ( 50, 51 ). Recently, Sgk1 (serum and glucocorticoid-induced kinase 1) has gained considerable interest with respect to ENaC regulation ( 24 ), since Sgk1 is one of the earliest proteins that are induced by aldosterone. It was also found to stimulate ENaC activity ( 6, 36 ) by increasing ENaC cell surface expression ( 3, 31 ) in Xenopus laevis oocytes and to promote transepithelial Na + transport in renal epithelial cells ( 2, 11, 20 ); a Sgk1 knockout model shows defects in the proper handling of the Na + balance when exposed to a low-Na + diet ( 54 ). It was recently proposed that Nedd4-2 is a potential target of Sgk1, since it contains two or three putative phosphorylation sites for Sgk1 phosphorylation. Indeed, when Sgk1 is coexpressed with Nedd4-2 in X. laevis oocytes, it is able to phosphorylate X. laevis Nedd4-2 on Ser 444 (corresponding to Ser 448 in human Nedd4-2) and, though to a lesser extent, on S338 (S342 in human). Such phosphorylation interferes with ENaC interaction, hence with ubiquitination and internalization, and may therefore represent one of the mechanisms by which Sgk1 controls cell surface expression of ENaC ( 7, 47, 49 ).
Although Nedd4-2 (and Nedd4-1 or WWP2) are potential susceptibility genes for hypertensive or hypotensive disorders, no genetic linkage has been established so far. Nedd4-2 is encoded on chromosome 18q21, GenBank Locus ID 23327, a gene that spans 350 kb and comprises 34 exons (Ref. 10 and Fig. 1 ). There is suggestive linkage between this chromosomal region and several hypo- or hypertensive pathologies, including essential hypertension ( 4, 29 ), postural change in systolic blood pressure ( 39 ), and orthostatic hypotensive disorder ( 8 ).
Fig. 1. Schematic view of the human (h) neuronal precursor cell expressed developmentally downregulated (Nedd4) 2 gene, hNedd4-2c, and hNedd4-2a (KIAA0439) mRNA. Indicated are the predicted exons spanning a region of 350 kb (chromosomal region 18q21), the putative initiation codons, and the stop codon. The various domains are indicated with different patterns.
In this study, we have searched for naturally occurring human Nedd4-2 variants in both controls and patients. Among several variants (see Table 2 ), we identified a nonsynonymous mutation changing proline-355 to leucine (Nedd4-2-P355L). We have characterized this mutant in X. laevis oocytes and found that it less potently inhibits ENaC compared with wild-type Nedd4-2. This difference is most likely related to altered phosphorylation of Nedd4-2, expected to interfere with the ENaC-Nedd4-2 interaction.
Table 2. Nedd mutations in healthy subjects and subjects with renal disease
METHODS
Subjects. The subjects investigated have been described previously ( 32, 37, 38, 55 ). Briefly, the study comprised 362 Caucasian patients with end-stage renal disease (ESRD; 102 on dialysis and 260 after kidney transplantation) from our Division of Nephrology and Hypertension of the University Hospital in Bern, Switzerland, and 494 subjects without renal disorders. More than 70% of the patients with ESRD suffered from arterial hypertension. From the 494 subjects without renal diseases, 193 had hypertension, 42 patients had diabetes, and 259 were healthy controls. The female-to-male ratio of the subjects studied was 45 to 55 and the age ranged from 17 to 92 yr. Genetic analysis of the Nedd4-2 gene was performed in all 856 subjects. All participants gave informed consent to the study, which was approved by the local ethical committee of the University Hospital of Berne.
DNA preparation and PCR analysis. Genomic DNA was isolated from peripheral blood using the Nucleon BACC3 DNA extraction kit (Amersham International, Buckinghamshire, UK). Mutation detection in the indicated exons of the Nedd4-2 gene was performed by single-strand conformational polymorphism (SSCP) of PCR products from exons 15, 16, 18, 19, 20, 21, 22, and 33, using the primers indicated in Table 1. All reactions were performed with 0.4 µM of each primer in a final volume of 25 µl containing 2 mM MgCl 2, 0.5 mM of each dNTP, and 0.5 units of Ampli Taq Gold polymerase (PE Biosystems, Foster City, CA). The DNA was amplified for 35 cycles with denaturation at 94°C for 20 s, annealing from 64 to 70°C for 30 s, and extension at 72°C for 45 s. PCR products were analyzed on 12% acrylamide gels containing 7.25% glycerol using a two-buffer system ( Fig. 2 A ). PCR sample buffer (4 µl) was loaded, and DNA was visualized by silver staining. Sequence changes were detected by double-band shifts on the gel. Variants were purified using a Qiaquick PCR purification column (Qiagen, Chatsworth, CA) according to the supplier's recommendations and sequenced by Microsynth (Balgach, Switzerland) using the same primers as for PCR ( Fig. 2 C ). The obtained sequences were compared with the Nedd4-2 gene sequence (GenBank no. AC011331 ). Identified variants were further analyzed by restriction digestion of PCR products according to standard methods ( Fig. 2 B ).
