Parathyroid hormone-mediated regulation of Na+-K+-ATPase requires ERK-dependent translocation of protein kinase Calpha.

Parathyroid hormone (PTH) inhibits Na+-K+-ATPase activity by serine phosphorylation of the alpha1 subunit through protein kinase C (PKC)- and extracellular signal-regulated kinase (ERK)-dependent pathways. Based on previous studies we postulated that PTH regulates sodium pump activity through isoform-specific PKC-dependent activation of ERK. In the present work utilizing opossum kidney cells, a model of renal proximal tubule, PTH stimulated membrane translocation of PKCalpha by 102 +/- 16% and PKCbetaI by 41 +/- 7% but had no effect on PKCbetaII and PKCzeta. Both PKCalpha and PKCbetaI phosphorylated the Na+-K+-ATPase alpha1 subunit in vitro. PTH increased the activity of PKCalpha but not PKCbetaI. Coimmunoprecipitation assays demonstrated that treatment with PTH enhanced the association between Na+-K+-ATPase alpha1 subunit and PKCalpha, whereas the association between Na+-K+-ATPase alpha1 subunit and PKCbetaI remained unchanged. A PKCalpha inhibitory peptide blocked PTH-stimulated serine phosphorylation of the Na+-K+-ATPase alpha1 subunit and inhibition of Na+-K+-ATPase activity. Pharmacologic inhibition of MEK-1 blocked PTH-stimulated translocation of PKCalpha, whereas transfection of constitutively active MEK-1 cDNA induced translocation of PKCalpha and increased phosphorylation of the Na+-K+-ATPase alpha1 subunit. In contrast, PTH-stimulated ERK activation was not inhibited by pretreatment with the PKCalpha inhibitory peptide. Inhibition of PKCalpha expression by siRNA did not inhibit PTH-mediated ERK activation but significantly reduced PTH-mediated phosphorylation of the Na+-K+-ATPase alpha1 subunit. Pharmacologic inhibition of phosphoinositide 3-kinase blocked PTH-stimulated ERK activation, translocation of PKCalpha, and phosphorylation of the Na+-K+-ATPase alpha1 subunit. We conclude that PTH stimulates Na+-K+-ATPase phosphorylation and decreases the activity of Na+-K+-ATPase by ERK-dependent activation of PKCalpha.

basolateral membrane protein provides the driving force for the vectorial transport of various solutes and ions, including sodium, from the tubular lumen to the renal vasculature. Regulation of proximal renal tubule sodium reabsorption by Na ϩ -K ϩ -ATPase activity is a major determinant of total body sodium homeostasis, extracellular fluid volume status, and blood pressure control. Abnormalities in the regulation of Na ϩ -K ϩ -ATPase activity have been implicated in the pathogenesis of some forms of hypertension associated with impaired salt excretion (2,3). Not surprisingly, Na ϩ -K ϩ -ATPase activity is highly regulated by many hormones through multiple signaling pathways, including those relying on protein kinase C (PKC) 1 activation (4).
