Phosphorylation of S955 at the Protein Kinase A Consensus Promotes Maturation of the Subunit of the Colonic H+,K+-ATPase
Juan Codina*,
Jingfang Liu*,
Anthony J. Bleyer*,
Raymond B. Penn and
Thomas D. DuBose, Jr.*
* Sections on Nephrology and Molecular Medicine, Department of Internal Medicine, Center for Human Genomics, Wake Forest University Health Sciences, Winston-Salem, North Carolina
Address correspondence to: Dr. Thomas D. DuBose, Jr., Department of Internal Medicine, Wake Forest University Health Sciences, Medical Center Boulevard, Winston-Salem, NC 27157. Phone: 336-716-2715; Fax: 336-716-2273; E-mail: tdubose{at}wfubmc.edu
Received for publication January 13, 2006.
Accepted for publication April 12, 2006.
All the subunits of the Na+,K+-ATPases and H+,K+-ATPases havea protein kinase A (PKA) consensus sequence near or in the ninthtransmembrane domain. The role of this domain in influencing subunit synthesis/degradation, plasma membrane localization,and 86Rb+ uptake has not been established for the subunit ofthe colonic H+,K+-ATPase. This study examined the effect ofmutating S955 (within the PKA consensus site of the subunitof the colonic H+,K+-ATPase [HK2]) to alanine (S955/A) or asparticacid (S955/D) on subunit expression and function. The resultsdemonstrate that a negatively charged amino acid at position955 of HK2 promotes higher expression levels of both whole-celland plasma membrane–localized HK2. Moreover, inhibitionof PKA reduced expression of wild-type HK2 and associated 86Rb+uptake. Last, the activity of the HK2 S955/A was rescued bytreatment with 4-phenylbutyric acid, a compound that was shownpreviously to restore function to the cystic fibrosis transmembraneconductance regulator.
The X+,K+-ATPase family of transporters consists of six different subunits and five different subunits (1–3). The commonfunction of these membrane transporters is to accomplish cellularuptake of K+ in exchange for either Na+ or H+. The , or catalytic,subunit consists of 10 transmembrane-spanning regions and bindsto the heavily glycosylated subunit at the fourth extracellularloop (1,4). The colonic H+,K+-ATPase also is an / heterodimer.The subunit (HK2) has a molecular weight of approximately 100kD, absorbs potassium from the distal nephron in exchange forH+ or Na+, and contains the binding sites for specific inhibitors.The abundance of HK2 mRNA and protein in the kidney is upregulatedpredominately in the outer medulla by chronic hypokalemia (5–7).HK2 integrity and function also are dependent on subunit assembly.The gastric H+,K+-ATPase (HK1) assembles with a unique subunitin the kidney and the stomach (G) (8), whereas 1-Na+,K+-ATPase(NK1) assembles specifically with 1-Na+,K+-ATPase (NK1) (9).HK2, in contrast, does not assemble with a unique subunit butrather assembles with NK1 in the renal medulla, the distal colon(10,11), and Sf9 cells (12).
In addition to these differences, members of the X+,K+-ATPasefamily of transporters exhibit different pharmacologic inhibitorprofiles. The Na+ pump is sensitive to ouabain (3,13) and insensitiveto Sch-28080, whereas gastric H+,K+-ATPase is insensitive toouabain but sensitive to Sch-28080 and omeprazole (8,14,15).In contrast, the colonic H+,K+-ATPase is insensitive to Sch-28080and partially sensitive to ouabain (16) or oligomycin (12).
Recent studies in our laboratory have demonstrated that HK2function is critically dependent on an intact carboxy-terminusfor 86Rb+ uptake, ATPase activity, and stable assembly of HK2/NK1(17,18). Chimeric constructs in which the carboxy-terminus ofHK2 is replaced by the carboxy-terminus of NK1 are fully functional,but chimeras that contain the carboxy-terminus of HK1 (17,18)are only partially functional. Moreover, only the HK2 carboxy-terminusand not the HK1 or NK1 carboxy-terminus associates with thetetraspanin protein CD63 (19), an interaction that seems toplay a role in the regulation of abundance of HK2 protein atthe plasma membrane (19).
