Acute Inhibition of Na/H Exchanger NHE-3 by cAMP
ROLE OF PROTEIN KINASE A AND NHE-3 PHOSPHOSERINES 552 AND 605*

Hui ZhaoDagger , Michael R. WiederkehrDagger , Lingzhi FanDagger , Roberto L. CollazoDagger §, Ladonna A. CrowderDagger , and Orson W. MoeDagger parallel

From The Department of Internal Medicine,  Department of Veterans Affairs Medical Center and Dagger  University of Texas Southwestern Medical Center, Dallas, Texas 75235

    ABSTRACT
Top
Abstract
Introduction
References

Regulation of the renal Na/H exchanger NHE-3 by protein kinase A (PKA) is a key intermediate step in the hormonal regulation of acid-base and salt balance. We studied the role of NHE-3 phosphorylation in this process in NHE-deficient AP-1 cells transfected with NHE-3 and in OKP cells expressing native NHE-3. A dominant-negative PKA-regulatory subunit completely abolished the effect of cAMP on NHE-3 activity demonstrating a role of PKA in the functional regulation of NHE-3 by cAMP. NHE-3 isolated from cAMP-treated cells showed lower phosphorylation by purified PKA in vitro suggesting that NHE-3 is a PKA substrate in vivo. Although changes in NHE-3 whole protein phosphorylation is difficult to detect in response to cAMP addition, the tryptic phosphopeptide map of in vivo phosphorylated NHE-3 showed a complex pattern of constitutive and cAMP-induced phosphopeptides. To test the causal relationship between phosphorylation and activity, we mutated eight serines in the cytoplasmic domain to glycine or alanine. Single or multiple mutants harboring S552A or S605G showed no PKA activation or reduced regulation by PKA activation. Ser-552 and Ser-605 were phosphorylated in vivo. However, multiple mutations of serines other than Ser-552 or Ser-605 also reduced the functional PKA regulation. We conclude that regulation of NHE-3 by PKA in vivo involves complex mechanisms, which include phosphorylation of Ser-552 and Ser-605.

    INTRODUCTION
Top
Abstract
Introduction
References

Mammalian plasma membrane Na/H exchangers (NHEs)1 use downhill inward Na+ gradients to extrude H+ from cells. Six genetic NHE isoforms have been identified with specific pharmacologic characteristics and tissue distributions (reviewed in Ref. 1). NHE-3 is limited to transporting epithelia such as the kidney and gastrointestinal tract (2, 3). In the kidney, NHE-3 is expressed exclusively on the apical membrane of the proximal tubule and the thick ascending limb where it mediates absorption of a significant fraction of the filtered NaCl and NaHCO3 (4-6). NHE-3 plays a critical role in renal regulation of extracellular fluid volume and acid-base balance (7). Acute hormonal regulation of NHE-3 involves multiple intracellular cascades including activation of adenylyl cyclase (1). The NHE-3 isoform when expressed in fibroblasts is inhibited by cAMP analogues or forskolin (8-10). Although the NHE-1 isoform is cAMP insensitive, chimeric insertion of the NHE-3 cytoplasmic domain confers cAMP sensitivity to the NHE-1 transporting domain (11). Partial deletions of the cytoplasmic domain of NHE-3 reduce although more drastic truncations abolish the effect of cAMP on NHE-3 activity (10, 11). The NHE-3 cytoplasmic domain is a substrate for protein kinase A (PKA) in vitro (10) and NHE-3 phosphorylation is increased by cAMP or forskolin addition in vivo (10, 12). Kurashima and co-workers (12) showed that Ser-605 and Ser-634 are both important for forskolin to inhibit NHE-3 activity, although only Ser-605 is phosphorylated in vivo. This study shows that PKA directly phosphorylates NHE-3 and inhibits its activity via complex mechanisms. Phosphorylation of Ser-552 and Ser-605 were increased by cAMP addition and appeared to be critical for functional inhibition of NHE-3, although other regions of the transporter are likely to be involved in PKA regulation.

    EXPERIMENTAL PROCEDURES

Cell Lines-- Rat NHE-3 was tagged (C-terminal) with hexahistidines (NHE-3/6H) by polymerase chain reaction, sequenced, and cloned into the mammalian expression plasmid pcDNA3 (Invitrogen, Carlsbad, CA). Plasmids were transfected into Na/H exchanger-deficient AP-1 cells (derived from Chinese hamster ovarian fibroblasts) (gift from Dr. Sergio Grinstein, Toronto, Ontario, Canada) using Lipofectin (Life Technologies, Inc.), and double-selected for G418 resistance (400 µg/ml) and H+ survival. Cells were maintained in minimum essential Eagle's medium (Sigma) with 10% fetal bovine serum (Life Technologies, Inc.) and 200 µg/ml G418. G418 was replaced by 100 units/ml penicillin and 100 µl/ml streptomycin two passages before experimentation. Individual clones were selected from pooled transfectants by limiting dilution. The function of NHE-3/6H was not different from nontagged NHE-3 (pCMV5/NHE3) expressed in AP-1 cells (10) in terms of basal activity, antigenic expression, Na, resting pHi and ethylisopropyl amiloride kinetics, and response to cAMP addition (data not shown). OKP cells (gift from K. Hruska, St. Louis, MO), were maintained in Dulbecco's modified Eagle's medium supplemented with 4.5 mg/ml glucose, 100 units/ml penicillin, and 100 µl/ml streptomycin. Confluent monolayers were rendered quiescent by serum removal (16-24 h, AP-1P cells; 36-48 h, OKP cells), and PKA was activated by the addition of cell permeant 8-Br-cAMP.