Several laboratories, including our own, reported that PTHstimulated PKC activation is critical for inhibition of Na ϩ -K ϩ -ATPase in proximal renal tubule cells (2, 16 -18). Other investigators have shown that membrane translocation of PKC␣ induced by nitric oxide and by phorbol 12-myristate 13-acetate is associated with inhibition of Na ϩ -K ϩ -ATPase activity in opossum kidney cells (OK), a model of renal proximal tubule (19). The PKC isoforms stimulated by PTH that regulate Na ϩ -K ϩ -ATPase have not been identified. We have previously demonstrated that PTH regulation of Na ϩ -K ϩ -ATPase activity is PKC-and ERK-dependent (17,18). However, the mechanisms for PTH-mediated ERK and PKC activation involved in Na ϩ -K ϩ -ATPase regulation have not been determined. Based on our previous work showing that PKC activates the ERK pathway, we postulated that PTH regulates Na ϩ -K ϩ -ATPase in OK cells by activation of ERK through a PKC␣-dependent mechanism. To address this hypothesis, we examined the ability of PTH to stimulate activation of specific PKC isoforms, the ability of PTH to regulate Na ϩ -K ϩ -ATPase after inhibition of specific PKC isoforms, and the role of PKC isoforms and ERK in the regulation of phosphorylation and activity of the sodium pump. In contrast to our original hypothesis, the results show that PTH regulates Na ϩ -K ϩ -ATPase in OK cells by ERK-dependent activation of PKC␣.  was purchased from Bachem Biosciences Inc. (King of Prussia, PA). Polyclonal antibodies against the Na ϩ -K ϩ -ATPase ␣ 1 subunit (for immunoprecipitation), recombinant active PKC␣, PKC␤1, and MEK-1, MEK-1 cDNA, PKC␣-specific small inhibitory RNA (siRNA), control nonspecific siRNA, and PKC activity kit were purchased from Upstate Biotechnology, Inc. (Waltham, MA). Monoclonal antibodies against Na ϩ -K ϩ -ATPase ␣ 1 subunit (for Western blots) were purchased from Sigma-RBI (Natick, MA). Antibodies against PKC ␣, ␤I, ␤II, and were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-phosphoserine antibodies were purchased from Zymed Laboratories Inc. (San Francisco, CA). Dr. D. Mochly-Rosen (Stanford University School of Medicine, Stanford, California) kindly provided PKC isoform-specific inhibitory peptides. The peptides were cross-linked via an N-terminal Cys-Cys bond to Drosophila Antennapedia homeodomain-derived carrier peptide (C-RQIKIWFQNRRMKWKK) (20 -24). U0126 was purchased from Promega. Wortmannin and LY294002 were purchased from Calbiochem-EMD Biosciences Inc (San Diego, CA). Bioporter transfection, Geneporter transfection, and Genesilencer siRNA transfection reagents were purchased from Gene Therapy Systems Inc. (San Diego, CA). Phosphate-buffered saline (PBS) without calcium and magnesium, pH 7.4, was purchased from Invitrogen. All other chemicals were purchased from Sigma unless otherwise specified.

Materials-PTH
Cell Culture-The OK cells are a continuous cell line derived from Virginia opossum and a widely used model for mammalian renal proximal tubule. These cells exhibit several characteristics of mammalian renal proximal tubules including a polarized morphology, basolateral expression of Na ϩ -K ϩ -ATPase, and regulation of Na ϩ -K ϩ -ATPase by PTH, cAMP-dependent protein kinase, and PKC (17). OK cells were maintained in minimal essential medium with Earl's salts (EMEM) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin at 37°C in 95% air, 5% CO 2 . The cells were fed twice a week and split once a week at a 1:4 ratio. All of the experiments were carried out using cells at 90 -95% confluence. Cells grown on 6-well culture plates were washed with serum-free medium 24 h prior to use.
Protein Kinase C Immunoblotting-Protein Kinase C translocation was determined by translocation of PKC isoforms to the membrane following the method of Clarke et al. (25). Briefly, cells grown on Falcon 100-mm tissue culture plates were treated with 10 Ϫ7 M PTH (bovine  in the presence or absence of 100 nM PKC isoform-specific peptide inhibitor or 10 Ϫ5 M U0126 (MEK-1 inhibitor) for 15 min. The cells were washed with ice-cold PBS without calcium and magnesium, pH 7.4, scraped, and homogenized into 1.5 ml of ice-cold PKC buffer containing 20 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 10 mM EGTA, 2 mM EDTA, 10 g/ml aprotinin, 20 g/ml leupeptin, and 50 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, pipetted into ultracentrifuge tubes, and sonicated on ice. The samples were centrifuged at 100,000 ϫ g for 1 h at 4°C in a Beckman SW55Ti rotor. The supernatant (cytosolic fraction) was transferred to a separate tube, and a sample was taken for protein estimation. An equal volume of 2ϫ Laemmli sample buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% ␤-mercaptoethanol, and 0.001% bromphenol blue was added. The solution was boiled for 5 min and stored at Ϫ70°C. To the pellet, 500 l of PKC buffer with 1% Triton X-100 was added, homogenized, and left on a rotator for 2 h at 4°C. The samples were centrifuged at 100,000 ϫ g as described above for 1 h. The supernatant (Triton-soluble fraction) was removed, a sample was taken for protein estimation, and an equal volume of 2ϫ Laemmli sample buffer was added, boiled for 5 min, and stored at Ϫ70°C. Western blot was performed as described previously (18).