All the subunits of the Na+,K+-ATPases (1, 2, and 3) and H+,K+-ATPases(HK1 and HK2) are highly similar in protein sequence, and allpossess a consensus sequence for protein kinase A (PKA)-mediatedphosphorylation (e.g., RRNSI in HK2) near the carboxy-terminus.On the basis of the topology predicted by the TM Predict Program(http://www.ch.embnet.org/software/TMPRED_form.html), this domainis in or near the ninth transmembrane domain. The transmembranelocation of the PKA domain is one of the arguments that havebeen used to question the physiologic relevance of the phosphorylationof NK1 by PKA (20). However, Vinceguerra et al. (21) demonstratedthat phosphorylation of NK1 by PKA is a required step for itsrecruitment into the plasma membrane. In cultured rat corticalcollecting duct and mouse mpkCCDC14 cells, the sodium-dependentrecruitment of NK1 to the plasma membrane requires phosphorylationof NK1 by a cAMP-independent, PKA-dependent mechanism (22).This study suggests that phosphorylation of NK1 by PKA occursin early stages of protein synthesis or / assembly and not inlater stages of plasma membrane recruitment.
To clarify the role of PKA in the maturation of HK2, we mutatedS955 to alanine (which cannot be phosphorylated) or asparticacid (which functions as a phosphorylated residue) and performedthe following experiments: (1) 86Rb+ uptake experiments to defineHK2 functionality at the plasma membrane, (2) biotinylationexperiments to assess HK2 expression at the plasma membrane,(3) immunoblots to quantify total HK2 protein abundance, and(4) metabolic labeling with [35S]methionine/cysteine to determinethe rates of synthesis and degradation of the various HK2 mutations.Our results demonstrate that S955 phosphorylation is necessaryfor full expression and functionality of HK2. We further demonstratethat PKA inhibition reduces wild-type HK2 expression and 86Rb+uptake and that 4-phenylbutyric acid (4-PBA) can rescue theHK2 S955/A mutant.
Generation of Constructs
Mutation of S955 of HK2 to alanine was accomplished with theQuikChange II Site-Directed Mutagenesis Kit (Stratagene, LaJolla, CA) using the primer 5'-GGAAAACCCGGAGGAATGCCATCTTTCAGCAGGG-3',which changes S955 (TCC) to alanine (GCC), and the completecDNA of wild-type HK2 (23) as template. The PCR product wasused to transform the Escherichia coli XL1 blue supercompetents(Stratagene), from which DNA was purified and sequence was confirmedby double-stranded DNA sequencing. Similarly, HK2(S955/D) wasgenerated using the primer 5'-GGAAAACCCGGAGGAATGACATCTTTC-AGCAGGG-3',which changes S955 (TCC) to aspartic acid (GAC). Sequences thatencoded wild-type and mutant HK2 were subcloned into pcDNA3.1(+)-Neothat contained an amino-terminal c-myc cassette (17,19) as describednext.
Cloning of the c-myc Epitope at the Amino-Terminus of HK2(S955/A) or HK2(S955/D)
In previous experiments, we cloned the c-myc epitope at theamino-terminus of HK2 in pcDNA3.1(+)-Neo (17,19). This constructwas linearized with BstEII/XbaI, dephosphorylated with calfintestinal phosphatase, and used as a plasmid to clone the insertthat was generated by digestion with BstEII/XbaI of HK2(S955/A)or HK2(S955/D) in pcDNA3.1(+)-Neo. Double-stranded DNA sequencingwas performed to verify the constructs.
Transient Transfection of HEK-293 Cells
HEK-293 cells were transiently transfected with LipofectaminePLUS method (Invitrogen, Carlsbad, CA) as described previously(11,18). The combination of plasmids that were used in everytransfection is indicated in the figure legends, and all theexperiments were performed 48 h after transfection.