Table 1. Sequences of primers for amplification of hNedd4-2 gene
Fig. 2. Identification of the P355L mutation in the Nedd4-2 gene. A : amplified PCR fragment from exon 15 was analyzed by single-strand conformational polymorphism (SSCP). Shown is a silver-stained acrylamide gel that displays control (Wt) and heterozygote mutant (Mut) DNA. B : amplified DNA was digested with Hpa II or Mse I and analyzed by SSCP. Arrows, heterozygote mutated DNA with slightly slower migration. C : automated sequencing of the amplified PCR product indicating the heterozygote mutation that changes proline-355 to leucine.
cDNA constructs. Human Nedd4-2c cDNA (GenBank no. AY312514; see Ref. 34 ) was cloned into the Not I/ Eco R V site of pSDeasySB ( 41 ). The human Nedd4-2a (KIAA0439) construct was described previously ( 25 ). Mutations were created by site-directed mutagenesis (Quickchange; Stratagene), and each mutant was verified by sequencing (Synergene Biotech, Schlieren, Switzerland). Human Sgk1 construct was prepared from an EST clone (BM460788 ), and a myc -tag (AEEQKLISEEDLLRKRREQLKHKLEQLRNSCA) was added at the NH 2 -terminus. The cDNA was cloned into Xba I/ Eco R V sites of pSDeasySB. -, -, and -subunits of human ENaC were provided by Dr. Pascal Barbry (Valbonne, France). The PY-motifs were mutated using site-directed mutagenesis, by changing tyrosine to alanine in the PY-motifs of,, and. A minigene human Nedd4-2 construct, containing exons 14-16 from either control or affected subjects, was made by PCR on genomic DNA using primes SPL-FWD and SPL-RWD ( Table 1 ). After TA cloning (TOPO cloning kit; Invitrogen, Basel, Switzerland) and sequencing to identify the wild-type and mutant cDNA, the fragments were cut and cloned into Xho I/ Eco R V of pcDNA3.1(-).
In vitro splicing assay. The Nedd4-2 minigene construct was transiently transfected into SW620 cells (human, Caucasian, colon, adenocarcinoma, ATCC CCL 227) using the FuGENE transfection reagent (Roche). After 48 h incubation, total RNA was isolated using the RNAeasy kit (Qiagen) and reverse transcribed using Superscript III RT (Invitrogen, Basel, Switzerland). Reversed transcription was followed by PCR using EX-SP-FWD/EX-SP-RWD primers. The amplified products were analyzed on agarose gels.
Expression in X. oocytes and electrophysiological measurements. Plasmid encoding ENaC, Sgk1, and Nedd4-2 proteins was linearized and in vitro transcribed, the cRNA was injected in X. laevis oocytes, and electrophysiological measurements were carried out after overnight incubation, as described previously ( 42 ). The following quantities of cRNA were injected: 3 ng for each ENaC subunit; 10 ng Sgk1 and Nedd4-2 as indicated legends for Figs. 1 - 11.
Fig. 11. Differential effect of Sgk1 on either N4-2 3 P (Nedd4-2-S342A-T367A-S448A)- or N4-2m 3 P-dependent regulation of ENaC. Oocytes were coinjected with cRNA encoding ENaC (3 ng·subunit -1 ·oocyte -1 ), either N4-2 3 P or N4-2m 3 P (3 ng/oocyte) with or without Sgk1 (10 ng/oocyte). Amiloride-sensitive Na + currents were measured and normalized to those of control oocytes (injected with ENaC alone: ENaC + H 2 O). ** P < 0.001, N4-2 WT vs. N4-2m (N4-2P355L) in the absence of Sgk1; n = 24 oocytes from 4 animals/condition. NS, not significantly different between N4-2 3 P or N4-2m 3 P with or without Sgk1 and between N4-2WT vs. N4-2m when Sgk1 was expressed.
Generation and purification of anti-Nedd4-2 phosphopeptide antibodies. The anti-Nedd4-2 phosphopeptide antibody was raised in rabbits using a keyhole limpet hemocyanin-coupled, synthetic phosphopetide (KPRSL-S Phos -SPTV) corresponding to amino acids 324-332 of mouse Nedd4-2 ( 25 ) as immunogen. The antiserum was further affinity-purified against the immunogenic peptide coupled to Actigel ALD (Sterogene, Carlsbad, CA) according to the manufacture's instructions. The resin was then packed in a Poly-Prep Chromatography Column (Bio-Rad) and washed with 10 bed volumes of PBS. The remainder of the procedure was performed at 4°C. Serum was loaded on the column two times, the column was washed with 10 bed volumes of PBS, and the antibodies were eluted with 10 bed volumes of 0.1 M glycine, pH 2.5, directly on a tube containing 1 bed volume of 2 M Tris·HCl, pH 8.0. The eluate was dialyzed overnight in PBS and subsequently concentrated in the dialysis tube with polyethylene glycol 20.000 (Sigma) until the volume was reduced to the initial volume of serum loaded on the column. The eluate was recovered, and albumin was added to a final 1% concentration. Before use, the antibody was mixed 1:1 (vol/vol) to a peptide resin slur containing the nonphosphorylated immunogenic peptide and rotated for 90 min at 4°C to eliminate antibodies that recognize nonphosphorylated mNedd4-2. The supernatant is the purified anti-Nedd4-2 phosphopeptide antibody.
