QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

Vol. 19, Issue 9, 3859-3870, September 2008

This Article Abstract Full Text (PDF) All Versions of this Article: E08-01-0019v1 19/9/3859    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Sarker, R. Articles by Li, X. PubMed PubMed Citation Articles by Sarker, R. Articles by Li, X.

Casein Kinase 2 Binds to the C Terminus of Na⁺/H⁺ exchanger 3 (NHE3) and Stimulates NHE3 Basal Activity by Phosphorylating a Separate Site in NHE3

Rafiquel Sarker^*, Mads Grønborg^,, Boyoung Cha^*, Sachin Mohan^*, Yueping Chen^*, Akhilesh Pandey, David Litchfield, Mark Donowitz^*,||,¶, and Xuhang Li^*,¶

*Division of Gastroenterology and Hepatology, Department of Medicine, Institute of Genetic Medicine, ^||Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada, N6A 5C1

Submitted January 9, 2008; Revised May 28, 2008; Accepted June 22, 2008
Monitoring Editor: M. Bishr Omary

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Na⁺/H⁺ exchanger 3 (NHE3) is the epithelial-brush border isoform responsible for most intestinal and renal Na⁺ absorption. Its activity is both up- and down-regulated under normal physiological conditions, and it is inhibited in most diarrheal diseases. NHE3 is phosphorylated under basal conditions and Ser/Thr phosphatase inhibitors stimulate basal exchange activity; however, the kinases involved are unknown. To identify kinases that regulate NHE3 under basal conditions, NHE3 was immunoprecipitated; LC-MS/MS of trypsinized NHE3 identified a novel phosphorylation site at S⁷¹⁹ of the C terminus, which was predicted to be a casein kinase 2 (CK2) phosphorylation site. This was confirmed by an in vitro kinase assay. The NHE3-S719A mutant but not NHE3-S719D had reduced NHE3 activity due to less plasma membrane NHE3. This was due to reduced exocytosis plus decreased plasma membrane delivery of newly synthesized NHE3. Also, NHE3 activity was inhibited by the CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole DMAT when wild-type NHE3 was expressed in fibroblasts and Caco-2 cells, but the NHE3-S⁷¹⁹ mutant was fully resistant to DMAT. CK2 bound to the NHE3 C-terminal domain, between amino acids 590 and 667, a site different from the site it phosphorylates. CK2 binds to the NHE3 C terminus and stimulates basal NHE3 activity by phosphorylating a separate single site on the NHE3 C terminus (S⁷¹⁹), which affects NHE3 trafficking.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Neutral NaCl absorption is a small intestinal and colonic absorptive process in which a brush-border (BB) Na⁺/H⁺ exchanger is coupled, at least functionally, to a BB Cl^–/HCO₃^– exchanger. In intestinal Na⁺ absorptive cells, the Na⁺/H⁺ exchanger involved is SLC9A3 (NHE3), and the anion exchanger is either SLC26A3 or 6 (DRA or PAT-1) (Lamprecht et al., 2002; Zachos et al., 2005). This process is highly regulated and a defining characteristic is that it is set at an intermediate level during the period between meals (called basal conditions), which allows both inhibition and stimulation to occur as part of digestion (Moe, 1999; Zachos et al., 2005; Donowitz and Li, 2007).

NHE3 is the highly regulated part of the neutral NaCl absorptive process. In all cells in which it has been studied, NHE3 regulation mimics the regulation of neutral NaCl absorption, being active under basal conditions and being both inhibited and stimulated in cell models by various mimics of digestive physiology. NHE3 is phosphorylated under both basal and regulated conditions (Yip et al., 1998; Wiederkehr et al., 1999; Wang et al., 2005). Identified sites of phosphorylation in the NHE3 C terminus include amino acids S⁵⁵⁴ and S⁶⁰⁷ (rabbit NHE3), which occurs both under basal conditions and increases after cAMP, and also S⁶⁶³, which is phosphorylated by serum and glucocorticoid-inducible kinase (SGK1) in acute stimulation by glucocorticoids (Wang et al., 2005).

The function of phosphorylation of NHE3 under basal conditions is not known, but cAMP-increased phosphorylation leads to NHE3 inhibition by steps that involve an initial inhibition of turnover number and then reduced plasma membrane expression due first to increased endocytosis and later reduced endocytic recycling (Moe, 1999). Phosphatase inhibitors also affect NHE3. Okadaic acid (PP1 and 2A inhibitor) stimulates NHE3 activity and genistein (Tyr kinase inhibitor) inhibits NHE3 (Levine et al., 1995). These results suggest that both S/T and Y phosphorylation stimulate NHE3 but do not indicate whether there are changes in phosphorylation of NHE3 or whether NHE3 activity is affected by changes in phosphorylation of regulatory proteins, although NHE3 has not been shown to be Tyr phosphorylated (Yip et al., 1998).

The current studies were based on the hypothesis that understanding of intestinal Na⁺ absorption requires understanding of both basal and regulated NHE3 activity at the level of the protein kinases involved. This study used a proteomics approach to identify and characterize the functional effects of a unique NHE3 phosphorylation site (S⁷¹⁹) that was subsequently identified as phosphorylated by casein kinase 2 (CK2).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Chemicals, Reagents, and Antibodies
Reagents were from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. 4,5,6,7-Tetrabromo-2-azabenzimidazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) were from Calbiochem (San Diego, CA). QuikChange site-directed mutagenesis kit and Pfu polymerase were from Stratagene (La Jolla, CA). EZ-Link Sulfo-NHS-SS-biotin and Sulfo-NHS-acetate were from Pierce Chemical (Rockford, IL). Glutathione-Sepharose 4B beads were from GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom). Restriction endonucleases were from New England Biolabs (Ipswich, MA). 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) was from Invitrogen (Carlsbad, CA). P81 phosphocellulose papers were from Whatman (Madistone, United Kingdom). Monoclonal mouse antibodies to the hemagglutinin (HA) epitope was from Covance Research Products (Princeton, NJ). Rabbit polyclonal anti-CK2, and CK2β antibodies, were from Abcam (Cambridge, United Kingdom). DNA primers were from Operon Biotechnologies (Huntsville, AL). Peptides were synthesized at the Johns Hopkins Synthesis and Sequencing Facility (Baltimore, MD).