Site-directed Mutagenesis-- Specific serines in the cytoplasmic domain of NHE-3 were mutated singularly or in combination using a modification of the double-stranded method of Deng and Nickoloff (15) (QuickChangeTM kit, Stratagene, La Jolla, CA). The 8 mutations were: S513G, S552A, S575A, S605G, S634A, S661A, S690G, and S804G. After annealing the pcDNA3/NHE-3/6H parent vector with mutagenic oligonucleotides, strands were extended with Pfu DNA polymerase. Host-derived wild type methylated templates were digested with DpnI and plasmid DNA's were isolated from transformed XL1-B Escherichia coli. Multiple mutations were created sequentially. Each mutation was confirmed by direct sequencing. New cell lines were generated as described above.

Na/H Exchanger Activity Assays-- NHE-3 activity was measured fluorimetrically in confluent cells on glass coverslips with the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(-6)-carboxyfluorescein as sodium-dependent cell pH recovery (dpHi/dt) after acid loading with the K/H ionophore nigericin (13). For sodium kinetics, sodium was replaced iso-osmotically with choline. Buffer capacity was calculated by measuring Delta pHi in response to a 20 mM NH4Cl pulse at the trough pH. The addition of 8-Br-cAMP did not alter 2',7'-bis-(2-carboxyethyl)-5-(-6)-carboxyfluorescein calibration or buffer capacity in AP-1 or OKP cells (data not shown). For 22Na uptake studies, monolayers on 24-well dishes were acidified by NH3/NH4+ prepulse/withdrawal (pulse in mM: 10 Hepes·HCl, pH 7.4, 30 NH4Cl, 100 choline Cl, 5 KCl; withdrawal in mM:10 Hepes·HCl pH 7.4, 130 choline chloride, 5 KCl), and 22Na flux was initiated at 20 °C (in mM: 130 choline chloride, 2 CaCl2, 1 MgCl2, 10 Hepes·HCl, pH 7.4, 1 ouabain, 1 22NaCl (1 µCi/ml), 50 nM ethyl-isopropyl-amiloride), and terminated by washing with phosphate-buffered saline (4 °C). Cells were lysed in 0.1 M NaOH for protein concentration determination (Bradford) and scintillation counting.

In Vivo Phosphorylation and Isolation of NHE-3-- NHE-3 phosphorylated in transfected AP-1 cells was isolated by either immunoprecipitation or nickel-affinity chromatography. After incubation in phosphate-free Dulbecco's modified Eagle's medium (30 min), cells were labeled with [32P]orthophosphate (200-330 µCi/ml; 120 min) and 8-Br-cAMP or vehicle was added. After washing with ice-cold Tris-buffered saline, cells were lysed with ice-cold RIPA buffer (in mM: 150 NaCl, 80 sodium fluoride, 50 Tris-HCl, pH 8.0, 5 EDTA, 1 EGTA, 25 sodium pyrophosphate, 1 sodium orthovanadate; Nonidet P-40 1% (v/v), deoxycholate 0.5% (w/v), SDS 0.1% (w/v); in µg/ml: 100 phenylmethylsulfonyl fluoride, 4 leupeptin, 4 aprotinin, 10 pepstatin). The slurry was cleared by centrifugation (109,000 × gmax @ 50,000 rpm; 25 min; 4 °C; Beckman TLX: TLA 100.3 rotor), and NHE-3 was immunoprecipitated with antiserum 1314 (14) (1:500 dilution) and protein A-Sepharose. After washing with RIPA buffer, the antibody-antigen complex was eluted in SDS buffer (5 mM Tris-HCl, pH 6.8, 10% (v/v) glycerol, 1% (w/v) beta -mercaptoethanol, 0.1% (w/v) SDS, 0.01% (w/v) bromphenol blue), resolved on SDS-PAGE and transferred to nitrocellulose membrane. For nickel-affinity purification of NHE-3, cells were lysed in buffer B1 (in mM: 8,000 urea, 100 Na2PO4, 10 imidazole, pH 8.0; in % (v/v): glycerol 30, Triton-X 100 0.5), sheared with a 25-gauge needle, combined with buffer B2 (1:1, v/v) (composition same as B1 except no glycerol), clarified by centrifugation (109,000 × gmax @ 50,000 rpm, 25 min, Beckman TLX: TLA 100.3 rotor), and equilibrated with Ni-NTA resin (Qiagen, Chatsworth, CA) by rocking for 1 h at room temperature. The resin was washed with 5× bed volumes of wash buffer (in mM: 8,000 urea, 100 Na2PO4, pH 8.0, 50 imidazole; in % (v/v): 0.5 Triton-X 100), and NHE-3/6His was eluted (in mM: 8,000 urea, 100 Na2PO4, 500 imidazole, pH 8.0) for SDS-PAGE. Immunoblots were performed with anti-peptide antiserum 1568 (1:2,000) (14) using enhanced chemiluminescence (Amersham Pharmacia Biotech), and the 32P content of NHE-3 was visualized by autoradiography on the same filters after decay of enhanced chemiluminescence. Both antigenic and 32P signals were quantified by densitometry, and changes in phosphorylation were normalized to the antigenic signal. In general, both methods of purification yielded similar results in terms of changes in phosphorylation.