Metabolic Labeling of HEK-293 Cells
Metabolic labeling of HEK-293 cells was performed as per Caplanet al. (24). Forty-eight hours after transfection, the mediumwas replaced with fresh medium (5 ml) without methionine/cysteine.One hour later, 40 µCi of EASYTAG Express Protein LabelingMix (PerkinElmer, Wellesley, MA) per 5 ml of medium (withoutmethionine/cysteine) was added. Cells subsequently were processedaccording to the experimental design outlined in the legendof Figure 3. In all instances, the cells were rinsed with PBSat 4°C, and the proteins were extracted with extractingbuffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 150 mM NaCl, 1 mMPMSF, 3 mM benzamidine, 1 µg/ml soybean trypsin inhibitor,and 1% CHAPS) for 1 h at 4°C. The insoluble material wasremoved by centrifugation, equal quantities (1 mg) of extractedprotein were incubated with the mAb 9E10 (5 µl, 4 h, 4°C)directed against the c-myc epitope, and protein A/G PLUS agarose(20 µl of packed resin) was added (Santa Cruz Biotechnology,Santa Cruz, CA) for 2 h at 4°C to precipitate the c-myc–HK2proteins. The resin was rinsed extensively with extracting buffer,and bound protein was extracted for 1 h at room temperaturewith Laemmli sample buffer (25). The resin was removed by centrifugation,and the extracted protein was applied to a 10% SDS-PAGE. The[35S]-labeled immunoprecipitated proteins were detected by fluorography,as described previously (16). The intensity of the bands wasquantified with the Scion Image Program (Scion Corp., Frederick,MD).
Figure 3. The PKA domain of HK2 is required for protein stability. (A) HEK-293 cells were transiently transfected with c-myc–HK2 plus NK1, c-myc–HK2(S955/A) plus NK1, c-myc–HK2 plus NK1, or pcDNA3.1(+)-Neo plus NK1. The cells were incubated for 1 h at 37°C in methionine/cysteine-free medium. The labeling was started by adding 40 µCi of EASYTAG Express Protein Labeling Mix (PerkinElmer). At various times (15, 30, 60, and 180 min), the cells of one dish of every group were rinsed with PBS at 4°C, and the proteins were extracted with extracting buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 150 mM NaCl, 1 mM PMSF, 3 mM benzamidine, 1 µg/ml soybean trypsin inhibitor, and 1% CHAPS) for 1 h at 4°C. The synthesized [35S]HK2 was immunoprecipitated, as described in Materials and Methods, with a mAb against the c-myc epitope, then separated on a 10% SDS-PAGE. The immunoprecipitated [35S]HK2 was detected by fluorography, and intensity of the bands on the autoradiography film were quantified using the Scion Image Program. The numbers under the bands indicate intensity of the bands as a result of scanning the autoradiography film. (B) HEK-293 cells were labeled for 1 h with EASYTAG Express Protein Labeling Mix, as described above. The medium was removed and replaced by fresh medium that contained methionine and cysteine. At various times (0, 1, 2, and 3 h), the cells were washed with PBS at 4°C; the proteins were extracted and immunoprecipitated with a mAb directed against the c-myc epitope. The intensity of the bands on the autoradiography film was quantified with the Scion Image Program and presented as the percentage remaining after the [35S] labeling. The experiments were repeated three times with similar results.
Biotinylation of Plasma Membrane HK2
To define cell surface expression of the various HK2 constructs,we used the biotinylation approach described by Wong et al.(26), which takes advantage of the fact that the biotinylatedproteins are not internalized at 4°C. After biotinylation,the cells were washed with cold PBS, and the remaining biotinwas quenched with 10 ml of PBS that containing 50 mM Tris-HCl(pH 8.0). The cells were lysed with lysis buffer (see Figure 4legend) and extracted for 1 h at 4°C. Equal quantities (100µg) of extracted protein were used to quantify the totalHK2 protein by immunoblot analysis. The rest of the extract(1 mg of protein) was incubated with 10 µl of streptavidinagarose (Pierce, Rockford, IL) for 4 h at 4°C with continuousshaking. The resin was removed by centrifugation, and immunoblotanalysis was used to quantify the disappearance of HK2 proteinfrom the supernatant, which was taken as an index of biotinylation.
