1Division of Digestive Diseases, Departments of Medicine and Physiology, Emory University School of Medicine, Atlanta, Georgia; and 2Department of Physiology, University of Tubingen, Germany
Submitted 6 December 2004 ; accepted in final form 6 May 2005
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ABSTRACT |
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intestinal epithelial cells; Na absorption; phosphorylation
In this study, we report that NHE3 is directly phosphorylated by SGK1 at Ser663. Mutation of Ser663 blocks stimulation of NHE3 by dexamethasone, suggesting that SGK1-dependent phosphorylation of NHE3 is a key step in glucocorticoid-induced activation of NHE3.
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MATERIALS AND METHODS |
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Cell culture and transfection. Intestinal epithelial cells (IEC6) (35) were cultured at 37°C in 95% air-5% CO2 in Dulbeccos modified Eagles medium (DMEM) supplemented with 5% fetal bovine serum, 0.1 U/ml insulin, 50 µg/ml streptomycin, and 50 U/ml penicillin. For transfection, cells grown to 70% confluence in 10 cm dishes were incubated for 16 h with 15 µg plasmid DNA and 40 µl lipofectamine 2000 (Invitrogen). IEC6 cells expressing NHE3 (IEC6/NHE3) were selected by resistance to G418 (400 µg/ml) and acid-loading in the presence of either 10 µM dimethyl amiloride or 30 µM HOE-694 (48). IEC6/NHE3 cells transfected with SGK1 or SGK1/K127M were selected by resistance to hygromycin (300 U/ml). Before being assayed, cells were rendered quiescent by serum starvation in DMEM for 1824 h and treated with 1 µM dexamethasone or ethanol for 4 or 24 h. For all the studies, transfected cells were used within 10 passages.
Western immunoblot analysis. Cells were lysed in a lysis buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.1 mM PMSF, 5 mg/ml aprotinin, 1 mM pepstatin, 1 mM iodoacetamide, 5 µg/ml leupeptin, and 1% Triton X-100), followed by centrifugation at 15,000 g at 4°C for 30 min as described in our previous study (47). Lysates were resolved by 10% SDS-PAGE, and Western immunoblot analysis was performed as previously described (25, 47). The anti-NHERF2 antibody, Ab2570, was previously described (46, 47). The HA-antibody was obtained from Covance. The anti-OK NHE3 antibody 5683 was provided by Dr. Orson Moe at the University of Texas Southwestern Medical Center (14).
In vitro phosphorylation assay.
Cells expressing NHE3 were lysed in the lysis buffer supplemented with phosphatase inhibitors composed of (in mM) 1 EGTA, 1 EDTA, 1 Na orthovanadate, 10 Na fluoride, 10 Na pyrophosphate, and 25 -glycerophosphate, as described above. NHE3 was purified by being immobilized on Ni-NTA resins (Qiagen). The immobilized NHE3 was incubated with 25 ng of the constitutively active SGK1 (Upstate) in 20 mM Tris, pH 7.4, 10 mM MgCl2, 5 µM ATP, 2 µM PKA inhibitor, 1 mM DTT, and 10 µCi [
-32P]ATP. In other experiments, the recombinant proteins corresponding to the carboxyl terminal (CT) domain of NHE3 were used as substrates (47). The phosphorylated samples were resolved by SDS-PAGE, followed by transfer onto nitrocellulose membrane. Phosphorylation levels were quantified by a densitometric analysis using the Typhoon phosphoimager (Amersham). To normalize the levels of phosphorylation to the amount of proteins, the amounts of NHE3 immunoprecipitated was determined by Western immunoblot using the anti-NHE3 antiserum 5683.
Site-directed mutagenesis. Site-directed mutagenesis was performed using the QuickChange site-directed mutagenesis kit according to the recommendation by the manufacturer (Stratagene). The presence of mutation was confirmed by nucleotide sequencing.