For studying surface NHE-3, only OKP cells were used. Repeated attempts to isolate surface NHE-3 from AP-1 cells yielded variable results ranging from low to no recovered biotinylated NHE-3 despite abundant total cellular NHE-3. Surface NHE-3 from four 100-mm dishes of confluent OKP monolayers were pooled to generate an adequate signal for one phosphopeptide map. Cells were labeled with 32P and incubated with 100 µM 8-Br-cAMP, surface proteins were labeled with biotin (in mM: 10 triethanolamine, pH 7.4, 2 CaCl2, 150 NaCl; 1.5 mg/ml NHS-SS-biotin, (Pierce)) (16) for 30 min at 4 °C. After quenching (in mM: 140 Na2HPO4, pH 7.4, 0.1 CaCl2, 1 MgCl2, 100 glycine), cells were lysed in RIPA buffer (in mM: 150 NaCl, 50 Tris-HCl, pH 7.4, 0.5 EDTA, 80 sodium fluoride, 25 sodium pyrophosphate, 1 sodium orthovanadate; in %: 1 (v/v) Triton X-100, 0.5 (w/v) deoxycholate, 0.1 (w/v) SDS; in µg/ml: 250 phenylmethylsulfonyl fluoride, 10 leupeptin, 10 aprotinin, 20 pepstatin) and equilibrated with streptavidin-agarose at 4 °C on a rotary mixer overnight. After washing with RIPA, biotinylated surface proteins were released from the biotin-streptavidin-agarose complex by 100 mM dithiothreitol at room temperature, and the liberated proteins retrieved as a supernatant after centrifugation. The supernatant was diluted 1:100 with RIPA buffer, spun-out with a Centricon-10 filter (Amicon, Beverly, MA), and reconstituted with RIPA without dithiothreitol. The plasma membrane 32P-labeled NHE-3 was immunoprecipitated as described above.

Specific Inhibition of PKA in Vivo-- AP-1 NHE-deficient cells were transfected with pcDNA-3/NHE-3 with or without the plasmid REVA/B (gift from Dr. Stanley McKnight, Seattle, WA) (17). REVA,B is a dominant-negative mutant regulatory subunit of PKA (PKA-RSU), which binds stoichiometrically to the catalytic subunit (PKA-CSU) but is devoid of both cAMP binding sites and, hence, does not release its pseudo substrate inhibition even in high ambient cAMP concentrations. 40% confluent AP-1 cells were transfected using LipofectAMINETM (Life Technologies, Inc.) with the following combination of plasmids: 1) pcDNA3 vector (20 µg); 2) pcDNA3/NHE-3/6H (10 µg), pcDNA3 (10 µg); 3) REVA/B (10 µg), pCDNA3 (10 µg); and 4) pcDNA3/NHE-3/6H (10 µg) and REVA/B (10 µg). A 5-min pulse of 5 µM 8-Br-cAMP was given to the cells 40 h post-transfection to allow the dominant-negative PKA-RSU to engage the native PKA-CSU. At 48-h post-transfection, cells were treated with either 100 µM 8-Br-cAMP or vehicle for 30 min and NHE-3 activity was measured by 22Na uptake.

In Vitro Phosphorylation of NHE-3 by PKA-- For sequential in vivo and/or in vitro or back phosphorylation experiments, AP-1 cells transfected with NHE-3/6H were treated with 100 µM 8-Br-cAMP or control medium as described above except no 32PO4 was added. NHE-3 was purified by nickel-affinity chromatography. The NHE-3 immobilized on Ni-NTA columns was partially renatured using an 8 to 0 M linear urea gradient (same as wash buffer above but with urea gradient) and retained as an insoluble complex on solid support. The beads were dialyzed in phosphate-buffered saline at 4 °C overnight and subjected to direct phosphorylation by purified PKA-CSU. The dialyzed NHE-3/Ni-NTA-agarose complex was washed with 1× kinase buffer (in mM: 50 Hepes-Tris, pH 7.0, 0.3 dithiothreitol, 5 MgCl2, 1 EGTA) and aliquoted in equal amounts into Eppendorf tubes. In vitro phosphorylation was initiated in each tube by addition of 1× kinase buffer supplemented with 50 µCi [gamma -32P]ATP, 0.1 mM ATP, 25 units of PKA-CSU, and rocked in room temperature for the designated time period. The reaction was stopped by addition of 5× reaction volumes of stop buffer (1% (w/v) SDS, 10 mM EDTA, 10 mM imidazole, pH 8.0), transferred to chromatography columns and washed with another 5× bed volume of stop buffer. The phosphorylated NHE-3 was eluted with 500 mM imidazole with 0.1% SDS and subjected to SDS-PAGE, transferred to nylon membranes, and autoradiography and immunoblot was performed as described above.