Figure 4. HK2(S955/A) localizes to the plasma membrane. HEK-293 cells that were transiently transfected with HK2 plus NK1, HK2(S955/A) plus NK1, HK2(S955/D) plus NK1, or pcDNA3.1(+)-Neo plus NK1 were washed with PBS at 4°C. Then 3 ml of PBS that contained 2 mg/ml EZlink sulfo-NHS-SS-biotin (Pierce) was added for 30 min at 4°C. The reaction was stopped by removing the biotin solution and rinsing the cells three times with 10 ml of PBS that contained 50 mM Tris-HCl (pH 8.0). The cells were lysed with lysis buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1 mM PMSF, 3 mM benzamidine, 1 µg/ml soybean trypsin inhibitor, and 1% CHAPS) and extracted for 1 h at 4°C, and the insoluble material was removed by centrifugation at 13,000 rpm for 10 min at 4°C. One aliquot (100 µg) of the total was used to quantify total HK2 protein by immunoblot analysis (A, top), and 20 µg was used to quantify total endogenous NK1 (A, bottom). The rest of the extract was incubated with 10 µl of streptavidin agarose (Pierce) for 4 h at 4°C with continuous shaking. The resin was removed by centrifugation, and immunoblot analysis was used to quantify HK2 or NK1 protein from the supernatant. The results of subunit unbound to the streptavidin resin are displayed in B. The bands were detected and quantified using the Odyssey Infrared Imaging System (Li-Cor) (27). Their intensity was multiplied by the volume of samples, and the unbound protein (as a percentage of total subunit expression) is indicated under each lane. Experiments were repeated three times with similar results.
Miscellaneous Reagents
Cell culture media were purchased from Invitrogen. The mAb againstthe c-myc epitope (9E10) and NK1 and against -actin were purchasedfrom Santa Cruz Biotechnology, the mAb against green fluorescenceprotein (GFP) was purchased from Covance (Princeton, NJ), andthe mAb against vasodilator-associated phosphoprotein was purchasedfrom BD Biosciences (San Jose, CA). The polyclonal antibodyagainst HK2 was raised and characterized in our laboratory (6).4-PBA was purchased from Sigma Chemical (St. Louis, MO). Biotinand streptavidin agarose were purchased from Aldrich Chemicals(St. Louis, MO). All other reagents were purchased from FisherScientific (Pittsburgh, PA) or Sigma Chemicals.
PKA Domain of HK2 Is Necessary to Sustain 86Rb+ Uptake
To define the role of the PKA domain in transport by HK2, wetransiently transfected HEK-293 cells with NK1 plus pcDNA3.1(+)-Neovector, wild-type HK2, HK2(S955/A), or HK2(S955/D). Figure 1demonstrates that HK2/NK1 supports ouabain-sensitive 86Rb+ uptake.Removal of the HK2 PKA domain by mutating S955 to alanine decreased86Rb+ uptake by approximately 50% compared with the control.Mutation of S955 to aspartic acid restored 86Rb+ uptake to valuesthat are equal to or slightly higher than the control group.Therefore, mutation analysis suggests that phosphorylation ofS955 is important in HK2 transporter function.
Figure 1. The protein kinase A (PKA) domain of -subunit of the colonic H+,K+-ATPase (HK2) influences 86Rb+ uptake. HEK-293 cells were transiently transfected with HK2 plus 1-subunit of the Na+,K+-ATPase (NK1), HK2 where S955 has been mutated to alanine [HK2(S995/A)] plus NK1, HK2 where S955 has been mutated to aspartic acid [HK2(S995/D)] plus NK1, or pcDNA plus NK1, as described in Materials and Methods. Transiently transfected HEK-293 cells were scraped, washed with PBS at 4°C, and resuspended in buffer A (145 mM NaCl, 1 mM KCl, 1.2 mM MgSO4, 2 mM Na2HPO4, 1 mM CaCl2, 200 µM bumetamide, 10 µM ouabain, and 32 mM HEPES [pH 7.4]) at 4°C and then equilibrated for 15 min with the same buffer in the presence or absence of 2 mM ouabain. 86Rb+ uptake was initiated by adding 10 µl of 86Rb+ (2 to 4 x 106 cpm in buffer A). After 15 min, the reaction was stopped by adding 1 ml of PBS that contained 1 mM BaCl2 (PBS-BaCl2) at 4°C. The cells were centrifuged at 5000 rpm for 2 min at 4°C. The pellet was washed with 1 ml of PBS-BaCl2 and centrifuged again. The pellet was resuspended in 400 µl of H2O; 200 µl was used to quantify 86Rb+ uptake by scintillation counting, and 100 µl was used to quantify proteins with the Lowry method (34). The difference in 86Rb+ uptake in the presence or absence of 2 mM ouabain was taken as ouabain-sensitive 86Rb+ uptake and expressed as pmol/mg protein per min. The wild and the different HK2 mutated at S955 were cloned in pcDNA3.1(+)-Neo, and NK1 was cloned in pcDNA3.1(+)-Zeo. The results represent the mean ± SEM of four independent experiments.