Mass Spectrometry Analysis and Database Searching
In-Gel Digestion. Immunoprecipitated NHE3 was first resolved by one-dimensional (1D) SDS-polyacrylamide gel electrophoresis (PAGE), and protein bands were visualized by Coomassie Blue staining. A protein band corresponding to NHE3 was excised from the gel and washed two times in MilliQ water (Millipore, Billerica, MA) and water:acetonitrile (1:1, vol/vol) for 15 min at room temperature. After washing the liquid was removed and replaced by 100% acetonitrile to shrink the gel pieces. After the gel pieces turned white, the acetonitrile was removed and replaced by 10 mM dithiothreitol in 100 mM ammonium bicarbonate for 45 min at 56°C to reduce Cys. The liquid was then removed and replaced by 55 mM iodoacetamide in 100 mM ammonium bicarbonate for 30 min at room temperature in the dark to alkylate the Cys. The gel pieces were then dehydrated in 100% acetonitrile and rehydrated in digestion buffer containing 12.5 ng/µl trypsin (modified sequence grade; Promega, Madison, WI) in 50 mM ammonium bicarbonate and incubated on ice for 45 min (Gronborg et al., 2004). The digestion buffer was then aspirated and replaced by 50 mM ammonium bicarbonate without trypsin and incubated overnight at 37°C. After digestion the supernatant was removed and the remaining peptides were extracted by incubating the gel pieces in 5% formic acid for 15 min and then adding the same volume of 100% acetonitrile for 15 min. The extraction was repeated twice and all three fractions were pooled and dried down in a vacuum centrifuge and resuspended in 10 µl of 5% formic acid.

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). After tryptic digestion of NHE3, the peptides were analyzed by nanoLC-MS/MS. The samples were loaded onto a 75-µm fused silica precolumn packed with 10-µm C₁₈ ODS-A (YMC, Kyoto, Japan) for washing and desalting in 95% mobile phase A (H₂O with 0.4% acetic acid and 0.005% heptafluorobutyric acid, vol/vol) and 5% mobile phase B (90% acetonitrile, 0.4% acetic acid, and 0.005% hepafluorobutyric acid in water, vol/vol). After washing and desalting, the sample was eluted from the precolumn by a linear gradient of 90% mobile phase A to 60% mobile phase A onto a 75-µm fused silica analytical column packed with Vydac C₁₈ resin. The peptides were eluted over a gradient of 34 min. The spectra were acquired on a QTOF API-US (Waters-Micromass, Manchester, United Kingdom). All data were obtained in positive ion-mode, and the data were analyzed using MassLynx 4.0 software (Waters-Micromass).

Database Searching. Database searching was done using the Mascot Search engine (Matrix Science, London, United Kingdom). The data were searched against the NCBInr database with a mass accuracy of 0.3 Da for the parent ion (MS) and 0.3 for the fragment ions (MS/MS). The peptides were constrained to be tryptic with a maximum of two missed cleavages. Fixed modifications were carbamidomethylation of Cys, whereas oxidation of Met and phosphorylation of Ser were considered as variable modifications.

Cell Culture, Plasmid Construction, and Transfection
PS120 fibroblasts, which lack all endogenous plasma membrane NHEs, were used for stable expression of rabbit NHE3-WT, NHE3-S719A, and NHE3-S719D), with either a triple-HA epitope tag at the N terminus (Murtazina et al., 2006) or a C-terminal vesicular stomatitis virus glycoprotein (VSV-G) epitope tag (Levine et al., 1995). The NHE3-S719A and NHE3-S719D mutations were made using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. The template for mutagenesis was the pcDNA3.1/Neomycin⁺ vector (Invitrogen) containing rabbit 3HA-NHE3. All PS120 cell lines were grown in DMEM supplemented with 25 mM NaHCO₃, 10 mM HEPES, 50 U/ml penicillin, 50 µg/ml streptomycin, 400 µg/ml G418 (Neomycin), and 10% fetal bovine serum in a 5% CO₂, 95% O₂ incubator at 37°C. Caco-2/bbe cell line originally derived from a human adenocarcinoma was obtained from M. Mooseker (Yale University, New Haven, CT) and grown in DMEM containing 25 mM NaHCO₃ supplemented with 0.1 mM nonessential amino acids, 10% fetal bovine serum, glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin, pH 7.4, in 5% CO₂ atmosphere at 37°C. Stably transfected PS120 cells were grown on glass coverslips up to 60–70% confluence and studied after serum starvation for 3–6 h. PS120/HA-NHE3 and HA-NHE3-S719A cell lines were selected for Na⁺/H⁺ exchange activity (3–4 passages) by exposing cells to an acid load as described previously (Levine et al., 1993). Caco-2/bbe cells were grown to confluence on small pieces of polycarbonate membranes glued to plastic coverslips (0.4-µm pore size, corning; called "filter slips") and polarized cells were used 14 d after reaching confluence.

Adenoviral Construct/Infection
Tripled HA-tagged rabbit NHE3 was engineered into Adeno-viral shuttle vector ADLOX.HTM under a cytomegalovirus promoter. Caco-2/bbe cells were treated with 6 mM EGTA in serum-free Caco-2 medium for 2 h at 37°C before viral infection to allow virus exposure to the basolateral surface. Appropriate amounts of viral particles were diluted (10⁹–10¹⁰ particles/ml) in serum-free Caco-2 medium and added to upper and lower chambers of EGTA-treated cells. Cells were infected by incubating at 37°C for 6 h and then replaced with normal growth medium. For transport assays or Western analyses, cells were used 48–60 h after infection; infection was typically day 11–12 after confluence and study was day 14. Generally, viral production was generated by transfection of CRE8 cells with the expressing constructs using Lipofectamine 2000 (Invitrogen), followed by infection of the cells with helper virus (5). Then, the crude adenoviruses were propagated by reinfection of CRE8 cells. Preparation of purified adenovirus with high titer was performed by CsCl gradient centrifugations. Viral titers were determined by a colorimetric method, following 1) the A₃₂₀ was below 0.02 and 2) the A₂₆₀/A₂₈₀ was between 1.2 and 1.3. Particle numbers were calculated by (A₂₆₀ value) x (1.1 x 10¹²) x dilution.