Phosphoamino Acid Analysis and Tryptic Phosphopeptide Mapping-- Purified phosphoproteins were eluted from the acrylamide gel with 50 mM NH4HCO3, trichloroacetic acid-precipitated, hydrolyzed by boiling in 6 M HCl, and the 32P-labeled amino acids were electrophoretically resolved in a Hunter TLC electrophoresis unit (HTLE 7000, CBS Scientific Co, Del Mar, CA) (first dimension: 2.2% formic acid, 7.8% acetic acid, pH 1.9; second dimension: 5% acetic acid, 0.5% pyridine, pH 3.5) along with cold standards (P-Ser, P-Thr, P-Tyr). Phosphoamino acids were identified by autoradiography and alignment with ninhydrin-stained phosphoamino acid standards (18). Tryptic phosphopeptide mapping was performed as described by Boyle (18). The purified in vivo phosphorylated NHE-3 was fractionated by SDS-PAGE and transferred to nitrocellulose membrane. Membrane pieces containing NHE-3 protein were localized by autoradiography, excised, and incubated in 100 mM acetic acid containing 0.5% (w/v) polyvinylpyrrolidone at 37 °C for 45 min. After washing with deionized water and 0.05 M NH4HCO3 solution, membranes were incubated in 15 µg TPCK (N-tyrosyl-L-phenylalanine chloromethyl ketone)-treated trypsin (Worthington) in 300 µl of 0.05 M NH4HCO3 in a 37 °C shaking waterbath. 15 µg of TPCK-treated trypsin was added after 2 and 12 h. After repeated deionized water washes and lyophilization, the dried sample was resuspended in 20 µl of electrophoresis buffer (per liter: 25 ml of 88% (w/v) formic acid, 78 ml of glacial acetic acid, 897 ml of deionized water, pH 1.95), spotted on a cellulose TLC plate (Merck, Darmstadt, Germany), and electrophoresis was performed on a Hunter thin-layer electrophoresis apparatus with the above buffer (30 min; 1.0 kV). Separation of the peptides in the second dimension was achieved by ascending chromatography (375:250:75:300 (v/v) n-butanol, pyridine, glacial acetic acid, deionized water). The phosphopeptides were visualized by either phosphorimaging or autoradiography, and the signal of individual phosphopeptides were quantified by densitometry. To eliminate variations in cpm's loaded and exposure times, the intensity of each phosphopeptide on a given TLC plate was normalized to the intensity of an invariant constitutive phosphopeptide B on the same plate. Quantitative differences between control and cAMP was assessed statistically by ANOVA.

    RESULTS

Effect of PKA Activation on NHE-3 Activity and Phosphorylation-- Fig. 1 shows that cAMP inhibited NHE-3 activity in a time- (Fig. 1A) and dose-dependent (Fig. 1B) fashion in both AP-1 and OKP cells. In both AP-1 (Fig. 2A) and OKP (Fig. 2B) cells, the Vmax was reduced by approximately 40% (con versus cAMP: AP-1P; 0.86 versus 0.55 pH units/min, p < 0.05, OKP; 1.05 versus 0.61 pH units/min, p < 0.05, t test) with no change in KNa (con versus cAMP: AP-1P;14 mM versus 12 mM, p = 0.52, OKP; 20 mM versus 16 mM, p = 0.67, t test). We have previously shown that 200 µM cAMP increases NHE-3 phosphorylation but lower doses were not examined (10). When we performed a dose response of the effect of cAMP on NHE-3 phosphorylation, we found no consistently detectable effect on NHE-3 whole protein phosphorylation in both AP-1 and OKP cells until 100 µM of cAMP was used (Fig. 3). Even at 100 µM, changes in total NHE-3 phosphorylation was variable. This was in sharp contradistinction to the effect of cAMP on NHE-3 activity, which was evident at doses as low as 1 and 10 µM for OKP and AP-1 cells, respectively.


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Fig. 1.   Time (A) and dose dependence (B) of cAMP addition on NHE-3 activity in AP-1 (solid bar) and OKP (open bar) cells. 8-Br-cAMP were added to cells, and NHE-3 activity was measured fluorimetrically as sodium-dependent cell pH recovery. Asterisk indicates significant difference (p < 0.05, ANOVA) from control. Bars and errors bars represent mean ± S.E.; n = 4 for each time point and dose. n = number of experiments for time dependence: 0 min, OKP n = 5, AP-1 n = 6; 10 min, OKP n = 4, AP-1 n = 3; 20 min, OKP n = 3, AP-1 n = 4; 30 min, OKP n = 7, AP-1 n = 9; 40 min, OKP n = 4, AP-1 n = 7. n = number of experiments for dose dependence: 0 µM, OKP n = 5, AP-1 n = 6; 10-6 M, OKP n = 4, AP-1 n = 5; 10-5 M, OKP n = 4, AP-1 n = 4; 10-4 M, OKP n = 7, AP-1 n = 9; 2-10-4 M, OKP n = 5, AP-1 n = 7.


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Fig. 2.   Effect of cAMP on NHE-3 sodium kinetics. open circle , control; bullet , 100 µM 8-Br-cAMP for 30 min. A, AP-1P cells (con versus 8-Br-cAMP: Vmax; 0.86 versus 0.55 pH units/min, p < 0.05, KNa; 14 mM versus 12 mM, p = 0.520). B, OKP cells (con versus 8-Br-cAMP: Vmax; 1.05 versus 0.61 pH units/min, p < 0.05, KNa; 20 mM versus 16 mM, p = 0.67). Symbols and errors bars represent mean ± S.E. n = number of experiments: n = 3 for each cell line treated with vehicle or 8-Br-cAMP for each [Na] except for [Na] = 5, n = 4 for each.