Biochemical analysis. Western blot analyses of X. laevis lysates were carried out as described previously ( 23 ), using either anti-c- myc (Santa Cruz Biotechnology), anti-Nedd4-2 ( 25 ), or anti-Nedd4-2 phosphopeptide antibody. Lysis of oocytes was carried out by washing oocytes one time with modified Barth's solution ( 42 ), followed by extraction in 25 µl/oocyte of Triton X-100 homogenization buffer (20 mM Tris·HCl, pH 7.4, 100 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 10 µg/ml aprotinin), and centrifuged for 10 min at 20,000 g (4°C). In the phosphorylation experiment ( Fig. 8 ), one-half of the lysates was treated with 1/10 vol 2 M Tris·HCl, pH 9.0, and 10 µl calf intestinal alkaline phosphatase (CIP; Roche) and incubated 3 h at 37°C. The enzyme was inactivated by adding 5 x sample buffer and heating for 5 min at 95°C. Coimmunoprecipitation analysis was performed as described by Kamynina et al. ( 23 ).
Fig. 8. Phosphorylation of Ser 448 differs between Nedd4-2-WT and Nedd4-2-P355L. A : oocytes were injected with cRNA encoding wild-type Nedd4-2 (N4-2) or Nedd4-2-P355L (N4-2m; 5 ng/oocyte) with and without serum- and glucocorticoid-induced kinase 1 (Sgk1; 10 ng); 10% of the lysate of 20 oocytes was analyzed by Western blot using an anti-Nedd4-2-pSer448, Nedd4-2, myc (Sgk1), or actin antibody. In lanes 1-6, lysates were treated with calf intestinal alkaline phosphatase (CIP). B : Nedd4-2-pSer448 and Nedd4-2 bands were quantified from 3 different experiments and are displayed as Nedd4-2-pSer448-to-CIP-treated Nedd4-2 ratio; n = 3 experiments/data point. * P < 0.05 between N4-2 and N4-2m. n.I., noninjected oocytes.
Quantification of gels and statistical analysis. Fluorograms of Western blots were scanned using an Epson 1680 Pro scanner and quantified using the B10-1D software (Vilbert Lourmat). Data are expressed as mean values ± SE and were analyzed by two-tailed Student's t -test for unpaired data.
RESULTS
Identification of naturally occurring human Nedd4-2 mutants. To search for genetic variants in the human Nedd4-2 gene, we analyzed 856 subjects with and without hypertension by PCR/SSCP ( Fig. 2 ). As outlined above, the Nedd4-2 gene is complex and consists of 350 kb (illustrated in Fig. 1 and described in Refs. 5, 10, and 22 ). Therefore, we focused in the present study on exons 15, 16, 18, 19, 20, 21, and 22, which encode regions of Nedd4-2 essential for the regulation of ENaC, including WW domains 3 and 4 that interact with ENaC ( 23 ) and two phosphorylation sites shown previously to be phosphorylated by Sgk1 ( 7 ). Whereas exons 15 and 18-22 were analyzed in all 856 subjects, exons 16 and 33, which account either for the putative phosphorylation site Thr 367 or the catalytic HECT domain, respectively, were screened in 200 patients with ESRD. We found five variants ( Table 2 ), two of which were nonsynonymous, one synonymous, and two located in introns. One of the nonsynonymous mutations was located in exon 19 and changed Ser 497 to Arg in WW domain 3 ( Table 2 ); the subject carrying this mutation was a healthy control without hypertension. The other mutation, located in exon 15, changed proline-355 to leucine ( Fig. 2 and Table 2 ). The two nonrelated patients carrying this mutation suffered from ESRD and were subjected to renal transplantation. The underlying renal diseases in these two patients were autosomal dominant polycystic kidney disease and Senior-Loken syndrome (juvenile nephronophthisis). We further characterized the mutation Nedd4-2-P355L observed in these two patients.
Human Nedd4-2-WT and human Nedd4-2-P355L RNA are spliced in a similar fashion. The mutation P355L is localized very close to the 3'-donor site of exon 15 and may therefore potentially influence the splicing at this site ( Fig. 3 ). We therefore investigated this possibility by generating a construct in pcDNA3.1 that contained the genomic sequence, including exons 14-16, in the wild-type or mutant form ( Fig. 3 A ). These constructs were transfected in SW620 cells. After 2 days, cells were lysed, RNA was extracted, and RT-PCR was carried out using two primers (EX-SP-FWD and EX-SP-RWD) covering the cloned sequence ( Fig. 3 A ). Whereas PCR of the plasmid yielded a fragment of 5108 bp ( Fig. 3 B, lanes 2 and 3 ), one of 302 bp (spliced transcript) was amplified by RT-PCR after transfection in the SW620 cells ( lanes 4 and 5 ), indicating that both constructs were properly spliced.