PKA Domain of HK2 Is Necessary to Sustain the Total Cellular Content of HK2 Protein
We performed experiments to assess the role of the HK2 PKA domainon the total cellular content of HK2 protein (Figure 2). Theresults demonstrate that in HEK-293 cells that were transientlytransfected with HK2 and carried the mutation S955 to alanine,cellular levels of HK2 protein were substantially lower thanthose of wild-type HK2 (i.e., in cells that were transfectedwith the wild-type construct). Conversely, expression of theS995/D mutant is similar to that of wild-type HK2.
Figure 2. The PKA domain of HK2 is necessary for cellular accumulation of HK2 protein. HEK-293 cells were transiently transfected, as described in Figure 1, and rinsed with PBS at 4°C, and proteins were extracted with 500 µl of extracting buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1 mM PMSF, 3 mM benzamidine, 1 µg/ml soybean trypsin inhibitor, and 1% CHAPS) for 1 h at 4°C. The insoluble material was removed by centrifugation at 13,000 rpm at 4°C for 10 min. The supernatant was kept, and the protein concentration was quantified by the Lowry method (34). Then 100 µg of protein was resolved on a 10% SDS-PAGE, transferred to a nitrocellulose membrane, and blotted against our anti-HK2 (6) (dilution 1:1000). The results are presented in the top panel. As a negative control, we used 25 µg of extracted protein to test expression of total endogenous NK1 by immunoblot analysis. The results of a representative experiment are displayed in the bottom panel. The immunoblot against NK1 was performed with a mAb commercially available from Santa Cruz Biotechnology. The experiments were repeated four times with similar results. Bands on the immunoblots were visualized and quantified using the Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE) (27).
Role of the PKA Domain of HK2 on Subunit Stability
One possible explanation for lower HK2(S955/A) protein levels(Figure 2) and function (Figure 1) is a lower rate of synthesisof HK2(S955/A) relative to that of HK2 or HK2(S955/D). It alsois possible that expressed HK2(S955/A) is degraded faster. Totest these possibilities, we performed metabolic labeling experimentsusing [35S]methionine/cysteine. In Figure 3A, the cells wereincubated at various intervals in methionine/cysteine-free mediumthat contained [35S]methionine/cysteine (40 µCi/5 ml).The synthesized subunit was monitored by autoradiography ofthe immunoprecipitated protein. The results demonstrate thatthe quantities of immunoprecipitated c-myc-HK2 and c-myc–HK2(S955/D)were very similar, whereas the abundance of c-myc–HK2(S955/A)was significantly less.
For assessment of the rate of degradation of expressed wild-typeor mutant HK2 proteins, cells were labeled with [35S]methionine/cysteine,then washed with medium that contained nonradiolabeled methionine/cysteine.Cells were harvested at time points up to 3 h for analysis ofc-myc–HK2 levels. The results (Figure 3B) demonstratethat the degradation rates of c-myc–HK2, c-myc–HK2(S955/A),and c-myc–HK2(S955/D) were very similar.
PKA Domain of HK2 Is Not Necessary for Localization of HK2 Protein at the Plasma Membrane
To assess subcellular distribution of HK2(S955/A), we performedbiotinylation experiments. We reasoned that if HK2, HK2(S955/A),and HK2(S955/D) migrate to the plasma membrane, then all subunitsshould be retained with similar efficiency by streptavidin agaroseafter biotinylation of the cell surface proteins at 4°C.However, if one of the mutants failed to migrate to the plasmamembrane, then it would not be retained efficiently in our biotinylationassay and would be quantified as an "unbound subunit" to thestreptavidin agarose. Figure 4 shows the results of a representativeexperiment. As in Figure 1, Figure 4A (top) demonstrates thatthe total cellular accumulation of HK2 and HK2(S955/D) exceededthat of HK2(S955/A). Figure 4B (top) depicts quantificationof the various forms of HK2 that did not bind to the streptavidinagarose and therefore are presumed to be of intracellular origin.The percentage of unbound HK2 is indicated. The results demonstratethat for all constructs, most (>90%) of the protein was boundto the resin. Figure 4B (bottom; used as control) demonstratesthat in all the transfections, endogenous NK1 is biotinylated(approximately 90%) and therefore in the plasma membrane.