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Fig. 3.   Dose-response of cAMP on NHE-3 activity and phosphorylation. AP-1P cells (closed symbols) and OKP cells (open symbols). The y-axis shows NHE-3 phosphorylation (square symbols) and activity (circles) expressed as percent of control. Changes in phosphorylation were normalized to NHE-3 antigen. NHE-3 activity was measured fluorimetrically as sodium-dependent cell pH recovery and is the same data as in Fig. 1B. Symbols and errors bars represent mean ± S.E. n = number of experiments for phosphorylation dose response: AP-1: control, n = 9; 1 µM, n = 3; 10 µM, n = 3, 100 µM. n = 9. OKP cells: control, n = 4; 1 µM, n = 3; 10 µM, n = 4, 100 µM. n = 6.

Role of PKA in Regulating NHE-3 Function-- One hypothesis is that the effect of cAMP may be independent of A kinase activity. Direct gating by cyclic nucleotides without involvement of protein kinase has been described for cation channels (19). To test this hypothesis, we inhibited endogenous PKA-CSU with a dominant-negative mutant regulatory subunit (PKA-RSU) in a transient transfection system rather than with pharmacologic inhibitors, which may have nonspecific effects. Because cells that did not take up REVA/B (plasmid for mutant PKA-RSU) also did not receive NHE-3, all 22Na uptake can be assumed to originate from cells that received both plasmids. Fig. 4 summarizes the data. Only cells transfected with NHE-3 expressed significant 22Na uptake indicating that the measured flux reflected NHE-3 activity and in the absence of REVA/B, NHE-3 activity was suppressed by cAMP addition as expected. In the presence of the dominant-negative PKA-RSU, cAMP had no effect on NHE-3 activity indicating that the cAMP effect on NHE-3 activity is mediated by PKA-CSU.


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Fig. 4.   Acute regulation of NHE-3 by cAMP: effect of dominant-negative regulatory subunit of PKA (REVA/B). NHE-deficient AP-1 cells were transfected with the following plasmids: pcDNA, expression vector; NHE-3; and REVA/B, dominant-negative regulatory subunit of PKA. Native PKA was activated by 100 µM 8-Br-cAMP for 30 min, and NHE-3 activity was measured by 22Na uptake and normalized to total cellular protein per well. Data from one experiment are shown. A total of three experiments were performed with similar results.

Phosphorylation of NHE-3 by PKA-- If PKA is necessary for the regulation of NHE-3 by cAMP, another hypothesis that can explain the dissociation between activity and whole protein phosphorylation is that PKA phosphorylates a protein other than NHE-3, which in turn modulates NHE-3 activity. NHE-RF for example is a phosphoprotein that regulates NHE-3 function (20-22). To address whether NHE-3 is phosphorylated by PKA in vivo, we used the indirect approach of back phosphorylation. If the PKA sites on NHE-3 are occupied by nonradioactive PO4 because of in vivo phosphorylation before cell lysis, these sites will be unavailable to accept 32PO4 in the subsequent in vitro reaction with purified PKA-CSU. When we harvested NHE-3 from either cAMP-activated or control cells and subjected purified NHE-3 to in vitro phosphorylation by PKA, we found a significantly smaller degree of 32PO4 incorporation into NHE-3 from cAMP-activated compared with control cells (Fig. 5). These findings are compatible with the hypothesis that PKA indeed directly phosphorylates NHE-3 in vivo. It is important to note that the current data do not rule out the fact that PKA may phosphorylate proteins other than NHE-3 and kinases other than PKA may also phosphorylate NHE-3.


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Fig. 5.   Sequential in vivo and in vitro phosphorylation of NHE-3. AP-1P cells were treated with 8-Br-cAMP or vehicle. 6His-tagged NHE-3 was purified, immobilized on Ni-NTA beads, and subjected to phosphorylation by purified PKA in the presence of [gamma -32P]ATP. The phosphorylated protein was eluted off the beads, fractionated by SDS-PAGE, transferred to filters, and analyzed with autoradiography and immunoblot. One experiment is shown. Three independent experiments showed similar results.