Measurement of Na⁺/H⁺ Exchange Activity
Na⁺/H⁺ exchange activity in PS120 and Caco-2 cells expressing HA-NHE3 was determined fluorometrically using the intracellular pH-sensitive dye BCECF, with PS120 cells grown to 60–70% confluence on glass coverslips (Levine et al., 1995) and Caco-2/bbe cells polarized on 0.4-µm polycarbonate membranes; they were studied 14 d after confluence as described previously (Janecki et al., 1998). Cells were loaded with 10 µM BCECF-AM in Na⁺/NH₄Cl solution (98 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 1 mM NaH₂PO₄, 25 mM glucose, 20 mM HEPES, and 40 NH₄Cl, pH 7.4) for 20 min at 37°C. During the dye loading and NH₄Cl prepulse, cells were treated with CK2 inhibitors, unless timing is described otherwise. The cells were initially perfused with TMA⁺ solution (130 mM tetramethylammonium chloride, 5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 1 mM NaH₂PO₄, 25 mM glucose, and 20 mM HEPES, pH 7.4), resulting in stable acidification of the cells. Then, Na⁺ solution (138 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 1 mM NaH₂PO₄, 25 mM glucose, and 20 mM HEPES, pH 7.4) was perfused. CK2 inhibitor was also present in TMA and Na⁺ solutions. To calibrate the relationship between the emission (530 nm) from the dual excitation (F_{490 and 440}) and pH_i, the K⁺/nigericin method was used. As described previously (Levine et al., 1995), Na⁺/H⁺ exchange rates (H⁺ efflux) were calculated as the product of Na⁺-dependent change in pH_i and the buffering capacity at each pH_i and were analyzed using the nonlinear regression data analysis program Origin, which allows fitting of data to a general allosteric model described by the Hill equation (v = V_max · [S]^napp/K' + [S]^napp, where v is velocity, [S] is the substrate concentration, n_app is the apparent Hill coefficient, and K is the affinity constant (K'(H⁺)_i), with estimates for V_max and K'(H⁺)_i and their respective errors (SE), as well as fitting to a hyperbolic curve. Data from each coverslip were calculated and analyzed as described above. For each independent experiment, results from all coverslips for each condition were analyzed together and represent an N = 1. To measure the Na⁺/H⁺ exchange activity in Caco-2/bbe cells, the cells were grown as polarized monolayers on pieces of polycarbonate membranes (0.4-µm pore size) attached over an aperture in rectangular plastic coverslips ("filter slip"), and cellular Na⁺/H⁺ exchange activity was determined fluorometrically by loading the intracellular pH-sensitive dye BCECF-AM for 60 min (Janecki et al., 1998). Filter slips were then mounted in a cuvette, placed in the fluorometer (Photon Technology International, Lawrenceville, NJ), and perfused at both apical and basolateral monolayer surfaces with TMA medium to allow rapid intracellular acidification. The TMA⁺ medium was subsequently replaced with Na⁺ medium at the apical monolayer surface, whereas TMA perfusion continued basally. Initial rates of Na⁺-dependent intracellular alkalinization (efflux of H⁺, in micromolar per second) were calculated for a given pH_i; 1 min of the initial rate of intracellular alkalinization was analyzed as pH/T. Means ± SE were determined from at least three experiments.

Expression and Purification of Fusion Proteins
Recombinant glutathione S-transferase (GST-CK2) was made in the (pGEX) vector. The 6x His-tagged fusion proteins made in the pET30a vector included four fragments of the NHE3 C terminus: F1 (amino acids [aa] 475-589), F2 (aa 590-667), F3 (aa 668-747), and F4 (aa 748-832) (Cha et al., 2006). His₆-tagged fusion proteins were expressed in Escherichia coli Rosetta 2 (DHE-3) cells, and crude cell extract was prepared following the Invitrogen protocol under native conditions. Proteins were affinity-purified with Ni²⁺-nitrilotriacetic acid resin as suggested by the manufacturer (QIAGEN, Valencia, CA).

In Vitro Phosphorylation Assay by CK2
In vitro phosphorylation experiments were carried out by incubating purified GST-CK2 fusion protein with synthetic peptide (100 µM) containing either S⁷¹⁹ or A⁷¹⁹ for 10 min at 30°C in a solution consisting of 50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 100 µM EGTA, and 25 µM [-³²P]ATP (final volume 50 µl). Two synthetic peptides (mimicking rabbit NHE3), 1) S⁷¹⁹ (wild-type), KERELELSDPEEAPD and 2) S719A, KERELELADPEEAPD were used in this experiment. Then, 40 µl of each reaction was spotted onto the center of a 2.5-cm P81 phosphocellulose paper, and reaction was terminated by immediately immersing the paper into 75 mM phosphoric acid. Phosphocellulose papers were washed three times in 75 mM phosphoric acid, rinsed briefly with acetone, and air-dried. Radioactivity was measured in a liquid scintillation counter. In this assay, kinase reactions without substrate or without CK2 were considered as background or blank.

Coimmunoprecipitation (IP) of NHE3 with CK2
IP was performed using lysates from PS120/NHE3 or PS120/NHE3-S719A cells, epitope tagged with HA, by using VSG-G as a negative control. Cells were grown in 10-cm dishes at 100% confluence and serum starved for 3 h; cell lysate was prepared in lysis buffer (60 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM KCl, 5 mM EDTA trisodium, 3 mM EGTA, 1 mM Na₃VO₄, and 1% Triton X-100 with protease inhibitor cocktail [Sigma-Aldrich]). Aliquots (1.5 mg of protein in 1 ml) of lysate were incubated overnight with 30 µl of either monoclonal anti-HA affinity agarose (Roche Diagnostics, Indianapolis, IN) or monoclonal anti-VSV-G antibodies conjugated to agarose beads at 4°C in a rotator (antibody conjugated agarose beads were prewashed with lysis buffer [as described above] before use). The antibody conjugated protein A-Sepharose beads were gently spun down and washed five times with lysis buffer. Bound proteins were eluted with 2x sample buffer, and proteins were separated by SDS-PAGE and transferred to nitrocellulose. The blots were probed with polyclonal anti-CK2 or anti-CK2β (Abcam) or monoclonal anti-HA primary antibody (Covance Research Products), followed by fluorescently labeled secondary antibody (IRDye 800 or 680) for visualization according to the manufacturer's protocol. Protein bands were visualized by the Odyssey system (LI-COR, Lincoln, NE).

In Vitro Binding Assays
Far Western Blot Analysis. An overlay approach was used to examine the direct interaction of purified recombinant His-tagged F1, F2, F3, and F4 fragments of NHE3 C terminus on blots with a purified GST-fusion protein encoding full-length CK2. Identification was with CK2 antibody. Recombinant F1, F2, F3, and F4 (3 µg) were separated by 14% SDS-PAGE, transferred to nitrocellulose, and blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS) (10 mM potassium phosphate buffer, pH 7.4, and 0.15 M NaCl) for 1 h. The blots were incubated with 5 ml of GST-CK2 (5 µg/ml) in 5% milk/PBS for 16–20 h at 4°C and then gently washed with 0.05% Tween/PBS three times for 5 min each. The blots were incubated with polyclonal CK2 antibody (1:1000) for 1 h, washed with 0.05% Tween/PBS three times for 5 min each, and incubated with fluorescently labeled, IRDye 800-conjugated goat anti-rabbit secondary antibody (Rockland Immunochemicals, Gilbertsville, PA) for 1 h. Finally, they were washed three times for 5 min each with 0.05% Tween/PBS and one time with PBS. Protein bands were detected by Odyssey system (LI-COR).

Pull-Down Assays. CK2-GST fusion proteins were purified using glutathione-Sepharose beads as described above and left on the beads for pull-down assays. Pull-down assays were performed by incubating 5 or 10 µl of CK2-GST beads with purified recombinant proteins, F1, F2, F3, and F4 fragments of C-terminal NHE3 (5 µg/ml) in binding buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% NP-40, 200 µM Na₃VO₄, and protease inhibitors) overnight at 4°C. Complexes were pelleted at 10,000 x g for 2 min and washed three times in 1 ml of lysis buffer. Bound proteins were eluted from the beads by heating in sample buffer for 5 min at 80°C. Proteins were separated in 14% SDS-PAGE, transferred to nitrocellulose membranes, and visualized by Ponceau S staining.