RT-PCR. Total RNA was prepared from IEC6 cells with the use of TRIzol (Life Technologies). Adult male Sprague-Dawley rat was anesthetized with pentobarbital sodium and total TNA was extracted from whole kidney homogenate with the use of TRIzol. Five micrograms of total RNA were used for the subsequent synthesis of cDNA using the First Strand Synthesis kit, as recommended by the manufacturer (Life Technology). For detection of NHE isoforms, the following primer pairs were used (8): NHE1: TCTGCCGTCTCAACTGTCTCTA and CCCTTCAACTCCTCATTCACCA; NHE2: GCAGATGGTAATAGCAGCGA and CCTTGGTGGGGGCTTGGGTG; and NHE3: GGAACAGAGGCGGAGGAGCAT and GAAGTTGTGTGCCAGATTCTC. Each reaction was performed from 1 µl of cDNA. Amplification was performed for 40 cycles at 94°C/60 s, 60°C/45 s, and 72°C/60 s.
In vitro back-phosphorylation assay.
An in vitro back-phosphorylation was performed as previously described to access dexamethasone-induced phosphorylation of NHE3 (53). IEC6 cells transfected with control NHE3 or NHE3/S663A were treated with dexamethasone for 024 h. Cell lysates were prepared and NHE3 was affinity purified as indicated above using Ni-NTA resins. The purified NHE3 was subjected to in vitro phosphorylation as described above using the purified SGK1 and [-32P]ATP, followed by SDS-PAGE and autoradiography.
Na+-dependent intracellular pH recovery. The Na+-dependent changes in intracellular pH (pHi) by NHE3 was determined with the use of the ratio-fluorometric, pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) as previously described (25). Briefly, cells were seeded on coverslips, grown to confluence, and then serum starved overnight. Cells were washed in Na buffer composed of (in mM) 130 NaCl, 20 HEPES, 5 KCl, 1 tetramethylammonium-PO4 (TMA-PO4), 2 CaCl2, 1 MgSO4, and 18 glucose, and then dye-loaded by incubation for 40 min with 6.5 µM BCECF-AM in the same solution. The coverslips were mounted on a perfusion chamber mounted on an inverted microscope, and were superfused with NH4 buffer (40 mM NH4Cl, 90 mM NaCl, 20 mM HEPES, 5 mM KCl, 1 mM TMA-PO4, 2 mM CaCl2, 1 mM MgSO4, and 18 mM glucose) and subsequently with TMA buffer (130 mM TMA-Cl, 20 mM HEPES, 5 mM KCl, 1 mM TMA-PO4, 2 mM CaCl2, 1 mM MgSO4, and 18 mM glucose). Na buffer containing 30 µM HOE-694 was then reintroduced to drive Na-dependent pH recovery. Calibration of the fluorescence signal was performed using the K+/H+ ionophore nigericin as described previously (25). The microfluorometry was performed on a Nikon TE200 inverted microscope with a Nikon CFI Super Fluor x40 objective, coupled to a Lambda 102 filter wheel controller equipped with a multiwavelength filter set designed for BCECF. Photometric data was acquired using the Metafluor software (Universal Imaging). Na+/H+ exchange rate was described by the rate of pHi recovery, which was calculated by determining slopes along the pHi recovery by linear least-squares analysis over a minimum of 9 s.
Surface biotinylation. Surface biotinylation of NHE3 was performed as previously described (1, 21). Briefly, cells grown in 10-cm petri dishes were rinsed three times in PBS, followed by borate buffer composed of (in mM) 154 NaCl, 7.2 KCl, 1.8 CaCl2, and 10 H3BO3, pH 9.0. Hereafter all of the procedures were performed at 4°C. Cells were then incubated for 40 min with 1.5 mg of NHS-SS-biotin (Pierce, Rockford, IL) in borate buffer. Unbound NHS-SS-biotin was quenched with Tris buffer (20 mM Tris, pH 7.4, 120 mM NaCl). Cells were then rinsed with PBS, scraped, solubilized in 1 ml of the lysis buffer described above, and sonicated for 20 s. The lysate was agitated for 30 min and spun to remove insoluble cell debris. An aliquot was retained as the total fraction representing the total cellular NHE3 antigen. The remainder was incubated with streptavidin-agarose (Pierce) for 1 h. The strepavidin-agarose beads were washed with the lysis buffer, and this represented the surface NHE3. Dilutions of the total and surface NHE3 were resolved by SDS-PAGE, immunoblotted with anti-NHE3 antibody, followed by densitometric analysis to quantify the amounts of the total and surface NHE3.