Phosphoamino Acid and Tryptic Phosphopeptide Mapping of in Vivo Phosphorylated NHE-3-- The most likely explanation for the dissociation between activity and whole protein phosphorylation is presence of constitutive phosphorylated residues on NHE-3 that are not regulated by cAMP addition. To address this, we performed tryptic phosphopeptide maps using varying doses of cAMP. We observed increased intensity of phosphopeptides D, E, F, and J after cAMP addition (Fig. 6A). Although there are variations from map to map, with over 30 maps performed on AP-1 cells, it is evident that phosphopeptide B is constitutively phosphorylated. Because the absolute signal of a specific phosphopeptide on a given map is affected by a variety of factors such as the efficiency of retrieval from the total protein as well as the exposure time; one way to summarize the results from all the maps quantitatively is to express the intensity of each phosphopeptide as a ratio to the constitutive phosphopeptide B on the same plate. The results from all the phosphopeptide maps performed on wild type cells are summarized in Fig. 6B. Phosphopeptides J and E were significantly increased by [cAMP] >=  1 and 10 µM, respectively, and phosphopeptide F was increased at 100 µM cAMP. Because NHE-3 is more abundant in intracellular compartments than in plasma membrane in AP-1 cells (12), we examined whether the measured inhibition of NHE-3 activity, which reflects plasma membrane NHE-3, is associated with changes in cell surface NHE-3 phosphorylation. Extremely low and variable levels of retrievable biotinylatable NHE-3 in AP-1 cells precluded a meaningful conclusion in these cells. However, when we isolated surface biotinylated NHE-3 from OKP cells, multiple cAMP-induced NHE-3 phosphopeptides were detected (Fig. 6C). The pattern of phosphorylation of OK surface NHE-3 was identical to that of total cellular NHE-3 (not shown). Next, we determined the phosphoamino acid composition of the in vivo phosphorylated protein in AP-1 cells to be almost exclusively serines (Fig. 7). Prolonged exposure revealed a small amount of phosphothreonine, which when quantified by scintillation represented 0.5-1% of the counts. Phosphoamino acid analysis of OK cells showed predominantly phosphoserines with 2% phosphothreonine (data not shown).


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Fig. 6.   Phosphopeptide map of NHE-3 in AP-1 and OKP cells: Response to 8-Br-cAMP. A, tryptic phosphopeptide maps of immunoprecipitated 32P-labeled NHE-3 from AP-1 cells treated with different [cAMP]. 32P-NHE-3 was digested with trypsin, resolved by electrophoresis and thin-layer chromatography, and visualized by autoradiography. Phosphopeptides are labeled by alphabet letters. B, quantitative summary of dose response of phosphopeptide maps of NHE-3 in AP-1 cells. Intensity of phosphopeptides C, D, E, F, and J were normalized to the intensity of the corresponding phosphopeptide B on the same thin-layer chromatography plate. Symbols and errors bars represent mean ± S.E. n = number of experiments: control, n = 9; 1 µM, n = 3; 10 µM, n = 3; 100 µM, n = 9. Asterisk indicates p = 0.05 when compared with the value of control by ANOVA. C, cAMP-induced phosphorylation of surface NHE-3 in OK cells. OK cells were pulsed with 32P, 100 µM 8-Br-cAMP was added, and surface NHE-3 was immunoprecipitated from the biotinylatable fraction of the cell lysate, digested with trypsin, and resolved in two dimensions. Two independent maps showed similar results.


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Fig. 7.   Phosphoamino acid analysis of NHE-3 phosphorylated in vivo. Cells were pulsed with [32P]orthophosphate, 100 µM 8-Br-cAMP was added, and the immunoprecipitated NHE-3 was subjected to acid hydrolysis, resolved on two-dimensional electrophoresis, and subjected to autoradiography for phosphoamino acid analysis. Three independent experiments showed predominantly phosphoserines.

Regulation of Na/H Exchange Activity in Mutant NHE-3 (NHE-3mut)-- If PKA phosphorylates NHE-3, the critical question is whether phosphorylation of NHE-3 is functionally important for its regulation. Because only serines are phosphorylated, we mutated 8 serines individually and in combination in the cytoplasmic domain that conform to the classical PKA consensus target motif (23) and screened for the ability of cAMP to regulate NHE-3 activity. Single amino acid mutations per se do not significantly affect whole cell NHE-3 protein expression (Fig. 8A). However, four or more point mutations did decrease NHE-3 expression with the lowest expression in the six-point mutant (Fig. 8B). NHE-3 transcript levels were not affected by single or multiple mutations (not shown). Fig. 9A shows tracings of several representative mutants, and Fig. 9, B and C, summarize the functional data from all the mutants. Among the single mutants, NHE-3mut/S552A failed to show a response to cAMP addition. NHE-3mut/S605G showed much reduced inhibition by cAMP (control versus cAMP p = 0.28). NHE-3mut/S634A showed a slightly attenuated but nonetheless intact response to cAMP. This finding suggests phosphorylation of Ser-552 and Ser-605 are key steps in modulating NHE-3 activity. One concern with this data is whether the lack of regulation is truly due to the mutant NHE-3 or because of cellular context. This is particularly critical for S552A because Kurashima and co-workers (12) found intact cAMP regulation with the same mutation. To determine that the lack of cAMP sensitivity actually stems from S552A rather than a missing cellular factor, we studied NHE-3mut/S552A against a heterogenous cellular background with two approaches. We isolated individual clones from the pooled NHE-3mut/S552A transfectants, and we transiently transfected NHE-3mut/S552A into AP-1 cells at different passages (Table I). As a positive control, we compared NHE-3mut/S552A to one of the cAMP-responsive single serine mutants NHE-3mut/S690G. As shown in Table I, all NHE-3mut/S552A-transfected cells remained cAMP insensitive, whereas all NHE-3mut/S690G-transfected cells maintained cAMP sensitivity. Despite the potentially diverse cellular context, cAMP responsiveness segregated with the mutation.


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Fig. 8.   Immunoblot of NHE-3 from AP-1 cells expressing NHE-3mut. 20 µg of membrane protein from each of the cell lines were resolved by SDS-PAGE and probed with anti-NHE-3 antiserum 1568. Single and multiple serine mutations are indicated on the figure. Single mutants (A) and multiple mutants (B). Two independent experiments showed similar results.