Immunoprecipitation-The whole cell lysate or crude membranes solubilized in immunoprecipitation (IP) buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM NaF, 1 mM EDTA, 1 mM EGTA, 5 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 1 mM sodium pyrophosphate, 1 g/ml aprotinin, 1 g/ml leupeptin, 100 l/ml phosphatase inhibitor mixture, 1% Triton X-100, 0.5% Nonidet P-40, and 0.5% SDS were centrifuged at 70,000 ϫ g for 1 h in a Beckman ultracentrifuge. 100 g of protein from the supernatant was precleared with Protein A-Sepharose beads for 2 h at 4°C. The beads were separated by centrifugation at 14,000 rpm for 1 min in a tabletop centrifuge (Spec-trafuge; National Labnet Co.). The supernatant was incubated overnight at 4°C with 50 ng of anti-PKC␣, PKC␤1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit polyclonal antibodies against the ␣ 1 subunit of Na ϩ -K ϩ -ATPase (Upstate Biotechnology, Inc.). Protein G-(PKC␣, or PKC␤1) or A-(Na ϩ -K ϩ -ATPase ␣ 1 subunit) Sepharose beads were added and incubated for 2 h at 4°C. The beads were washed three times with IP buffer by centrifugation at 14,000 rpm for 1 min in a tabletop centrifuge. An equal volume of 2ϫ Laemmli sample buffer was added and boiled for 5 min. The beads were centrifuged as above, and the proteins in the supernatant were separated by 10% SDS-PAGE transferred to nitrocellulose membranes and probed with Na ϩ -K ϩ -ATPase ␣ 1 subunit or anti-PKC isoform-specific antibodies.
Determination of PKC Activity-The OK cells were incubated in the presence or absence of 10 Ϫ7 M PTH for 15 min. The cells were washed two times with PBS, pH 7.4, and lysed in immunoprecipitation buffer. PKC␣ or ␤1 was immunoprecipitated from 100 g of whole cell lysate proteins as described above. The beads were washed three times with IP buffer, and PKC activity was determined according to the manufacturer's protocol (Upstate Biotechnology, Inc.). Briefly 10 l each of assay dilution buffer (20 mM MOPS, pH 7.2, 25 mM ␤-glycerol phosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, and 1 mM CaCl 2 ), substrate mixture (500 M PKC substrate), cAMP-dependent protein kinase/calmodulin kinase inhibitor mixture (2 M protein kinase A inhibitor peptide PKI, and 20 M R24571 in assay dilution buffer), PKC lipid activator (0.5 mg/ml phosphatidyl serine and 0.05 mg/ml diacylglycerol in assay dilution buffer), and magnesium/ATP mixture (75 mM MgCl 2 , 500 M ATP and 100 Ci of [␥-32 P]ATP) were added to the protein G-Sepharose beads and incubated at 30°C for 10 min. After 10 min a 25-l sample was slowly transferred to the center of a P81 phosphocellulose paper and incubated at room temperature for 30 min. The P81 papers were washed three times with 0.75% phosphoric acid and once with acetone, dried, and transferred to scintillation vials. Bound radioactivity was quantitated by addition of 3 ml of scintillation fluid and reading in a scintillation counter (Amersham Biosciences). A substrate control was measured to correct for nonspecific binding along with the samples. Control counts were subtracted from the sample counts to calculate the PKC activity according to the manufacturer's protocol. The activity is expressed as pmol of phosphate incorporated into the PKC substrate peptide/min/mg protein.
Membrane Preparation-The cells were treated with 10 Ϫ7 M PTH (bovine,  in the presence or absence of PKC isoform-specific peptide inhibitors for a specified time (see "Results"), washed twice with PBS, and lysed in 50 mM mannitol, 5 mM Tris, pH 7.4. The lysate was homogenized using a high speed polytron type homogenizer (3 strokes, 15 s) and incubated with 10 mM MgCl 2 for 20 min on ice, followed by centrifugation at low speed (3000 rpm for 10 min) to remove cell debris. The supernatant was centrifuged at 20,000 ϫ g for 20 min. The pellet containing crude membrane material was resuspended in the homogenizing buffer.