Cell Surface Biotinylation
NHS-SS-biotin was used to determine the percentage of total NHE3 on the plasma membrane; rates of exocytosis and endocytosis; and with ³⁵S labeling, delivery rates of newly synthesized NHE3 to the plasma membrane, as described previously (Cha et al., 2006). PS120 cells stably expressing HA-NHE3-WT and HA-NHE3-S719A were grown to 70–80% confluence in 10-cm Petri dishes. The cells were then serum starved for 4 h. Cells were rinsed three times with ice-cold phosphate-buffered saline (150 mM NaCl and 20 mM Na₂HPO₄, pH 7.4) and once in borate buffer (154 mM NaCl, 1.0 mM boric acid, 7.2 mM KCl, and 1.8 mM CaCl₂, pH 9.0). For surface labeling of NHE3, cells were incubated with 0.5 mg/ml NHS-SS-biotin (biotinylation solution; Pierce Chemical) for 20 min and repeated once. After labeling, cells were washed three times with the quenching buffer (20 mM Tris and 120 mM NaCl, pH 7.4) to scavenge the unreacted biotin. Cells were washed three times with ice-cold phosphate-buffered saline and solubilized with 0.8 ml N⁺ buffer (60 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM KCl, 5 mM Na₃EDTA, 3 mM EGTA, and 1% Triton X-100). Lysate, representing the total postnuclear fractions, was incubated with streptavidin-agarose beads for 16 h (overnight). After avidin-agarose precipitation, the supernatant was retained as the intracellular fraction. The avidin-agarose beads were washed five times in N⁺ buffer to remove nonspecifically bound proteins. The avidin-agarose bead bound proteins, representing plasma membrane NHE3, were solubilized in equivalent volumes of loading buffer (5 mM Tris-HCl, pH 6.8, 1% SDS, 10% glycerol, and 1% 2-mercaptoethanol), boiled for 5 min, size-fractionated by SDS-PAGE (10% gels), and then electrophoretically transferred to nitrocellulose. After blocking with 5% nonfat milk, the blots were probed with a monoclonal anti-HA antibody. Western analysis and the quantification of the surface fraction were performed using the Odyssey system and Odyssey software (LI-COR). Multiple volumes for each total, surface, cytosol sample were used with linear regression with intensity of signal to obtain a single value for each sample. Percentage of surface NHE3 was calculated from the ratio [(surface NHE3 signal/total NHE3 signal) x dilution factor of surface and total NHE3 samples] and expressed as percentage of total NHE3.

Metabolic Pulse-Chase Labeling
To measure the newly synthesized NHE3 that was delivered to the plasma membrane, PS120/NHE3-WT and NHE3-S719A cells in Met/Cys-free DMEM medium for 1 h at 37°C were pulse labeled in the same medium containing Tran³⁵S-Label reagent (Met/Cys, 0.3 mCi/ml; Valeant Pharmaceuticals, Costa Mesa, CA) for 30 min. The extracellular pulse media were quickly removed by washing with cold DMEM. Cells were then chased for 0, 30, 60, 120, and 180 min at 37°C in PS120 media containing 10x nonradiolabeled Cys and Met. At each time point, cells were cooled to 4°C, and cell surface biotinylation was performed as described above. Subsequently, the cells were lysed, and biotinylated surface proteins were isolated by streptavidin beads and IP with anti HA-antibody. Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose. Detection of ³⁵S-labeled NHE3 and NHE3-S719A mutant were after 24–48 h by using the Storm 860 PhosphorImager system (GE Healthcare). Intensity measurements of ³⁵S-labeled NHE3-WT and NHE3-S719A mutant were quantified with MetaMorph software (Molecular Devices, Sunnyvale, CA). Western analysis was then performed on the same samples using anti-HA antibody by the Odyssey system.

Exocytic Insertion of NHE3 into the Plasma Membrane
To measure exocytic insertion of NHE3 (also called endocytic recycling), N-hydroxysuccinimide (NHS)-reactive sites on proteins on the PS120 cell surface were first blocked by pretreatment with sulfo-NHS-acetate with some slight modifications (Cha et al., 2006). Cells were rinsed twice with PBS-Ca-Mg (PBS with 0.1 mM Ca²⁺ and 1 mM Mg²⁺) at 4°C. The apical surface was then exposed to 1.5 mg/ml sulfo-NHS-acetate in 0.1 M sodium phosphate, pH 7.5, and 0.15 M NaCl (3 times 40 min at 4°C) to saturate NHS-reactive sites on the cell surface. After quenching (PBS containing 1 mM MgCl₂, 0.1 mM CaCl₂, and 100 mM glycine) for 20 min at 4°C, the cells were rinsed with PBS and then incubated in serum-free normal PS120 media at 37°C for 10 min to allow intracellular NHE3 to reach the plasma membrane (exocytosis). Cells were then treated with 0.5 mg/ml sulfo-NHS-SS-biotin as described above and lysed with N⁺ buffer. The biotinylated fraction, which represents newly inserted surface proteins, was precipitated by streptavidin-agarose, and the precipitate was subjected to SDS-PAGE and Western blotting with anti-HA antibody, as described above.

Endocytosis of NHE3
Endocytosis was measured by a protocol described previously (Cha et al., 2006). PS120 cells expressing NHE3-WT and NHE3-S719A mutant were surface-labeled with sulfo-NHS-SS-biotin at 4°C. Cells were then warmed to 37°C for 0, 5, 10, 15, and 30 min, respectively, and rinsed with cold phosphate-buffered saline twice at 4°C. Surface biotin was cleaved by rocking cells with 150 mM reduced glutathione (GSH) containing buffer at 4°C for 30 min. The biotin bound to freshly endocytosed proteins is protected from the GSH cleavage. Cells were solubilized in N⁺ buffer, and biotinylated proteins were retrieved with streptavidin-agarose affinity precipitation, and internalized and total cellular NHE3 was detected by Western analysis. Intensities of each band were quantitated using Odyssey software. Endocytosis was normalized to surface NHE3 or NHE3-719S present initially.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Identification of a Unique NHE3 Phosphorylation Site
NHE3 under basal conditions is phosphorylated. To identify the phosphorylation sites in NHE3, VSV-G–tagged rabbit NHE3 (E3V) expressed in PS120 cells was immunoprecipitated by monoclonal antibodies against the VSV-G epitope and resolved by 1D SDS-PAGE. The immunoprecipitated NHE3 was visualized by Coomassie Blue stain (Figure 1A) and further confirmed by SDS-PAGE and Western blot with anti-VSV-G antibody (Figure 1B). NHE3 was excised from the gel and digested by trypsin. The generated tryptic peptides were then analyzed by liquid chromatography tandem mass spectrometry (nanoLC-MS/MS), which identified a total of 13 unique peptides corresponding to 30% of the protein sequence (Figure 1C). As shown in Figure 1D, two of the identified peptides, one doubly charged (m/z 500.28) and one triply charged peptide (m/z 636.33), showed a characteristic loss of phosphoric acid (loss of 98 Da), which corresponds to the conversion of either phosphoserine or phosphothreonine into dehydroalanine or dehydroaminobutyric acid, respectively. Analysis of the doubly charged phosphopeptide identified the specific phosphorylation site to be S⁵⁵⁴ (₅₅₂RGpSLAFIR₅₅₉; Figure 1D, top), which is a known protein kinase A (PKA) phosphorylation site. Complete y- and b-ion series were identified, both confirming S⁵⁵⁴ as being phosphorylated by the conversion of phosphoserine into dehydroalanine. MS/MS analysis of the triply charged phosphopeptide identified S⁷¹⁹ (₇₁₅ELELpSDPEEAPDYYEAEK₇₃₂) as a unique phosphorylation site in NHE3, not previously identified (Figure 1D, bottom). Phosphorylation of S⁷¹⁹ was also confirmed by a series of y- and b-ion series as described above. We focused further attention on this newly identified S⁷¹⁹ phosphorylation site.