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Fig. 9.   Effect of cAMP on NHE function NHE-3muts in mutants with serine mutations. Point mutants of NHE-3 was expressed in AP-1 NHE-deficient fibroblasts. NHE-3 activity in control and 8-Br-cAMP (100 µM, 30 min)-treated cells were measured fluorimetrically as sodium-dependent cell pH recovery. Asterisk indicates p < 0.05 by unpaired t test (control versus cAMP in same cell line). A, representative tracings from six selected clones; B, summary of all single mutants. n = number of experiments: WT, n = 21; S513G, n = 4; S552A, n = 11; S575A, n = 9; S605G, n = 9; S634A, n = 4; S661A, n = 6; S690G, n = 4; S804G, n = 6. C, summary of all multiple mutants. n = number of experiments: WT, n = 21, S513G/S552A, n = 4; S513A/S552A/S661A, n = 3; S661A/S690G/S804G, n = 4; S513G/S552A/S575A/S605G, n = 5; S552A/S575A/S661A/S690G/S804G, n = 5; S513G/S552A/S575A/S661A/S690/S690G/S804G, n = 5.

                              
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Table I
NHE-3 activity in AP-1 cells transfected rat NHE-3
22Na flux pmol/mg/10 seconds; mean ± S.D. from four to six experiments.

The results were less clear in the multiple mutants. All the multiple mutants harboring Ser-552 were not regulated by cAMP addition confirming the importance of Ser-552. However, NHE-3mut/S575A/S661A/S690G/S804G which contains neither Ser-552 nor Ser-605, also showed no inhibition by cAMP addition.

Tryptic Phosphopeptide Map of NHE-3mut/S552A and NHE-3mut/S605G-- If Ser-552 and Ser-605 are indeed critical for inhibition of NHE-3 activity as phosphoserines, they should be phosphorylated in vivo by cAMP addition. We compared phosphopeptide maps of these two NHE-3mut's to wild type NHE-3 in response to cAMP. Fig. 10 shows the phosphopeptide maps of wild type NHE-3, NHE-3mut/S552A, and NHE-3mut/S605G. Phosphopeptide J, which was increased by 8-Br-cAMP in wild type NHE-3 is absent in NHE-3mut/S552A, and phosphopeptide E, which was also increased by 8-Br-cAMP in wild type NHE-3, was absent from NHE-3mut/S605A indicating that both Ser-552 and Ser-605 are phosphorylated in vivo.


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Fig. 10.   Phosphopeptide map of in vivo phosphorylated wild type and mutant NHE-3. Cells were pulsed with 32P and treated with 8-Br-cAMP. NHE-3 was immunoprecipitated, and the tryptic peptides were resolved by electrophoresis and thin-layer chromatography and visualized by autoradiography. A, WT versus NHE-3mut/S552A. n = 5; B, WT versus NHE-3mut/S605G, n = 3.


    DISCUSSION

We confirmed the previous finding of inhibition of Vmax by cAMP in native NHE-3 in renal epithelial cells (24-26) and NHE-3 heterologously expressed in fibroblasts (8-12). The lack of detectable NHE-3 whole protein phosphorylation is comparable with the report by Kurashima and co-workers (12) although we observed discernible but variable changes in NHE-3 whole protein phosphorylation with 100 µM 8-Br-cAMP. As some cation channels are directly gated by cyclic nucleotides (19), we queried if such can be a model for NHE-3 regulation by cAMP. When we inactivated the catalytic subunit of PKA, 8-Br-cAMP no longer regulated NHE-3 activity indicating that the cAMP effect is kinase-dependent. Also congruent with A kinase-mediated action is the fact that unlike the cyclic nucleotide-gated channels (27), the primary sequence of NHE-3 does not conform to the beta -roll/alpha -helix motif derived from the catabolite gene activator protein model (28). In addition, a recombinant fusion protein of maltose-binding protein/NHE-3 cytoplasmic domain failed to bind 3H-cAMP (data not shown).

The cytoplasmic domain of NHE-3 contains numerous PKA consensus target sites and NHE-3 is rapidly phosphorylated by PKA in vitro (10). Although NHE-3 is a phosphoprotein in vivo (10, 12), there is no a priori reason to assume that PKA directly phosphorylates NHE-3 in vivo. The basis of the back phosphorylation assay rests on the presumption that certain common sites are preserved for in vivo and in vitro kinase reactions and multiple kinases do not converge on the exact same serine residues. Our results suggest that PKA likely phosphorylates NHE-3 in the cell. It is possible that the regulation of NHE-3 by cAMP involves phosphorylation of proteins other than NHE-3 by PKA and that PKA may activate other kinases that phosphorylate NHE-3. Kurashima and co-workers (12) showed that the lack of change in total NHE-3 protein phosphorylation in vivo is because of multiple constitutively phosphorylated sites masking the change in the regulated sites. We also found that phosphopeptides B, C, and D are largely unregulated in vivo. In contrast, phosphopeptide E (Ser-605) and J (Ser-552) were increased at 1 and 10 µM 8-Br-cAMP, respectively. The dose-response profiles of phosphopeptides E and J actually correlate well with that of changes in NHE-3 activity in response to cAMP.