In Vitro Phosphorylation of Na ϩ -K ϩ -ATPase ␣ 1 Subunit by PKC␣ and PKC␤1-Crude membranes were prepared from OK cells as described above. Crude membranes were solubilized in IP buffer, and Na ϩ -K ϩ -ATPase ␣ 1 subunit was immunoprecipitated from 100 g of membrane protein as described above. Protein A-Sepharose beads containing the immunoprecipitated Na ϩ -K ϩ -ATPase ␣ 1 subunit were incubated in 40 l of PKC phosphorylation buffer containing 100 mM HEPES, pH 7.4, 6.5 mM CaCl 2 , 4 mM dithiothreitol, 50 mM MgCl 2 , 5 mM ATP, 1 mM IP20 (cAMP-dependent protein kinase inhibitory peptide), PKC activator (1 mg/ml phosphatidyl serine, Promega), and 100 nCi of [␥-32 P]ATP at 30°C for 30 min in the presence or absence of constitutively active recombinant PKC␣ or ␤1. At the end of the reaction, the beads were washed three times with IP buffer as described above, and an equal volume of 2ϫ Laemmli sample buffer was added and boiled for 5 min. The proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membrane, and autoradiographed. To determine equal loading of protein, Western blot analysis using Na ϩ -K ϩ -ATPase ␣ 1 subunit antibodies was performed.
Determination of Na ϩ -K ϩ -ATPase Activity as K ϩ -stimulated pN-PPase Activity-K ϩ -stimulated pNPPase activity was measured as described previously (26) following the method of Hird et al. (27) with slight modifications as described by Tran and Farley (28). Briefly, Na ϩ -K ϩ -ATPase activity as K ϩ -stimulated pNPPase activity is measured by assessing its ability to cleave p-nitrophenyl phosphate to pnitrophenol in the presence of high molar concentration of potassium. 20 l of the membrane (25-30 g of protein) was added to 0.88 ml of assay buffer (30 mM histidine, 150 mM KCl, 20 mM MgCl 2 , 2 mM EGTA, pH 7.4, and 10 mM p-NPP). The assay mixture was incubated for 15 min at 37°C. The reaction was stopped by the addition of 100 l of 5 N NaOH containing 0.1% Triton X-100. A standard curve was constructed by serial dilutions of p-nitrophenol, and the color was read at 410 nm. The samples and the standards were centrifuged at 1500 ϫ g for 15 min before spectrophotometric analysis. Ouabain-sensitive pNPPase activity was determined by subtracting the activity measured in the presence of 10 mM ouabain in the assay medium from total activity measured in absence of ouabain. The assay was run in triplicate, and activity is expressed as moles of p-nitrophenol released/mg protein/h. 86 Rb Uptake-Ouabain-sensitive 86 Rb uptake was measured as an index of Na ϩ -K ϩ-ATPase-mediated ion transport as described previously (18) following the method of Okafor et al. (29). OK cells were pretreated with 5 M monensin for 30 min in the presence or absence of PKC inhibitory peptides. The cells were exposed to PTH for 5 min prior to adding trace amount of 86 Rb (ϳ1 Ci/ml 86 RbCl) in Dulbecco's modified Eagle's without serum. Half the cells received ouabain (final concentration, 1 mM) 15 min prior to the start of 86 Rb uptake. 86 Rb uptake was carried out for 10 min at room temperature after which the cells were washed five or six times with ice-cold PBS. The cells were lysed overnight in 0.5 N NaOH containing 0.1% Triton X-100 at 37°C. An aliquot (100 l) of the lysate was used to measure radioactivity. The difference between the 86 Rb uptake measured in the presence of 1 mM ouabain and the absence of ouabain was used as a measure of Na ϩ -K ϩ -ATPase-mediated transport activity. The uptake data are expressed as nmol of rubidium ( 86 Rb) accumulated/mg protein/min, and the results are plotted as percentages of control.