That the difference in the rate of loss of [35S]methionine/cysteine-labeledHK2(S955/A) and wild-type HK2 or HK2(S955/D) is minimal (Figure 3B)and migration of HK2 to the plasma membrane does not seem tobe influenced by S955 modification (Figure 4) suggest that anegatively charge amino acid at position 955 is required inthe early stages of HK2 synthesis. A subpopulation of nascentHK2 protein, perhaps misfolded and cleared with rapid kinetics,is not represented in the fully mature proteins that were immunoprecipitatedin experiments in Figure 3. Once HK2 transitions through thisearly stage, it can migrate to the plasma membrane, where itis stable and fully functional.
Inhibition of the PKA Activity Decreases 86Rb+ Uptake by the HK2/NK1 Complex
To define the physiologic relevance of the PKA activity on 86Rb+uptake by the HK2/NK1 complex, we co-transfected HEK-293 cellswith HK2/NK1 with either the rabbit muscle heat-stable inhibitorof the PKA gene (PKI; accession number M23079), expressed asGFP chimera (PKI-GFP) (27), or with (control) GFP. The resultsof a representative experiment are displayed in Figure 5. Expressionof PKI-GFP decreased ouabain-sensitive 86Rb+ uptake of HK2/NK1complex (Figure 5A) without altering the basal activity of thecells that were transfected with pcDNA/NK1. Figure 5B demonstratesthat HK2 protein in cell lysate decreased when the cells wereco-transfected with PKI (top; compare lanes 1 and 2). This reductionin HK2 protein was associated with reduced phosphorylation ofthe intracellular PKA substrate vasodilator-associated phosphoprotein(represented by the abundance of the slower migrating, approximately50-kD band [27]) in cells that were co-transfected with PKI-GFP(compare lane 2 versus lane 1 and lane 4 versus lane 3). Theseresults are consistent with our interpretation that PKA activityis required for efficient processing of the HK2/NK1 complexand 86Rb+ uptake.
Figure 5. Inhibition of the PKA activity decreases 86Rb+ uptake by HK2/NK1. HEK-293 cells were co-transfected with HK2/NK1/pEGFP-N1, HK2/NK1/PKI, pcDNA/NK1/pEGFP-N1, or pcDNA/NK1/PKI. (A) Transfection of cells with rabbit muscle heat-stable inhibitor of the PKA gene (PKI) decreased the ouabain-sensitive 86Rb+ uptake of cells that were co-transfected with HK2 plus NK1 but did not alter the basal activity of cells that were co-transfected with pcDNA plus NK1. (B) Aliquots of the cells that were used to perform the 86Rb+ uptake were extracted with 1% CHAPS; the insoluble material was removed by centrifugation; and the supernatant was used to quantify HK2, vasodilator-associated phosphoprotein (VASP), GFP/PKI-GFP, and -actin protein. The top panel demonstrates that HK2 is abundantly expressed in cells that were co-transfected with HK2/NK1 and the plasmid pEGFP; however, expression is decreased in cells that were co-transfected with HK2/NK1 and PKI-GFP. Lanes 3 and 4 depict expression in cells that were co-transfected with empty pcDNA plasmid plus either GFP or PKI-GFP–encoding constructs. The second panel depicts the profile of VASP under the various transfection conditions and indicates a reduction in phospho-VASP levels (the slower migrating of the two bands) in cells that co-expressed PKI-GFP, consistent with a reduction in basal PKA activity. The third panel depicts expression of GFP and PKI-GFP in the various groups. In the fourth panel, quantification of the -actin demonstrates similar loading in all the lanes. The experiments were repeated three times with similar results.
4-PBA Increases 86Rb+ Uptake by HK2(S955/A)
On the basis of the results presented in Figures 1 through 5,we reasoned that S955 phosphorylation is necessary for promotionof maturation HK2 in the early stages of protein processing.To corroborate this possibility independently, we used 4-PBA,a compound that is known to increase plasma membrane immunolocalizationand excretion to the medium of partially misfolded uromodulinin patients with medullary cystic kidney disease type 2 andfamilial juvenile hyperuricemic nephropathy (28). Figure 5 showsthe results of a representative experiment; adding 4 mM 4-PBAfor 24 h to the medium did not change 86Rb+ uptake of the cellsthat were transfected with NK1 plus either wild-type HK2 orHK2(S955/D) but restored 86Rb+ uptake in cells that were transfectedwith NK1 plus HK2(S955/A).