Statistics. Densitometric analyses were performed on the Typhoon phosphoimager (Amersham) using the ImageQuant program. Statistical significance was assessed by ANOVA. Results were considered statistically significant when P < 0.05. If not otherwise specified, results are presented as the means ± SE.
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RESULTS |
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Phosphorylation at S663 of NHE3 is necessary for dexamethasone-induced activation. We next studied whether the S663A mutation affects the activation of NHE3 by dexamethasone. In IEC6 cells, dexamethasone treatment for 2 h had no apparent effect on NHE3 activity (Fig. 4A). However, consistent with previous studies in Caco-2 cells (41), the NHE3 activity in IEC6 cells was significantly stimulated after 4 h or 24 h with dexamethasone. The mutation of S663A in NHE3 did not affect the basal Na+/H+ exchange activity of NHE3 indicating that this mutation did not alter the carrier activity or trafficking of NHE3 under the basal conditions. However, we found that dexamethasone for 24 h (Fig. 4B) or 4 h (data not shown) did not activate the transport activity in the presence of the S663A mutation. To show that the lack of response to dexamethasone is specific to the S663A mutation, we determined the effect of dexamethasone on NHE3/S607A. As noted earlier, Ser607 of rabbit NHE3 (identical to Ser605 in rat NHE3) is a phosphorylation site by PKA and the mutation at Ser607 renders NHE3 unresponsive to PKA (24, 52). Unlike the S663A mutant, it is shown in Fig. 3C that the S607A mutation did not affect SGK1-mediated phosphorylation of NHE3 protein. In accord with the in vitro phosphorylation, NHE3/S607A was stimulated to the same extent by dexamethasone as the control NHE3. These results strongly suggested that dexamethasone activated NHE3 via a change in phosphorylation of NHE3 at S663.
To determine whether dexamethasone-dependent induction of NHE3 activity correlates with phosphorylation of NHE3, we determined the time course of dexamethasone-induced phosphorylation of NHE3. Previous studies (53) have shown that NHE3 exhibits a high level of basal phosphorylation in vivo making it difficult to resolve the change in phosphocontent of NHE3. To overcome this problem, we employed an in vitro back-phosphorylation assay to determine the phosphorylation states of NHE3 in response to dexamethasone (53). In this technique, endogenous phosphates were incorporated into NHE3 during the dexamethasone treatments and the phosphorylation sites cannot be phosphorylated by SGK1 under in vitro conditions. On the other hand, in untreated cells, these sites are not occupied by unlabeled phosphate residues and these sites should be available to SGK1 to phosphorylate in vitro. Thus a decrease in back-phosphorylation reflects an increase in endogenous phosphorylation. Cells were treated with dexamethasone for the indicated times and NHE3 proteins were isolated under conditions preventing dephosphorylation. The purified NHE3 was then incubated with purified SGK1 and [-32P]ATP to incorporate radioactive phosphate into sites that were not phosphorylated in situ. Figure 5A shows the levels of back-phosphorylation of NHE3 purified from IEC6 cells that were treated with dexamethasone for different time durations. Normalization of the levels of back-phosphorylation to the amount of NHE3 proteins (Fig. 5A) revealed that dexamethasone significantly blocked subsequent back-phosphorylation of NHE3 by SGK1, indicating that NHE3 was phosphorylated in situ during the dexamethasone treatment. The phosphorylation occurred at as early as 2 h, at which time NHE3 activity was not increased in response to dexamethasone. In contrast to the control NHE3, the phosphorylation level of NHE3/S663A (Fig. 5B) was low even in the absence of dexamethasone, consistent with the earlier results. More importantly, the phosphorylation levels of NHE3/S663A were not changed throughout the dexamethasone treatment. Taken together, these data demonstrate that dexamethasone induces phosphorylation of NHE3 at S663A and this change in phosphorylation precedes the increase in NHE3 activity.