View larger version (51K): [in this window] [in a new window]   Figure 1. A novel phosphorylation site at NHE3-S719 was identified by nanoLC-MS/MS analysis of immunopurified NHE3. (A) Approximately 2 µg of NHE3 was purified by IP using anti-VSV-G monoclonal antibody from PS120/NHE3-VSV-G cells. Note duplicate lanes of purified NHE3 were separated by SDS-PAGE and stained with Coomassie Blue. The amount of NHE3 was estimated by comparison with amounts of BSA loaded on the same gel. (B) The purified NHE3 was confirmed by Western blot analysis using anti-VSV-G antibody. 1% of the total purified NHE3 was used in the analysis (lane 2). Anti-FLAG antibody was used as a negative control for IP (lane 1). (C) Sequence coverage of NHE3 by nanoLC-MS/MS analysis: 50% coverage of the entire C terminus (underlined) was achieved. The two phosphorylation sites found were S554 and S719, which are shown in a black square/oval, respectively. Peptides identified (16) by mass spectroscopy are in bold, with overlapping peptides separated by a vertical line. (D) Mass spectroscopy: MS/MS analysis of the identified two peptides, one with a doubly charged phosphopeptide identified the phosphorylation site at S554 (top) and the other with a triple charged phosphopeptide identified the S719 phosphorylation site (bottom).

In Vitro Phosphorylation of NHE3 S⁷¹⁹ by Recombinant CK2

View larger version (32K): [in this window] [in a new window]   Figure 2. Recombinant protein kinase CK2 phosphorylates S719 of NHE3 in vitro. (A) The newly identified S719 phosphorylation site is conserved across mammalian species in both NHE3 and NHE5 (the NHE isoform that is most related to NHE3). Please note that the S719 is surrounded on both N- and C-terminal sides by negatively charged amino acids. *, identity; :, homologous residues; ., nonconserved residues. (B) An in vitro kinase reaction was carried out at 30°C with [32P-]ATP containing purified recombinant CK2 at several amounts (375, 75, and 7.5 ng of protein) and 100 µM synthetic peptides containing S719 or A719 of NHE3. After 10 min, each reaction done in duplicate was terminated. Average radioactivity counts of two separate experiments are shown.

View larger version (33K): [in this window] [in a new window]   Figure 3. S719A but not S719D mutation significantly reduced NHE3 activity in PS120 cells. Basal transport activity of NHE3-WT and NHE3-S719A and NHE3-S719D mutants were measured in stably transfected PS120 cells. Cells were loaded with BCECF-AM dye in Na+/NH4Cl medium before being mounted in a spectrofluorometer cuvette. NHE3 activity was then assessed as the rate of Na+-dependent cell pH recovery from an acid load. Results shown are from three to four experiments. Kinetic parameters from three to four experiments are shown as mean ± SEM.

View larger version (13K): [in this window] [in a new window]   Figure 4. Na+/H+ exchange activities of NHE3-WT is sensitive to CK2-specific inhibitors but NHE3-S719A mutant in resistant to DMAT inhibition. PS120/HA-NHE3-WT and HA-NHE3-S719A cells were grown on glass coverslips at 60–80% confluence. Cells on the coverslips were simultaneously loaded with BCECF dye and treated (except control) with CK2 inhibitors DMAT (A) or TBB (B) (at indicated concentrations, 0–30 µM) in Na+/NH4Cl medium for 20 min before being mounted in a spectrofluorometer cuvette. TBB and DMAT were also present in TMA and Na+ solutions. Na+/H+ exchange rates (H+ efflux) are shown from a single experiment (in triplicate), which was repeated three times with similar results. (C) Similar studies on NHE3 activity were performed with TBB and DMAT (30 µM) in Caco-2/HA-NHE3 cells. Results shown are means ± SEM of initial rates, with results expressed as percentage of each experiment's untreated controls. N is number of experiments. P values are comparison of percentage of inhibition by DMAT and TBB.

Inhibition of NHE3 activity was also demonstrated in an intestinal epithelial cell model, Caco-2/BBE. In Caco-2/HA-NHE3-WT cells, both TBB and DMAT inhibited NHE3 activity (Figure 4C). However, 30 µM TBB had a significantly larger inhibitory effect on NHE3 compared with DMAT (when both effects were expressed as percentage of inhibition of basal NHE3 activity), also suggesting a role for both CK2 and CK1 (inhibited by high concentration of TBB but not DMAT) in NHE3 regulation in Caco-2 cells.

CK2 Associates with NHE3
CK2 has been shown to bind to interacting proteins in addition to phosphorylating them (Shi et al., 2002; Bildl et al., 2004; Treharne et al., 2007). NHE3 is regulated as part of multiprotein complexes. To determine whether CK2 associated with NHE3 as part of basal function, coimmunoprecipitation was performed using PS120/HA-NHE3 cells (Figure 5A). Cell lysate prepared from PS120 cells stably expressing HA-tagged NHE3 was incubated with anti-HA conjugated to agarose beads. As a negative control, equal volume of the same cell lysate was incubated with anti-VSV-G agarose (NHE3 was not VSV-G epitope-tagged in these studies). As shown in Figure 5A, CK2 but not CK2β coimmunoprecipitated with NHE3. The relationship between NHE3 and CK2 was further examined, asking whether the CK2 phosphorylation of NHE3 at S⁷¹⁹ affected their coprecipitation. HA-NHE3 was IP from total lysates of PS120/HA-NHE3-WT and HA-NHE3-S719A cells, respectively. As shown in Figure 5B, CK2 was coimmunoprecipitated equally with NHE3-WT and the NHE3-S719A mutant, indicating that CK2 phosphorylation of NHE3 is not required for the binding of CK2 with NHE3. This also suggests that the binding of CK2 occurs at sites other than at which NHE3 is phosphorylated.