4-PBA Increases Accumulation of HK2(S955/A)
To test whether the increase in 86Rb+ uptake that was inducedby 4-PBA in the cells that were co-transfected with HK2(S955/A)plus NK1 (Figure 6) was due to an increase of specific activityor an increase in plasma membrane HK2(S955/A) protein, we examinedthe effects of 4-PBA on HK2 expression and localization (Figure 7).The results demonstrate that treatment with 4-PBA increasesthe total content of HK2(S955/A) protein (Figure 7, top left).Furthermore, all the HK2(S955/A) protein is retained by streptavidinagarose in the biotinylation experiments, demonstrating thatthe protein has migrated to the plasma membrane, and reductionof membrane translocation does mediate the reduction in activityassociated in the HK2(S955/A) mutant. As a control in the sameexperiment, we quantified -actin. The results displayed in theFigure 7 (bottom) demonstrated that -actin was not retainedby the streptavidin agarose. Therefore, our results with 4-PBAdemonstrate that the use of a compound that is known to preventmisfolding of proteins increases cellular expression of HK2(S955/A)to augment 86Rb+ uptake.
Figure 6. 4-Phenylbutyric acid (4-PBA) increases 86Rb+ uptake by HK2(S955/A). HEK-293 cells were transiently transfected with HK2 plus NK1, HK2(S955/A) plus NK1, HK2(S955/D) plus NK1, or pcDNA3.1(+)-Neo plus NK1. 86Rb+ uptake experiments were performed 48 h later as described in Materials and Methods and Figure 1. 4-PBA (4 mM) was added to the cells for 24 h before the 86Rb+ uptake experiments. , no 4-PBA; , cells treated with 4 mM 4-PBA. The experiment was repeated three times with similar results.
Figure 7. 4-PBA increases HK2(S955/A) protein. HEK-293 cells were transiently transfected with HK2(S955/A) plus NK1 and incubated for 48 h in the absence or presence of 4 mM 4-PBA. Biotinylation and generation of cell lysates were performed as described in the legend of Figure 4. A total of 100 µg of cell lysate was resolved by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The top part of the membrane (approximately 100-kD region) was cut and blotted with anti-HK2 antibody, whereas the lower part (approximately 45-kD region) was blotted with anti–-actin. The results (top left) demonstrate that addition of 4-PBA increases cellular content of HK2(S955/S) protein. The rest of the sample (500 µg) was incubated for 4 h at 4°C with streptavidin agarose. The resin was removed by centrifugation, and supernatant (100 µg of protein) was separated on an SDS-PAGE and tested for the presence of HK2(S955/A) by immunoblot analysis (top left). The results demonstrate that mostly of the HK2(S955/A) protein was absorbed by the streptavidin agarose in both groups of cells. These results are consistent with the interpretation that 4-PBA by increasing the stability of HK2(S955/A) increased its plasma membrane localization and 86Rb+ uptake displayed in Figure 6. The results, displayed in the bottom panel, show that -actin was not retained by the streptavidin agarose, demonstrating that only the plasma membrane proteins were retained by the streptavidin agarose when the biotinylation was performed at 4°C. –, HEK-293 cells that were cultured in absence of 4 mM 4-PBA; +, HEK-293 cells that were cultured in presence of 4 mM 4-PBA. The experiment was repeated three times with similar results.
Our results demonstrate that a negative charge at position 955of HK2 is critical for expression and function of HK2. Mutationof S955 to alanine produces a decrease in cellular content ofHK2 (Figures 2 and 4), a decrease in [35S]methionine/cysteineincorporation to HK2 after a pulse label (Figure 3A), and adecrease in 86Rb+ uptake (Figure 1) but only minimal changein the rate of subunit degradation (Figure 3B). Cellular contentand 86Rb+ uptake of HK2 also can be decreased by expressionof PKI-GFP (Figure 5), and cellular content of HK2(S955/A) (Figure 7,top) and 86Rb+ uptake (Figure 6) is increased by addition ofthe chemical chaperon 4-PBA. Collectively, our data suggestthat proper conformation of HK2, at early stages of proteinsynthesis, is dependent on a negative charge at position 955.The absence of a negative charge at this position can be overcomeby the chemical chaperone 4-PBA.