PKC␣ siRNA Transfection-200 pmol of SMART-Pool PKC␣ or nonspecific control small inhibitory RNA (Upstate Biotechnology, Inc.) was transfected into OK cells using Genesilencer Transfection reagent according to the manufacturer's protocol. Briefly, 5 l of Genesilencer reagent was diluted with 25 l of serum-free EMEM. In another tube 25 l of siRNA diluent was diluted with 15 l serum-free EMEM, and 200 pmol of siRNA was added. The siRNA containing mixture was mixed with diluted Genesilencer reagent and incubated at room temperature for 15 min. The siRNA, Genesilencer reagent mixture was added onto growing cells in serum-free EMEM and incubated at 37°C in 95% air, 5% CO 2 for 6 h, following which 1 ml of EMEM containing 20% serum was added and further incubated for 24 h at 37°C in 95% air, 5% CO 2 .
MEK-1 cDNA Transfection-A cDNA for constitutively active MEK-1 (Upstate Biotechnology, Inc.) was transfected into OK cells using Geneporter transfection reagent according to the manufacturer's protocol. Briefly, the cDNA and the Geneporter reagent were diluted separately in serum-free medium. The diluted cDNA was mixed with diluted Geneporter reagent and incubated at room temperature for 30 min. The culture medium from the cells was replaced with the mixture containing Geneporter and MEK-1 cDNA and incubated for 24 h at 37°C in 95% air, 5% CO 2 , following which the cells were lysed, and the membrane and cytosolic fractions were separated and processed for Western blot analysis. The supernatant proteins were separated by 10% SDS-PAGE and analyzed by Western blot using phospho-ERK antibodies to document successful transfection of MEK-1.
Uptake of Constitutively Active Recombinant MEK-1-Wild type OK cells were washed with Dulbecco's modified Eagle's medium without fetal calf serum 24 h prior to uptake of active recombinant MEK-1. 1 g/ml GFP-linked constitutively active recombinant MEK-1 was mixed with 5 l of dried Bioporter film in a 1.5-ml tube and incubated for 5 min at room temperature. The mixture was then transferred to OK cells and incubated for 4 h at 37°C in a humidified 95% air, 5% CO 2 incubator. After 4 h, the cells were washed thrice with 1ϫ PBS and then lysed in 50 mM mannitol, 5 mM Tris-HCl buffer, pH 7.4. The crude membranes were prepared as described above. The supernatant proteins were separated by 10% SDS-PAGE and analyzed by Western blot using phospho-ERK antibodies to document successful uptake of MEK-1. Protein concentration was measured by the BCA method (Sigma) using bovine serum albumin as standard.
Densitometry-The Western blots were scanned and analyzed using Personal densitometer (Molecular Probes) and expressed as arbitrary densitometric units.
Statistics-The data are shown as the means Ϯ S.E. All of the experiments were repeated at least three times unless otherwise stated to document reproducibility. The p values are calculated using Sigma-Stat software utilizing a paired t test. A p value less than 0.05 was a priori considered statistically significant.

Identification of PKC Isoforms Activated by PTH in OK
Cells-Consistent with previous reports (19,30,31) preliminary studies demonstrated the expression of PKC␣, ␤I, ␤II, and but not of PKC␦, ␥, and ⑀ in OK cells (data not shown). To determine which PKC isoforms were activated by PTH, OK cells were treated with PTH (10 Ϫ7 M) for 15 min. Activation of specific PKC isoforms was determined by measuring the translocation to the membrane by Western blot analysis (19) and by measuring the enzyme activity of immunoprecipitated PKC isoforms. As shown in Fig. 1 (top panel), PTH stimulated translocation of PKC␣ and PKC␤1 to the membrane fraction, whereas no translocation of PKC␤II or was observed. The bar diagram shows the change in membrane expression as determined by densitometric analysis of Western blots from three separate experiments. PTH increased the membrane abundance of PKC␣ by 102 Ϯ 16%, whereas PKC␤1 was increased by 41 Ϯ 7%. To determine whether translocation of PKC␣ and PKC␤1 was associated with an increase in the activity, the activity of the PKC isoforms was measured. As shown in Fig. 2, PTH increased PKC␣ activity but had no effect on PKC␤1 activity.