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DISCUSSION |
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In this work, we used IEC6 cells as a cell model system to study the regulation of NHE3. IEC6 cells have an epitheloid origin from rat small intestine (35). Previous studies (34, 35) showed that IEC6 cells retained some characteristics of normal intestinal cells, but exhibit a flattened appearance and the lack of immunological determinants specific for differentiated villus cells. Consistently, IEC6 cells lacked the expression of NHE3, but expressed NHERF1 (data not shown) and NHERF2, two major scaffold proteins present in intestinal villus cells. To our knowledge, the regulation of NHE3 activity has not been studied in IEC6 cells. However, previous studies using non-epithelial PS120 and AP1 cells, epithelial Caco-2 and OK cells, or intestinal brush border vesicles have shown remarkable similarity in the regulation of NHE3, although some difference in NHE3 regulation has also been noted (13, 14, 21, 52). Our previous studies showed that SGK1 mediated the dexamethasone-mediated activation of NHE3 in both OK cells and PS120 fibroblasts, and this activation was facilitated by the presence of NHERF2. We demonstrated the presence of NHERF2 in IEC6 cells and the stimulation of NHE3 activity by dexamethasone. In addition, NHE3 was acutely inhibited by Ca2+ ionophore, further demonstrating the validity of IEC6 cells as a cell culture model system to study the regulation of NHE3. However, it is conceivable that there could be subtle difference in the regulation of NHE3 in IEC6 cells compared with normal polarized epithelium.
SGK1 was first shown to stimulate sodium reabsorption in aldosterone-responsive segments of the outer medulla of the kidney by specifically targeting the epithelial Na channel (ENaC) (11, 29, 33). It has been suggested that SGK1 regulates ENaC via phosphorylation of Nedd42 that affects binding of Nedd42 to the PY-motif of ENaC (15, 38, 39). Although direct phosphorylation of ENaC is not yet demonstrated, a mutation of the SGK1 consensus motif in the -subunit of ENaC abolished the stimulatory effect of SGK1, suggesting a potential role of direct phosphorylation by SGK1 (17). Similarly, our data suggest that mutation of the SGK1 consensus motif at Ser663 abolished the dexamethasone-induced activation of NHE3-mediated transport. However, it seems unlikely that SGK1 affects the interaction of NHE3 via Nedd4 or Nedd42 because NHE3, with an exception of NHE3 from human, lacks a PY motif (20), and the importance of the PY motif in human NHE3 is yet to be proven.
The substrate specificity of SGK1 is similar to that of Akt1 and the putative phosphorylation site is determined to be RxRxxS/T based on studies using random peptides (22, 32). On the basis of this, some substrates that contain this motif have been identified (10, 12, 36, 51). The presence of a putative SGK1 phosphorylation site in NHE3 strongly suggests a role of phosphorylation in NHE3 activation. Our results indicated that S663 in NHE3 is the major site of phosphorylation by SGK1. The Ser residue is strictly conserved in all NHE3 proteins from multiple species and the mutation to Ala attenuated the extent of phosphorylation, The RxRxxS/T motif encompasses the consensus phosphorylation site, RxxS/T, by PKA, which also regulates NHE3 activity. PKA-mediated signaling results in inhibition of NHE3 and two independent studies identified Ser552 (identical to Ser555 in rabbit NHE3 or Ser560 on OK NHE3) and Ser605 (identical to Ser607 in rabbit NHE3 or Ser613 in OK NHE3) (24, 52). However, our data showed that the S607A mutation in rabbit NHE3 did not reduce the SGK1-mediated phosphorylation of NHE3 and did not affect the activation of NHE3 activity by dexamethasone. In the case of renal outer medullary K+ channel (ROMK), it has recently been shown that phosphorylation at Ser44 led to a drastic reduction in K+ current and surface expression of the ROMK protein in Xenopus oocytes (44). Unlike ROMK, the S663A mutation in NHE3 did not alter basal Na+/H+ exchange activity, suggesting the probable differences in the underlying mechanisms regulating NHE3 and ROMK by SGK1.