View larger version (35K): [in this window] [in a new window]   Figure 5. NHE3 physically associates with CK2. (A) NHE3-WT Coimmunoprecipitates CK2. PS120/HA-NHE3-WT cells were grown in 10-cm dishes and serum starved for 3 h. Then, 1.5 mg of total cell lysate was mixed with anti-HA agarose or anti-VSV-G agarose (negative control) overnight at 4°C in a rotator. After five gentle washes, proteins were eluted by adding 50 µl of SDS loading buffer. Proteins were separated by SDS-PAGE, blotted to nitrocellulose membrane, and probed with anti-HA, anti-CK2, and anti-CK2β antibodies, as indicated. A negative control was PS120/HA-NHE3 IP with anti-VSVG antibodies. Post-IP refers to supernatant after removal of IP. (B) NHE3-S719A coimmunoprecipitates CK2 in PS120/HA-NHE3-S719A cells. Cells were grown in 10-cm dishes and serum starved for 3 h. Cell lysate was prepared in lysis buffer including 1 mM Na3VO4, and 1% Triton X-100 with protease inhibitor cocktail. Then, 1.5 mg of total cell lysate was mixed with anti-HA agarose overnight at 4°C in a rotator. After five gentle washes, proteins were eluted by adding 50 µl of SDS loading buffer. Proteins were separated by SDS-PAGE, blotted to nitrocellulose membrane and probed with anti-HA, anti-CK2, and anti-CK2 antibodies, as indicated. PS120/NHE3-VSVG (not HA-tagged) cells were studied on parallel as a negative control.

CK2 Directly Binds the NHE3 C Terminus In Vitro

View larger version (56K): [in this window] [in a new window]   Figure 6. Binding of CK2 with C-terminal region of NHE3 at aa 590-667. (A) Pull-down by CK2 recombinant protein of different fragments of NHE3 C terminus (fragments F1–4). Five or 10 µl of glutathione-Sepharose beads with GST-CK2 attached (top) were incubated overnight with 3 µg/ml His6-tagged purified F1 (aa 475-589), F2 (aa 590-667), F3 (668-747), and F4 (748-832) proteins separately. After washing, proteins were eluted and separated with SDS-PAGE (14% gel) and transferred to nitrocellulose membranes. Protein bands were visualized by Ponceau S staining. Only the F2 fragment was detected in the pull-down by Ponceau S shown in the bottom panel. (B) Far Western blot (overlay assays) showed direct binding of NHE3-F2 domain with CK2. Purified GST-CK2 fusion protein (5 µg/ml) was incubated with an immunoblot that contained His6-F1, F2, F3, and F4 fragments of NHE3 C terminus (3 µg of each), and they were separated by SDS-PAGE and transferred to nitrocellulose. Input of the four fragments transferred to nitrocellulose membrane were detected by Ponceau S is shown in the top panel. Binding of CK2 was only to the F2 fragment as detected by immunoblot with anti-CK2 antibody (bottom).

View larger version (37K): [in this window] [in a new window]   Figure 7. Amount of plasma membrane NHE3-S719A is reduced as determined by surface biotinylation assay. PS120 cells stably expressing HA-NHE3-WT and HA-NHE3-S719A were grown in 10-cm dishes and biotinylated with NHS-SS-biotin. Cells were then lysed in equal volumes of HEPES buffer containing 1% Triton X-100 at 4°C. Of the 0.8 ml of cell lysate, 0.72 ml was incubated with streptavidin-agarose beads to isolate the biotinylated proteins. Amount of NHE3 present in the biotinylated fractions was considered as surface NHE3. Biotinylated protein was eluted from the beads with 80 µl of sample buffer. The surface protein samples were 9 times concentrated compared with the total cell lysate sample. Multiple dilutions of total, surface and nonavidin precipitated (referred to as cytosolic) fractions were loaded (volume) in SDS- gels. NHE3 protein abundance was quantified by immunoblot with anti-HA antibody (A) by using anti-GAPDH as a loading control. The intensity of each band was quantitated using Odyssey software and the amount of NHE3 calculated in each fraction using linear regression of the different sample volumes and intensity. Bar graphs show the calculated mean ± SEM amount of surface NHE3 as percentage of total NHE3 (described in Materials and Methods) from similar experiments (B). NHE3-S719A mutant had 50% less surface NHE3 compared with wild-type (p < 0.01).

View larger version (34K): [in this window] [in a new window]   Figure 8. CK2 inhibitor DMAT reduces surface expression of NHE3 in PS120 cells. PS120 cells expressing NHE3 were grown in 10-cm dishes, cells were serum starved for 2 h and then treated with DMSO (control) or 100 µM DMAT for 30 min at 37°C, before biotinylation as in Figure 7. Detection and quantitation of total and surface amount of NHE3 with DMSO and DMAT treatments are shown in A. Surface expression of NHE3 was determined as a percent of total NHE3. Results are mean ± SEM from three experiments. (B) DMAT treated cells had 40% less surface NHE3 expression than DMSO-treated cells. The p value is comparison of surface NHE3 of wild-type NHE3 exposed to DMAT versus just DMSO (p < 0.01).

View larger version (54K): [in this window] [in a new window]   Figure 9. Delivery of newly synthesized NHE3 is decreased in NHE3-S719A mutant, whereas plasma membrane retention is normal. (A–C) Metabolic pulse-chase/surface biotinylation experiments were performed to determine the surface delivery of newly synthesized NHE3. PS120 cells expressing HA-NHE3-WT and HA-NHE3-S719A were starved of Met/Cys for 60 min and then pulse labeled with 0.3 mCi/ml [35S]Met/Cys for 30 min at 37°C. The cells were then chased in normal medium supplemented with unlabeled Cys and Met at 37°C for 0, 30, 60, 120, 150, and 180 min. After completion of each time, cells were chilled to 4°C, surface biotinylated, and lysates were precipitated with streptavidin and separated by SDS-PAGE. Then, biotinylated surface[35S]NHE3-WT and NHE3-S719A were detected by autoradiography and immunoblotted using HA-antibody (A). The intensity of 35S surface NHE3-WT and NHE3-S719A were quantitated by MetaMorph, normalized to amount of surface NHE3, and plotted versus time. (B) A single experiment. (C) Mean ± SEM of four experiments. Rate of delivery of newly synthesized NHE3-S719A mutant was decreased compared with wild-type NHE3. Wild-type NHE3 and NHE3-S719A both had similar retention after peak delivery of newly synthesized NHE3 (see C). All points in C except O were significantly different between wild-type NHE3 and NHE3-S719A (p at least <0.05).