If phosphorylation of Ser-552 and Ser-605 are important, then elimination of phosphorylation should abolish functional regulation. All the single and double point mutants showed comparable antigen expression and baseline transport activity but the quadruple mutants showed decreased and the six-point mutant showed markedly attenuated expression of both NHE-3 protein and activity. Because NHE-3 transcript levels are not affected by the mutations, one has to postulate that the multiple serine mutations significantly affected translation and/or protein half-life. This study does not address the mechanism of these changes. Ser-552 and Ser-605 were both phosphorylated in vivo and when mutated singly to nonphosphorylatable residues, functional regulation by PKA was abrogated in both mutants. Kurashima and co-workers (12) have reported the importance of Ser-605 and Ser-634 in the functional regulation of NHE-3 by PKA although Ser-634 appeared not to be a phosphoserine. Our phosphopeptide map and functional response of the NHE-3mut/S605A is very similar to the previous report. A major disparate finding is that we found Ser-552 to be critical for PKA regulation, whereas Kurashima and co-workers (12) found normal functional regulation of NHE-3mut/S552A by cAMP. In addition, Cabado and co-workers (11) from the same group found that truncation of the cytoplasmic domain at amino acid 579 rendered NHE-3 nonresponsive to cAMP. The difference may reside in the fact that Ser-552, which is phosphorylated in our cells, was not phosphorylated in the previous report as is evident by the absence of phosphopeptide J (12). It is conceivable that multiple PKA target phosphoserines may participate in mediating the functional inhibition of NHE-3 and different host cells may use different sets of serines. The basis for the differential NHE-3 phosphorylation is unclear, because both laboratories used AP-1 cells from the same source. Because AP-1 cells are mutants derived and selected from the parental Chinese hamster ovary cell, repeated passages and clonal expansion might have created different phenotypes.

One possible interpretation for the lack of regulation of NHE-3mut/S552A is that some cellular cofactor(s) are missing in that particular AP-1 recipient that harbors the mutant NHE-3 and that the cAMP-insensitive phenotype stems from the cell rather than NHE-3mut/S552A. Such an explanation is unlikely for several reasons. All pooled transfectants of single or multiple mutant NHE-3s were generated about the same time from the same parental AP-1 cells. In addition, when we tried to examined NHE-3mut/S552A in as heterogenous an AP-1 cellular background as we can create, we found that the cAMP-nonresponsive phenotype prevailed.

The lack of regulation of NHE-3mut/S575/S661/S690/S804 is harder to explain because none of the serines were functionally important when examined as single mutants. One can speculate on several explanations. One is that there are multiple regulatory phosphoserines with P-Ser-552 and P-Ser-605 having the most dominant effects with lesser contribution from the other phosphoserines. Although singular mutations of serines other than Ser-552 and Ser-605 do not affect the overall response, mutation of four minor phosphorylation sites may be sufficient. A second possibility is that these serines participate in specific protein-protein interactions with regulatory factors such as NHE-RF (20, 21, 29-31), E3KARP (22), or calmodulin (32) that modulate NHE-3 function, and this interaction is disrupted by the mutations. A third possibility is that the mutations distort the protein structure to such an extent that it nonspecifically alters NHE-3 function and its regulation. Crystallographic evidence indicates that one can significantly alter the protein structure of a domain of a protein by a single amino acid mutation from tyrosine to phenylalanine, which essentially only removes a single hydroxyl group (33). The present paper does not distinguish the three stated possibilities. We did however observe intact regulation of all mutants including the four- to six-point mutants by acute hyperosmolality (25-40% inhibition) (data not shown). This is compatible with the ability of hyperosmolality to inhibit NHE-3 activity despite radical truncations of the cytoplasmic domain (34).

In summary, we hypothesize that the functional regulation of NHE-3 by PKA does not converge on single covalent modifications such as phosphorylation of a specific serine. We submit a model where PKA phosphorylates NHE-3 on Ser-552 and Ser-605 in addition to other serines. Phosphorylation of Ser-552 and Ser-605 each play a major although not exclusive role in the functional regulation of NHE-3, which involves specific interaction of various regions of the cytoplasmic domain of NHE-3 with other regulatory cofactors.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Stan McKnight for providing reagents, to Dr. Melanie Cobb and Dr. Robert Alpern for helpful discussions, and to Dr. Michel Baum and Dr. Robert Alpern for careful reading of the manuscript.

    FOOTNOTES

* This work was supported by the Research Service of the Department of Veterans Affairs and the National Institutes of Health (DK48482).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.

§ Recipient of a National Institutes of Health Training Grant (T32 DK07257-17).

parallel To whom correspondence should be addressed: Dept. of Internal Medicine, 5323 Harry Hines Blvd., Dallas, TX 75235-8856. Tel.: 214-648-3152; Fax: 214-648-2071; E-mail: omoe{at}mednet.swmed.edu.

The abbreviations used are: NHE, Na/H exchanger; PKA, protein kinase A; PAGE, polyacrylamide gel electrophoresis; CSU, catalytic subunit; RSU, regulatory subunit; TPCK, N-tyrosyl-L-phenylalanine chloromethyl ketone; ANOVA, analysis of variance; WT, wild type.
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Abstract
Introduction
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