Whether the beneficial effect of 4-PBA on the maturation ofHK2(S955/A) is direct or indirect was not tested in our study.Singh et al. (29) identified 85 differentially expressed proteinsin IB3–1 cystic fibrosis bronchial epithelial cells thatwere treated with 4-PBA and consisted primarily of "chaperones,catalytic enzymes, and proteins comprising structural elements,cellular defense, protein biosynthesis, trafficking activity,and ion transport."
The observation that 4-PBA increases the transport of HK2(S955/A)also is consistent with recent studies that examined wild andmutated forms of uromodulin (also known as Tamm-Horsfall glycoprotein).Uromodulin is the most abundant protein excreted in human urine.Hart et al. (28) demonstrated that patients with medullary cystickidney disease type 2 and familial juvenile hyperuricemic nephropathyhave mutations in the uromodulin gene. Two of these mutations(mutant F1, which contains a 27-bp deletion that results inthe in-frame deletion of amino acids 177 to 185, and mutantF2, which contains a missense mutation that changes a conservedC148 to tyrosine) were expressed in our laboratory in stablytransfected HEK-293 and thick ascending limb of Henle’sloop cells. The results demonstrate that wild-type uromodulinis excreted into the cell culture medium (30). In comparison,the excretion of mutated uromodulin was much less than thatof wild type. Incubation with 4-PBA also enhanced plasma membraneimmunolocalization and extracellular excretion of both wild-typeand mutated uromodulin, and the levels of excretion of mutateduromodulin in the presence of 4-PBA exceeded the level of excretionfor wild-type uromodulin in the absence of 4-PBA (30).
Previous studies from our laboratory demonstrated that the entirecarboxy-terminus of HK2 is necessary for 86Rb+ uptake (17).The carboxy-terminus deletion mutant of HK2 (or HK2) associatedpoorly with NK1 and did not accumulate efficiently at the plasmamembrane (17). Furthermore, 4-PBA does not improve 86Rb+ uptakeof cells that were transfected with HK2 plus NK1 (results notshown). Therefore, it seems that the beneficial effect of 4-PBAon protein maturation requires the HK2 carboxy-terminus andis obtained only when the protein is partially or reversiblymisfolded.
The view that a negatively charged amino acid at position 955is essential for the maturation of HK2 is consistent with thestudies of Vinciguerra et al. (21), which demonstrated thatproteasomal activity was necessary for degradation of "an inhibitor."The degradation of the "inhibitor" could allow PKA to shiftfrom an inactive to an active state and phosphorylate NK1 andpromote its recruitment to the plasma membrane.
Three PKA-activating mechanisms have been described. The firstuses the cAMP that is generated by activating adenylyl cyclasein the plasma membrane (31). The second uses cAMP that is generatedby activating soluble adenylyl cyclase. Although the latteris present in the male reproductive tract, it also is operativein kidney and localized to epithelial cells of distal tubules,the thick ascending limb of Henle’s loop, and collectingducts (32,33). Recently, Dulin et al. (22) described PKA activationby a cAMP-independent mechanism. Determining which of thesemechanisms regulates the PKA activity that is responsible forphosphorylation of HK2 will be necessary for a full understandingof how HK2 expression is regulated at the plasma membrane.
Our findings suggest that phosphorylation of S955 of HK2 functionsas a "switch" to promote the maturation of HK2 protein and representsa limiting factor in transport function.
Acknowledgments
This work was supported, in part, by National Institute of Diabetesand Digestive and Kidney Diseases grant R01 DK-30603 (T.D.D.)and NHLBI58506 (R.B.P.).
We thank Dr. Paul Dawson (Wake Forest University School of Medicine)for valuable suggestions about metabolic labeling of HEK-293cells and biotinylation of plasma membrane proteins.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Subunit of the Colonic H+,K+-ATPase
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Abstract
Introduction
subunits of the Na+,K+-ATPases and H+,K+-ATPases have a protein kinase A (PKA) consensus sequence near or in the ninth transmembrane domain. The role of this domain in influencing 
subunits (1–3). The common function of these membrane transporters is to accomplish cellular uptake of K+ in exchange for either Na+ or H+. The 
HK
, no 4-PBA; 
, cells treated with 4 mM 4-PBA. The experiment was repeated three times with similar results.