View larger version (28K): [in this window] [in a new window]   Figure 10. NHE3 exocytosis but not endocytosis is reduced in the NHE3-S719A mutation. (A) Exocytosis: To measure the endocytic recycling (exocytosis), cell surface NHE3-WT or NHE3-S719A were blocked by sulfo-NHS acetate at 4°C for 60 min. Cells were then washed and temperature increased to 37°C in serum-free PS120 medium for 10 min to allow arrival of new NHE3 to the surface. As controls, one set of cells were kept at 4°C throughout, and one set of cells were handled as the others but without sulfo-NHS-acetate blocking. NHE3 was identified by IB. Total amount of biotinylated surface NHE3 without sulfo-NHS acetate in cells kept at 4°C is shown in lane 1. Amount of surface NHE3 present after blocking with sulfo-NHS-acetate (0 min) is shown in lane 2. Amount of NHE3-WT and NHE3-S719A at the plasma membrane in 10 min at 37°C is shown in lane 3. After 10 min at 37°C, there was much less newly arrived surface NHE3-S719A than NHE3-WT. (B and C) Endocytosis: PS120/HA-NHE3-WT and PS120/HA-NHE3-S719A cells were exposed to sulfo-NHS-SS-biotin at 4°C for 40 min. After washing out of nonbound biotin, biotinylated cells were warmed to 37°C for indicated times. Then, surface biotin was removed by exposure to reduced glutathione. Internalization of NHE3 from the cell surface was detected as the biotinylated NHE3 protected from the reductive cleavage by reduced-glutathione at 4°C. The internalized biotinylated NHE3 was precipitated with strepavidin beads, separated, and immunoblotted. Samples without reductive cleavage were used as controls to indicate surface NHE3 and to normalize endocytosed NHE3 (lane 1, labeled C top row, in which 50% of sample was loaded on gel). Second row (lane 1, labeled C) was total NHE3 (6% of total sample was loaded on gel). (C) Internalized surface NHE3 amount at different times was quantitated using Odyssey software and expressed as a percentage of initial surface amount, after normalization to total amount of NHE3. Zero time internalization was used as background and subtracted from each time point. Results shown are mean ± SEM of three experiments. Internalization rate of surface NHE3 was similar in NHE3 wild type (open circles) and NHE3-719A mutant cells (closed circles).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CK2 phosphorylation of NHE3 regulates exchanger activity by increasing the percentage of total NHE3 on the plasma membrane by affecting its trafficking. CK2 inhibitors reduced NHE3 activity by 40% and reduced the percent NHE3 on the plasma membrane by 50%, consistent with the effects being largely by affecting trafficking. The CK2 effects on trafficking consist of increased endocytic recycling (exocytosis) with no effect in rate of endocytosis or total NHE3 expression. In addition, CK2 phosphorylation of NHE3 was necessary for normal delivery of newly synthesized NHE3 to the plasma membrane. Quantitative effects were somewhat different studying the S719A mutant, which reduced NHE3 activity by 70% while reducing plasma membrane amount NHE3 by 60%. Possibilities for the difference between CK2 drug inhibitors and mutation of S⁷¹⁹ include that S719A affects NHE3 activity by a mechanism in addition to serving as the CK2 phosphorylation site or that chronic effects of the S719A mutation lead to further secondary changes that do not necessarily directly involve CK2.

The search for unrecognized phosphorylation sites of NHE3 was based on our belief that in spite of NHE3 phosphorylation sites having been identified for cAMP and SGK1, it was likely that there were additional sites present (Donowitz and Li, 2007). This was because, under basal conditions, the S/T phosphatase inhibitor okadaic acid stimulated NHE3 (Levine et al., 1995), implying a role for NHE3 phosphorylation in stimulating NHE3, which cAMP inhibits. In addition to the opposite effects on NHE3 activity of phosphorylation by CK2 versus cAMP, another important difference of the effects of CK2 versus PKA and SGK1, is that the latter are brought into the NHE3 complex by binding NHERF1 (PKAII) and/or NHERF2 (PKAII, SGK1), whereas CK2 is brought into the complex by directly binding NHE3.

A role in NHE3 regulation for CK2 was not surprising. It is a ubiquitous kinase with at least the widely quoted 300 recognized substrates, including all classes of proteins that are found in most cellular compartments (Litchfield, 2003; Olsten and Litchfield, 2004; Meggio and Pinna, 2005). Although nuclear proteins seem to be the most affected (Faust and Montenarh, 2000), CK2 regulates multiple transport proteins in many cell compartments (Table 1).

Although CK2 generally is thought to function in a constitutive manner, as with the identified NHE3 regulation, there is emerging evidence that it also takes part in regulated functions (Olsten et al., 2005; Theis-Febvre et al., 2005; Allen et al., 2007). Multiple mechanisms have been identified by which CK2 regulates transporter function. In its interactions with the small-conductance Ca²⁺ activated potassium channels, CK2 is in a complex with the channel, calmodulin (CaM), and PP2A (Allen et al., 2007). It regulates channel gating by phosphorylating CaM in the multiprotein complex leading to reduction of the Ca²⁺ sensitivity of the channel (Arrigoni et al., 2004). Cystic fibrosis transmembrane conductance regulator (CFTR) binds and is regulated by CK2 (Treharne et al., 2007). CK2 binds wild-type CFTR at approximately aa 505 (very close to the common mutation Phe 508) and phosphorylates CFTR at S⁵¹¹, which seems necessary for cAMP gating of the channel. Binding of CK2 to CFTR requires the presence of the S⁵¹¹. That is, the substrate phosphorylation site is necessary for nearby CK2 binding. CK2 phosphorylates S¹⁴⁸⁰ of the N-methyl-D-aspartate (NMDA) receptor, which leads to reduced binding of this transporter to postsynaptic density 95/disc-large/zona occludens domains of PSD95 and SAP102, as well as reduced NMDA surface expression. Thus, there are examples of CK2 phosphorylating transporters and also binding to and phosphorylating the same part of the protein (CFTR). However, unique aspects of transporter regulation by CK2 demonstrated for NHE3 include regulation of basal transport function, contribution to trafficking with effects on exocytosis and delivery of newly synthesized NHE3 to the plasma membrane, and binding to one site but phosphorylating a separate domain in the protein.

We showed here that CK2 phosphorylation of NHE3-S719 stimulates NHE3 activity. Intriguingly, we demonstrated previously that NHE3 truncated at aa 690 (NHE3-690), in which S⁷¹⁹ was also removed, exhibits a threefold increase in activity due to a threefold increase in the amount of NHE3 at the cell surface. If S⁷¹⁹ were required for the basal stimulation of NHE3, removal of S⁷¹⁹ would have been expected to decrease the basal NHE3 activity. Why did we observe an increase of activity in NHE3-690 instead? We previously identified two endocytosis domains in the C terminus of NHE3: one located at aa 690-756 (which includes S⁷¹⁹) and the other at aa 757-832 (Akhter et al., 2000). Regulation of WT NHE3 is primarily through trafficking, which is dictated by the balance or interplay between exocytosis (via mechanisms such as CK2 mediated S⁷¹⁹ phosphorylation) and endocytosis (via endocytic signals as mentioned above) (Donowitz and Li, 2007). These results indicate that NHE3 aa, which normally stimulate NHE3 activity (S⁷¹⁹), are interspersed with domains that normally inhibit NHE3 (two endocytosis signals) and show that there is intermixing of stimulatory and inhibitory domains in the NHE3 C terminus. How these interspersed signals functionally interact will be important to understand in the future.