Fig. 3. Splicing between exon 15 and 16 is equivalent between Nedd4-2-wild type (WT) and Nedd4-2-P355L. A : scheme of utilized construct. B : genomic DNA fragment comprising exons 14-16, either wild type or mutated, was transiently transfected in SW620 cells (colon cell line). After 48 h incubation, RNA was extracted from cells, and RT-PCR was carried out using EX-SP-FWD and EX-SP-RWD primers. As a control, PCR was also carried out directly on the plasmid DNA. Amplified DNA was separated on an agarose gel and visualized with ethidium bromide and ultraviolet light.
Human Nedd4-2-P355L is less efficient than wild-type human Nedd4-2 in downregulating ENaC in X. laevis oocytes. One of the best characterized functions of Nedd4-2 is the regulation of ENaC via WW-domain-PY-motif interaction when coexpressed in X. laevis oocytes. This prompted us to investigate if the P355L mutation affected the ability of Nedd4-2 to control ENaC activity. We expressed wild-type or mutant Nedd4-2 together with human ENaC in Xenopus oocytes. In most experiments, a novel form of Nedd4-2, Nedd4-2c, was used, which we recently cloned from a human kidney cDNA library (GenBank accession no. AY312514; see Ref. 34 ). Nedd4-2c comprises a complete C2-domain resulting from alternative splicing at the 5'-end and exon 16, which is spliced out in some of the previously described forms of human Nedd4-2 (e.g., KIAA0439; see Ref. 25 ). This exon encodes a third putative phosphorylation site for Sgk1 (T367), described previously by Snyder and collaborators ( 47 ). We injected increasing amounts of Nedd4-2-WT or Nedd4-2-P355L (Nedd4-2m) cRNA together with ENaC cRNA in X. laevis oocytes and monitored amiloride-sensitive Na + currents by the two-electrode voltage-clamp method, a measure of ENaC activity ( Fig. 4 C ). In agreement with our previous reports ( 25 ), increasing amounts of Nedd4-2-WT lead to diminished 95% inhibition at maximal Nedd4-2 cRNA levels. In contrast, Nedd4-2-P355L consistently inhibited ENaC to a lesser extent. Similar expression of Nedd4-2-WT and Nedd4-2-P355L proteins was verified by Western blotting the lysates of the same oocytes using an anti Nedd4-2 antibody ( Fig. 4 A and Ref. 25 ) and quantification of the detected Nedd4-2 protein on the blot ( Fig. 4 B ). To further support the notion that Nedd4-2-P355L inhibited ENaC less efficiently, we looked at the mean absolute currents of 86 different experiments that we carried out under various conditions [rat or human (h) ENaC, hNedd4-2a or hNedd4-2c, different concentrations of injected Nedd4-2 cRNA]. As can be seen in Fig. 4, D and E, this large number of experiments confirmed that the Nedd4-2 mutant had a smaller impact on ENaC than the wild-type protein. When various Nedd4-2-WT-to-Nedd4-2-P355L ratios were injected with ENaC, we observed that increasing proportions of Nedd4-2-P355L yielded higher ENaC currents, corroborating the idea that this mutant is a weaker inhibitor of ENaC compared with wild-type Nedd4-2 ( Fig. 5 ).
Fig. 4. Nedd4-2-P355L is less efficient in regulating ENaC than wild-type Nedd4-2 in Xenopus laevis oocytes. Oocytes were injected with cRNA encoding ENaC (3 ng·subunit -1 ·oocyte -1 ) and the indicated amounts of Nedd4-2 cRNA and incubated overnight at 19°C. They were analyzed the next day. A : top, immunoblot analysis using an anti-Nedd4-2 antibody indicating levels of expression of either hNedd4-2-WT (N4-2) or hNedd4-2-P355L (N4-2m) with increasing amounts of Nedd4-2 cRNA injected (ng/oocyte indicated). Bottom, actin. B : quantification of the Nedd4-2-to-actin ratio ( n = 3 individual experiments). C : corresponding normalized amiloride-sensitive Na + currents. Currents were normalized to control oocytes (0 ng Nedd4-2 or Nedd4-2m). Open circles (thin line), Nedd4-2; triangles (bold line), Nedd4-2m. * P < 0.05 and ** P < 0.001, Nedd4-2m vs. Nedd4-2; n = 30 oocytes from 3 animals for each data point. D : absolute mean currents of 86 experiments. Each experiment comprises 6 oocytes/condition. Pooled are experiments using wild-type (Nedd4-2) and mutant (Nedd4-2m) forms of hNedd4-2a or hNedd4-2c, with various concentrations. Experiments used either rat or human epithelial Na + channel (ENaC). E : summary of the data in D. * P < 0.05 between wild-type and mutant Nedd4-2; n = 86 different experiments.