SGK1 has now been implicated in the activation of several channels and transporters, including the renal outer medullary K+ channel ROMK, the cystic fibrosis transmembrane conductance regulator, Na+ dicarboxylate cotransporter NaDCT-1, Na+-K+-2Cl cotransporter, Na+-K+-ATPase, and NHE3 (7, 11, 31, 37, 40, 44, 46). In some cases, translocation of channels or transporters to the plasma membrane mediated by SGK1 has been suggested as a possible mechanism, but how SGK1 may mediate translocation of protein(s) is yet to be elucidated. Our data showed that the increase in NHE3 phosphorylation precedes the stimulation of NHE3 activity after a treatment with dexamethasone. For instance, at 2 h with dexamethasone treatment, NHE3-mediated transport activity was not affected despite the increase in NHE3 phosphorylation (Figs. 4A and 5A). Phosphorylation of NHE3 occurring before the activation of NHE3 activity is consistent with our conclusion that phosphorylation at S663 of NHE3 is a key event in NHE3 regulation by dexamethasone. The present study showed that dexamethasone induced translocation of NHE3 into the plasma membrane, but not NHE3/S663A, suggesting that phosphorylation at S663 is closely associated with the translocation of NHE3 protein into the plasma membrane. Our preliminary studies indicate the effect of dexamethasone on NHE3 can partially be disrupted by nocodazole, indicating a microtubule-dependent mechanism (D. Wang and C. C. Yun, unpublished data). Furthermore, there was a substantial decrease in NHE3 phosphorylation at 24 h compared with 12 h, presenting the possibility that NHE3 might be subjected to dephosphorylation once inserted or translocated to the plasma membrane. Whether the phosphorylation at S663 triggers the translocation by enhancing the association with the trafficking assembly remains an intriguing prospect.
As we suggested earlier (46), the glucocorticoid-mediated activation of NHE3 consists of both genomic and non-genomic regulation. This is based on, in addition to the known transcriptional induction of NHE3 and SGK1, the difference in the magnitude of activation in cells endogenously expressing NHE3 compared with the heterologous expression of NHE3. For instance, in PS120 fibroblasts and IEC6 cells transfected with NHE3, the augmentation in NHE3 activity ranges 3050% compared with 100% or more in Caco-2 or OK cells with endogenous NHE3 expression (3, 46). We attributed this difference to the presence or the lack of transcriptional induction of NHE3 mRNA in the respective cell lines. If the role of SGK1 is to phosphorylate NHE3, it seems paradoxical why a minimum of 34 h is required for NHE3 activity to be stimulated. One possibility is that the limiting step might be the induction of SGK1. On the basis of previous studies (9, 30), it is anticipated that dexamethasone initiates transcriptional, translational, and posttranslational modification of SGK1, resulting in eventual phosphorylation of NHE3 at S663. However, SGK1 mRNA level is rapidly induced by dexamethasone although the rate of posttranscriptional modification is not clear (2, 9, 30). An alternative mechanism may involve induction of other regulatory or adaptor proteins by dexamethasone. These regulatory proteins may interact with NHE3 in a phosphorylation-dependent manner and this interaction is necessary for translocation of NHE3 to the surface. Additional studies are needed to elucidate this hypothesis.
In summary, our work demonstrated that SGK1 specifically phosphorylates NHE3 at Ser663 in response to dexamethasone and mutation of S663 abolishes the stimulatory effect of dexamethasone on NHE3 transport activity. The stimulation of NHE3 activity was accompanied by increased NHE3 protein abundance at the plasma membrane. These findings suggest that SGK1 plays a pivotal role in activation of NHE3 in response to dexamethasone by phosphorylating NHE3 that stimulates insertion NHE3 protein into the plasma membrane.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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