In addition to data with mutation of the NHE3 CK2 phosphorylation site S⁷¹⁹, elucidation of the functional role of CK2 phosphorylation of NHE3 was partially based on study of two pharmacologic inhibitors with high specificity for CK2. DMAT has an IC₅₀ value of 0.14 µM for CK2, with no effect on CK1 at concentrations up to 100 µM (at least 700x less sensitive), whereas TBB has an IC₅₀ value of 0.5 µM for CK2 but inhibits CK1, with an IC₅₀ value of 25 µM (50x less sensitive) (Sarno et al., 2002, 2005; Ruzzene et al., 2002; Pagano et al., 2004). Although there are several additional putative CK2 phosphorylation sites in the NHE3 C terminus, the fact that there was no significant effect on NHE3-S719 activity of DMAT at up to 100 µM was consistent with there being only a single NHE3 CK2 phosphorylation site that is linked to regulation of basal NHE3 activity. In contrast, the fact that TBB at high concentrations inhibited NHE3-S719A suggests that a kinase in addition to CK2 contributes to basal NHE3 activity. We suggest that, given that CK1 is inhibited by high concentrations of TBB and that the NHE3 C terminus contains several putative CK1 phosphorylation sequences, it is likely that CK1 also contributes to basal NHE3 activity.

This study identified a newly recognized phosphorylation site in the NHE3 C terminus and showed that it was phosphorylated by CK2 and contributed to setting basal NHE3 activity by increasing the amount of NHE3 on the plasma membrane. Moreover, we identified the specific aspects of NHE3 trafficking that were CK2 dependent, which included exocytosis and delivery of newly synthesized NHE3. In contrast, total NHE3 expression, endocytosis and plasma membrane retention were not CK2 dependent. These studies indicate that the NHE3 C terminus not only acts as a scaffold for some of its regulatory proteins but also there seems to be organization of these proteins (in this case, CK2 binding at one site and phosphorylation at another site) along the C terminus to control NHE3 activity.

	ACKNOWLEDGMENTS

 
We acknowledge the expert editorial assistance of H. McCann. This study was supported in part by the National Institute of Health, National Institute of Diabetes and Digestive and Kidney Diseases grants R01-DK26523, R01-DK61765, K01-DK62264, P01-DK072084, and R24-DK64388 (The Hopkins Basic Research Digestive Diseases Development Core Center), and the Hopkins Center for Epithelial Disorders and NIH Roadmap Grant U54RR020839.

	Footnotes

 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08-01-0019) on July 9, 2008.

^¶ These authors contributed equally to this work.

Present address: Department of Neurobiology, Max-Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.

Address correspondence to: Mark Donowitz (mdonowit{at}jhmi.edu) or Xuhang Li (xuhang{at}jhmi.edu)

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Akhter, S., Cavet, M. E., Tse, C. M., and Donowitz, M. (2000). C-Terminal domain of Na+/H+ exchanger isoform 3 are involved in the basal and serum-stimulated membrane trafficking of the exchanger. Biochemistry 39, 1990–2000.[CrossRef][Medline]

Allen, D., Fakler, B., Maylie, J., and Adelman, J. P. (2007). Organization and regulation of small conductance Ca2+-activated K+ channel multiprotein complexes. J. Neurosci 27, 2369–2376.[Abstract/Free Full Text]

Arrigoni, G., Marin, O., Pagano, M. A., Settimo, L., Paolin, B., Meggio, F., and Pinna, L. A. (2004). Phosphorylation of calmodulin fragments by protein kinase CK2. Mechanistic aspects and structural consequences. Biochemistry 43, 12788–12798.[CrossRef][Medline]

Bildl, W. et al. (2004). Protein kinase CK2 is coassembled with small conductance Ca(2+)-activated K+ channels and regulates channel gating. Neuron 43, 847–858.[CrossRef][Medline]

Bolanos-Garcia, V. M., Fernandez-Recio, J., Allende, J. E., and Blundell, T. L. (2006). Identifying interaction motifs in CK2beta–a ubiquitous kinase regulatory subunit. Trends Biochem. Sci 31, 654–661.[CrossRef][Medline]

Bone, D. B., Robillard, K. R., Stolk, M., and Hammond, J. R. (2007). Differential regulation of mouse equilibrative nucleoside transporter 1 (mENT1) splice variants by protein kinase CK2. Mol. Membr. Biol 24, 294–303.[CrossRef][Medline]

Cai, Y. et al. (2004). Calcium dependence of polycystin-2 channel activity is modulated by phosphorylation at Ser812. J. Biol. Chem 279, 19987–19995.[Abstract/Free Full Text]

Cha, B., Tse, M., Yun, C., Kovbasnjuk, O., Mohan, S., Hubbard, A., Arpin, M., and Donowitz, M. (2006). The NHE3 juxtamembrane cytoplasmic domain directly binds ezrin: dual role in NHE3 trafficking and mobility in the brush border. Mol. Biol. Cell 17, 2661–2673.[Abstract/Free Full Text]

Donowitz, M., and Li, X. (2007). Regulatory binding partners and complexes of NHE3. Physiol. Rev 87, 825–872.[Abstract/Free Full Text]

Donowitz, M., and Welsh, M. J. (1986). Ca2+ and cAMP in regulation of intestinal Na, K and Cl transport. Annu. Rev. Physiol 48, 135–150.[Medline]

Donowitz, M., Wicks, J., Madara, J. L., and Sharp, G. W. (1985). Studies on role of calmodulin in Ca2+ regulation of rabbit ileal Na and Cl transport. Am. J. Physiol 248, G726–G740.[Medline]

Faust, M., and Montenarh, M. (2000). Subcellular localization of protein kinase CK2. A key to its function? Cell Tissue Res 301, 329–340.[CrossRef][Medline]

Filhol, O., Martiel, J. L., and Cochet, C. (2004). Protein kinase CK 2, a new view of an old molecular complex. EMBO Rep 5, 351–355.[CrossRef][Medline]

Glavy, J. S., Horwitz, S. B., and Orr, G. A. (1997). Identification of the in vivo phosphorylation sites for acidic-directed kinases in murine mdr1b P-glycoprotein. J. Biol. Chem 272, 5909–5914.[Abstract/Free Full Text]

Gronborg, M., Bunkenborg, J., Kristiansen, T. Z., Jensen, O. N., Yeo, C. J., Hruban, R. H., Maitra, A., Goggins, M. G., and Pandey, A. (2004). Comprehensive proteomic analysis of human pancreatic juice. J. Proteome Res 3, 1042–1055.[CrossRef][Medline]

Janecki, A. J., Montrose, M. H., Zimniak, P., Zweibaum, A., Tse, C. M., Khurana, S., and Donowitz, M. (1998). Subcellular redistribution is involved in acute regulation of the brush border Na+/H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger. J. Biol. Chem 273, 8790–8798.[Abstract/Free Full Text]

Jones, S., and Yakel, J. L. (2003). Casein kinase ii (protein kinase ck2) regulates serotonin 5-ht(3) receptor channel function in ng108̵