Fig. 5. Regulatory influence of Nedd4-2-P355L over Nedd4-2-WT action on ENaC. Various ratios of cRNA encoding Nedd4-2-P355L (m) and Nedd4-2-WT (w) were injected in oocytes, and amiloride-sensitive Na + currents were measured. Currents are normalized to those of control oocytes injected with cRNA encoding ENaC only. Each oocyte was injected with a total of 3 ng Nedd4-2 cRNA and with cRNA encoding ENaC (3 ng·subunit -1 ·oocyte -1 ), and the ratios (w/m) are indicated. * P < 0.05 and ** P < 0.001 vs. Nedd4-2-WT (6w); n = 36 oocytes from 6 animals/data point.
It is well established that Nedd4-2 exerts its effect on ENaC via WW domain-PY-motif interaction ( 19, 23, 46, 50 ). To ensure that the difference between Nedd4-2-WT and Nedd4-2-P355L is also depending on this interaction, we expressed ENaC channels with all PY-motifs disrupted by mutating the tyrosine residues to alanine. As expected and reported previously ( 1 ), such channels displayed much higher activities compared with wild-type channels ( Fig. 6, compare ENaC with ENaC- PY), likely because of the loss of all binding sites for endogenous Nedd4-2. When Nedd4-2-WT or Nedd4-2-P355L (Nedd4-2m) was coexpressed with wild-type ENaC, the activity was significantly decreased, with Nedd4-2-WT being more efficient in inhibiting ENaC activity than Nedd4-2-P355L, as observed before. However, no effect either by Nedd4-2 or by Nedd4-2-P355L was observed with ENaC- PY, suggesting that both forms of Nedd4-2 regulate ENaC via WW-domain-PY-motif interaction ( Fig. 6 ).
Fig. 6. Differential inhibition of Nedd4-2-WT or Nedd4-2-P355L depends on integrity of ENaC PY-motifs. Oocytes were coinjected with 3 ng each of cRNA encoding the ENaC subunits, either wild-type ENaC (ENaC) or PY-motif mutated (ENaC PY), and 5 ng Nedd4-2-WT (N4-2) or Nedd4-2-P355L (N4-2m). Amiloride-sensitive Na + currents were measured and normalized to the control condition (ENaC). * P < 0.05 between ENaC + N4-2 and ENaC + N4-2m; n = 24 oocytes from 4 animals/data point. N.S., not significant between ENaC- PY, ENaC- PY + N4-2, and ENaC- PY + N4-2m.
Both Nedd4-2-WT and Nedd4-2-P355L interact with ENaC in X. laevis oocytes. The reduced effect of Nedd4-2-P355L on ENaC activity could be the result of either 1 ) reduced enzymatic Nedd4-2 activity or 2 ) reduced interaction with ENaC. To date, no quantitative enzymatic assay is available for Nedd4/Nedd4-like ubiquitin-protein ligases. We therefore investigated if the interaction between ENaC and Nedd4-2 is changed in this mutant. We carried out coimmunoprecipitation experiments between ENaC and either Nedd4-2-WT or Nedd4-2-P355L in X. laevis oocytes, as described previously ( 7, 23 ). We coexpressed Flag-tagged rat ENaC subunits with human Nedd4-2 (wild type or mutant), metabolically labeled the oocytes overnight with [ 35 S]methionine, solubilized a membrane fraction, and precipitated the ENaC subunits with anti-Flag antibodies. The precipitated proteins were visualized by SDS-PAGE autoradiography. As shown in Fig. 7, the ENaC subunits were precipitated each time ENaC was expressed ( Fig. 7 ). When either Nedd4-2-WT or Nedd4-2-P355L was added, a supplementary band at 120 kDa was coimmunoprecipitated, corresponding to the predicted molecular weight of Nedd4-2 ( Fig. 7 ), whereas no Nedd4-2 band was observed when ENaC was omitted. We observed no obvious difference in the amount of coimmunoprecipitated hNedd4-2-WT or hNedd4-2-P355L, suggesting that both forms of Nedd4-2 bind ENaC with comparable apparent affinity.
Fig. 7. Nedd4-2-WT and Nedd4-2-P355L coimmunoprecipitate equally well with ENaC. Oocytes were injected with cRNA encoding FLAG-tagged rat ENaC (3 ng·subunit -1 ·oocyte -1 ), together with Nedd4-2 or Nedd4-2-P355L (Nedd4-2m; 6 ng/oocyte) or as a control without FLAG-tagged rat ENaC. Top : after overnight incubation with [ 35 S]methionine, membrane fractions were solubilized and immunoprecipitated (IP) with anti-FLAG (ENaC) antibodies. Immunoprecipitated material was analyzed by SDS-PAGE and autoradiography ( top ). ENaC subunits and Nedd4-2 are indicated. Middle : immunoblot (IB) of membrane fraction using an anti-Nedd4-2 antibody. Bottom : immunoblot with anti-actin antibody. Position of Nedd4-2, ENaC subunits are indicated. NI, noninjected oocytes; 40 oocytes have been used for each condition.
The level of Ser 448 phosphorylation is different between Nedd4-2-WT and Nedd4-2-P355L. We and others have shown that the aldosterone-induced Sgk1 kinase increases ENaC activity by phosphorylating Nedd4-2 on two sites, primarily on Ser 444 and less efficiently on Ser 338 in X. laevis Nedd4-2 (corresponding to Ser 448 and Ser 342 in human Nedd4-2; see Refs. 7 and 47 ), causing reduced interaction between ENaC and Nedd4-2. In addition, a third putative Sgk1-dependent phosphorylation site (Thr 367 ) has been described by Snyder et al. ( 47 ). We therefore wished to know if the level of phosphorylation induced by Sgk1 varied between wild-type and mutant Nedd4-2. Because we had previously shown that Sgk1 phosphorylates primarily Nedd4-2 on Ser 448, we focused on this residue and raised a phosphopeptide-specific antibody against this site. Figure 8 A shows that this antibody is specific for phosphorylated Nedd4-2, since treatment with CIP completely removed the signal for Nedd4-2, whereas another anti Nedd4-2 antibody ( 25 ) still recognized the protein ( Fig. 8 A, compare lanes 2-5 with 8-11 ). The Nedd4-2 bands were quantified in three different experiments, and the ratio between pSer 448 -Nedd4-2 and total Nedd4-2 was calculated ( Fig. 8 B ). Thereby, we normalized against the CIP-treated total Nedd4-2 pool ( lanes 1-6, second panel), since our Nedd4-2 antibody appears to recognize the phosphorylated Nedd4-2 (in the presence of Sgk1) less well ( Fig. 8 A, second panel, compare Nedd4-2 in presence or absence of Sgk1 in -CIP; lanes 8-11 ). Remarkably, when Sgk1 was absent, Nedd4-2-P355L (Nedd4-2m) was stronger phosphorylated than Nedd4-2-WT (compare lane 8 with 9 in Fig. 8 A and quantification in Fig. 8 B ), which likely is explained by an endogenous Sgk-like activity in X. laevis oocytes. When we coexpressed Sgk1, the phosph-Ser 448 signal was increased for Nedd4-2-WT by 4.5-fold, whereas the signal for Nedd4-2-P355L increased only 2-fold ( Fig. 8 A and quantification in 8 B ), yielding the same phosphorylation levels as Nedd4-2-WT. The observed difference in basal phosphorylation may explain why the Nedd4-2 mutant is less efficient than Nedd4-2 wild type in inhibiting ENaC activity, since such phosphorylation is expected to interfere with the Nedd4-2-ENaC interaction. In parallel, we studied the functional effect of Sgk1 on ENaC activity in oocytes expressing either wild-type or mutant Nedd4-2 together with ENaC by measuring amiloride-sensitive Na + currents ( Fig. 9 ). As can be seen in Fig. 9 A, Sgk1 increased, whereas Nedd4-2-WT and (to a lesser extent) Nedd4-2-P355L (Nedd4-2m) decreased, ENaC activity. When Sgk1 was coexpressed with Nedd4-2 or Nedd4-2-P355L, the inhibition was reversed, and stimulation of ENaC was observed, as reported previously ( 7 ). However, compatible with the phosphorylation experiments in Fig. 8, Sgk1 increased the currents in the presence of Nedd4-2-WT by nearly 20-fold ( Fig. 9, A and B ), whereas it affected the current with Nedd4-2-P355L less strongly (7.5-fold), yielding currents that were not distinguishable between wild-type and mutant Nedd4-2 ( Fig. 9 ). If differential level of Ser 448 phosphorylation is the explanation for the difference in ENaC inhibition between Nedd4-2 and Nedd4-2-P355L, one may expect that mutation of Ser 448 will abolish such dissimilarity. Consequently, we mutated this site to alanine both in wild-type and mutant Nedd4-2 and monitored ENaC activity. We verified that both proteins were expressed at the same level ( Fig. 10, A and B ). As predicted, the difference between Nedd4-2 and Nedd4-2-P355L was smaller in the S448A background ( Fig. 10 C ). Surprisingly, we now found an inverse relationship, i.e., Nedd4-2-P355L-S448A was slightly more potent in ENaC inhibition than Nedd4-2-S448A ( Fig. 10 C ). Consistent with the important role of S448 in Sgk1 phosphorylation, coexpression of Sgk1 displayed a weaker increase of the ENaC currents compared with the condition in which S448 was not mutated ( Fig. 10 D; compare columns N4-2/N4-2m ± Sgk1 with N4-2 S448A/N4-2m S448A ± Sgk1).
Fig. 9. Differential effect of Sgk1 on either wild-type or mutant Nedd4-2-dependent regulation of ENaC. A : oocytes were coinjected with cRNA encoding ENaC (3 ng·subunit -1 ·oocyte -1 ), Nedd4-2-WT (N4-2), or Nedd4-2-P355L (N4-2m; 5 ng/oocyte), with or without Sgk1 (10 ng/oocyte). Amiloride-sensitive Na + currents were measured and normalized to those of control oocytes (injected with ENaC alone: ENaC + H 2 O). * P < 0.01, Nedd4-2-WT vs. Nedd4-2-P355L in the absence of Sgk1; n = 24 oocytes from 4 animals/data point. NS, not significantly different between N4-2 vs. N4-2m when Sgk1 was coexpressed. B : ratios of currents from experiment in A, between conditions either expressing or not expressing Sgk1 (ENaC alone, ENaC + Nedd4-2, or ENaC + Nedd4-2m).
Fig. 10. N4-2 S/A (Nedd4-2-S448A) is less efficient in regulating ENaC than N4-2m S/A (Nedd4-2 P355L-S448A) in X. laevis oocytes. Oocytes were coinjected with cRNA encoding ENaC (3 ng·subunit -1 ·oocyte -1 ) and the indicated amounts of Nedd4-2 S/A cRNA or Nedd4-2m S/A and incubated overnight at 19°C. They were analyzed the next day. A : top, immunoblot analysis using an anti-Nedd4-2 antibody, indicating levels of expression of either hNedd4-2 S/A or hNedd4-2 P355L S/A with increasing amounts of different mutant Nedd4-2 cRNA injected (ng/oocyte as indicated). Bottom, actin. B : quantification of the different Nedd4-2 mutation-to-actin ratios ( n = 3 individual experiments). C : corresponding normalized amiloride-sensitive Na + currents. Currents were normalized to control oocytes (0 ng N4-2 S/A or N4-2m S/A). Thin line, Nedd4-2 S/A; bold line, Nedd4-2m S/A. * P < 0.05 and ** P < 0.001, N4-2m S/A vs. N4-2 S/A; n = 30 oocytes from 3 animals for each data point. D : differential effect of Sgk1 on either N4-2 S/A- or N4-2m S/A-dependent regulation of ENaC. Oocytes were coinjected with cRNA encoding ENaC (3 ng/subunit) and either N4-2 S/A or N4-2m S/A (3 ng/oocyte) with or without Sgk1 (10 ng/oocyte). Amiloride-sensitive Na + currents were measured and normalized to those of control oocytes (injected with ENaC alone: ENaC + H 2 O). * P < 0.05, N4-2 S/A vs. N4-2m S/A and ** P < 0.001, N4-2 vs. N4-2m in the absence of Sgk1; n = 24 oocytes from 4 animals/condition. NS, not significantly different between N4-2 vs. N4-2m when Sgk1 was expressed.
P355L affects other putative Sgk1 phosphorylation sites as well. The observation that mutation of S448 did not completely abolish but rather inversed the difference between wild type and Nedd4-2-P355L suggested that this mutation also affects other regions in Nedd4-2. Because P355 lies between the two other putative Sgk1 phosphorylation sites, S342 and T367, we tested if the mutation also had an impact on these sites and inactivated them together with S448A either in the wild-type or in the P355L background (Nedd4-2 P or Nedd4-2m P). As before, the effect of these mutations on ENaC currents was measured in oocytes. When Sgk1 was coexpressed with such Nedd4-2 mutants, no stimulation of ENaC activity was observed, consistent with the idea that Sgk1 stimulates ENaC via phosphorylation of Nedd4-2 on the three putative phosphorylation sites (S342, T367, and S448; Fig. 11, compare columns N42 ± Sgk1 with N42 P ± Sgk1). Moreover, in this background, the mutation P355L did not affect the ability of Nedd4-2 to regulate ENaC currents (compare columns N4-2 P with N4-2m P), suggesting that this mutation indeed also affects the other putative Sgk1 phosphorylation sites.
DISCUSSION
Our data reveal the existence of a naturally occurring human Nedd4-2 variant (P355L) that affects the property to regulate the epithelial Na + channel ENaC when expressed in X. laevis oocytes. This mutant, found in 2 out of 856 subjects, inhibited ENaC less efficiently compared with wild-type Nedd4-2. This difference can be explained by the fact that, in Nedd4-2-P355L, Ser 448 is stronger phosphorylated by an endogenous kinase and therefore expected to display weaker interaction with ENaC. In addition, this mutation also has an impact on the other Sgk1-dependant phosphorylation sites, S342 and T367.
One of the major difficulties when one analyzes the Nedd4-2 gene is its complexity. As shown in Fig. 1 and reported previously ( 5, 10, 22 ), the gene contains 34 exons spanning over a region of 350 kb. The gene encodes a large number of alternative transcripts, varying not only at the 5'-end but also generating proteins that either do or do not include the C2 domains ( 5, 10, 22 ). Other splice variants vary within the protein and either lack WW domains or a putative phosphorylation site for Sgk1 ( 12, 25, 47 ). We therefore limited our search for mutations in the regions with potential relevance for the regulation of ENaC, i.e., the WW domains 3 and 4, shown to interact with ENaC ( 23 ), or the regions close to or including the Sgk1-dependent phosphorylation sites ( 7 ). These regions are encoded by exons 15, 18, 19, 20, 21, and 22. Genomic DNA of some patients was also screened for the catalytic HECT domain (exon 33) or in exon 16 of Nedd4-2. Overall, we have identified five variants, two of which were nonsynonymous changes, one synonymous, and two mutations located in introns (see Table 2 ). This number of variants seems to be low compared with the study by Dunn et al. ( 10 ) in which 38 variants were found in 48 Caucasians. However, their mutations were mostly located either in 5'-noncoding regions or within introns.
Here, we have focused on the properties of Nedd4-2-P355L, one of the two nonsynonymous mutants, and analyzed it with respect to ENaC regulation in X. laevis oocytes. The oocyte system has proven to be a powerful and reliable tool for the study of ion channels and plasma membrane proteins in general (for a review, see Ref. 45 ). Our analysis showed that the Nedd4-2 mutant was less efficient in downregulating ENaC activity, which we tend to explain by stronger phosphorylation of Ser 448 in the mutant. We do not know the nature of the endogenous kinase that is involved in this phosphorylation, but it may involve Sgk1, or one of its isoforms, Sgk2 and -3, since they all have similar consensus sequences for phosphorylation ( 27, 28, 40 ) and are activated by the phosphatidylinositol 3-kinase pathway, known to sustain ENaC activity in X. laevis oocytes ( 53 ). Moreover, both Sgk2 and -3 have been shown to increase ENaC activity in X. laevis oocytes ( 13 ). Notably, the two other putative Sgk1-dependent phosphorylation sites (S342 and T367), which are situated in close proximity to P355, appear to be affected as well by the P355L mutation, although to a lesser extent. The observation that Nedd4-2-P355L-S448A is more efficient in ENaC inhibition than Nedd4-2-S448A, and the fact that additional mutation of S342 and T367 eliminates this difference, suggests that one or both of these sites are less phosphorylated in the presence of P355L. This remains to be investigated further.
In our previous studies, we found that Sgk1-dependent phosphorylation of S448 impairs the interaction of ENaC with Nedd4-2 ( 7 ). Although Nedd4-2-P355L is stronger phosphorylated on Ser 448 than wild-type Nedd4-2, we are not able to detect any differences between mutant and wild-type Nedd4-2 in our coimmunoprecipitation experiments ( Fig. 7 ). This might be explained by the small change in impact on ENaC activity and phosphorylation state of S448 that we observed between wild-type and P355L mutant. Alternatively, we cannot exclude that the enzymatic activity of Nedd4-2 is also affected by phosphorylation.
It may come as a surprise that the mutation of Pro 355 to leucine does affect the phosphorylation level of Ser 448 to such an extent. However, mutations involving a proline may significantly change the overall conformation of the enzyme. Moreover, we do not know how Sgk1 is interacting with Nedd4-2. This may be via PY-motif-WW domain interaction, as proposed previously ( 7, 47 ), or by other mechanisms ( 21 ) and may be affected by this mutation.
What is the biological relevance of such a Nedd4-2 mutation? The two patients with the Nedd4-2-P355L mutation could not be investigated with respect to abnormal renal sodium and potassium handling and blood pressure-regulating hormones. Both patients had been on dialysis and had been transplanted before the mutations were found. The underlying clinical entities in the native kidneys, autosomal polycystic kidney disease and juvenile nephronophthisis (Senior-Loken syndrome), affect by an inherited mechanism the tubular interstitial area of the kidney. These are two complex renal diseases without apparent link to dysfunction of ENaC. Moreover, it has to be noted that this is a relatively rare mutation that, when expressed in X. laevis oocytes, displays subtle effects on Nedd4-2 function (i.e., weaker downregulation of ENaC resulting from elevated phosphorylation level of Ser 448 ). Admittedly, these differences may be inherent to the oocyte system and will have to be demonstrated in an epithelium. Therefore, it is too early to speculate about a pathophysiological role for this mutation, and the potential role for the induction of or predisposition to hypertension remains to be established.
In summary, we have shown that the naturally occurring P355L mutant of human Nedd4-2 inhibits ENaC less efficiently in X. laevis oocytes. This appears to be caused by enhanced phosphorylation on Ser 448 by an endogenous yet undefined kinase expected to interfere with ENaC-Nedd4-2 interaction. The function of the other putative Sgk1-dependent phosphorylation sites appears also to be affected. To our knowledge, these data represent the first report on functional aspects of a human Nedd4-2 variant and help to elucidate the structure-function relationship of the concerted action of Nedd4-2, ENaC, and Sgk1.
GRANTS
This work was supported by Swiss National Science Foundation grants to O. Staub (31-64052.00), F. J. Frey and B. M. Frey (31-61505.00), and J. Loffing (32-061742.00), Leenaards Foundation (O. Staub), the Roche Research Foundation (O. Staub), and the EMDO foundation (J. Loffing).
ACKNOWLEDGMENTS
We thank Dr. Pascal Barbry for providing human ENaC cDNA. We are grateful to Drs. Laurent Schild, Dmitri Firsov, Hugues Abriel, Phil Shaw, Jean-Daniel Horisberger, and Laura Stanasila for critically reading the manuscript.
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作者单位：1 Division of Nephrology and Hypertension, Department of Clinical Research, University of Bern, CH-3010 Bern; 2 Department of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland; and 3 Faculty of Medicine X. Bichat, Institut National de la Santé et de la Recherc