Association of PKC Isoforms with the Na ϩ -K ϩ -ATPase ␣ 1 Subunit-The ability to phosphorylate the Na ϩ -K ϩ -ATPase ␣ 1 subunit suggests that specific PKC isoforms should be physically associated with this substrate. To determine whether the Na ϩ -K ϩ -ATPase ␣ 1 subunit physically associated with either of the PKC isoforms, we immunoprecipitated Na ϩ -K ϩ -ATPase ␣ 1 subunit from vehicle-and PTH-treated OK cells and analyzed the immunoprecipitates for the presence of PKC␣ and PKC␤1 by Western blot analysis. As shown in Fig. 4A, immunoprecipitation with the Na ϩ -K ϩ -ATPase ␣ 1 subunit antibody of untreated cells produced faint staining for the PKC␣ isoform and marked staining for the ␤1 isoform. PTH significantly enhanced the association between PKC␣ and the Na ϩ -K ϩ -ATPase ␣ 1 subunit, whereas association between PKC␤1 and Na ϩ -K ϩ -ATPase ␣ 1 subunit did not change with PTH treatment. Reciprocal immunoprecipitation assays with anti-PKC␣ and anti-PKC␤1 antibodies and Western blot analysis for Na ϩ -K ϩ -ATPase ␣ 1 subunit confirmed the increased association with PKC␣ upon treatment with PTH (Fig. 4A). Fig. 4B shows densitometric data from three independent experiments, indicating a significant increase in the association of PKC␣ with Na ϩ -K ϩ -ATPase ␣ 1 subunit.
Role of PKC␣ in PTH-mediated Phosphorylation of Na ϩ -K ϩ -ATPase ␣ 1 Subunit and Inhibition of Na ϩ -K ϩ -ATPase Activity-The preceding data strongly point toward PKC␣ as the isoform mediating PTH regulation of Na ϩ -K ϩ -ATPase. To determine whether PKC␣ is responsible for phosphorylation of the Na ϩ -K ϩ -ATPase ␣ 1 subunit in intact cells, OK cells were pretreated with 100 nM PKC␣ inhibitory peptide for 30 min followed by treatment with 10 Ϫ7 M PTH for 15 min. Na ϩ -K ϩ -ATPase ␣ 1 subunit was immunoprecipitated from the membrane fraction and analyzed by Western blot for phosphorylation using antiphosphoserine antibodies. As shown in Fig. 5, PTH induced a 4-fold increase in the phosphorylation of Na ϩ -K ϩ -ATPase ␣ 1 FIG. 2. Effect of PTH on activation of PKC␣ and PKC␤I. OK cells were treated with 10 Ϫ7 M PTH for 15 min. PKC␣ and PKC␤I were immunoprecipitated from whole cell lysates, and PKC activity was measured as described under "Experimental Procedures." Each bar represents the mean activity Ϯ S.E. (pmol of 32 P i incorporated into PKC substrate peptide/mg protein/min) from three independent experiments. The results demonstrate that PTH activates PKC␣. *, p Ͻ 0.05.

FIG. 3.
In vitro phosphorylation of Na ؉ -K ؉ -ATPase ␣ 1 subunit by PKC␣ or PKC␤I. The Na ϩ -K ϩ -ATPase ␣ 1 subunit was immunoprecipitated from OK cell crude membrane fractions and subjected to in vitro phosphorylation by recombinant active PKC␣ or PKC␤I. A representative autoradiograph of Na ϩ -K ϩ -ATPase ␣ 1 subunit phosphorylation from three independent experiments is shown (upper panel). The lower panel is a Western blot for the Na ϩ -K ϩ -ATPase ␣ 1 subunit showing equal loading in each condition. The graph presents quantitative data as the means Ϯ S.E. in arbitrary densitometry units from three independent phosphorylation experiments. The figure shows that PKC␣ associates with Na ϩ -K ϩ -ATPase ␣ 1 subunit upon activation by PTH. *, p Ͻ 0.05. WB, Western blot.

FIG. 4.
Association between Na ؉ -K ؉ -ATPase ␣ 1 subunit, PKC␣, and PKC␤I. OK cells were treated with 10 Ϫ7 M PTH for 15 min. The Na ϩ -K ϩ -ATPase ␣ 1 subunit, PKC␣, or PKC␤I was immunoprecipitated from crude membrane fractions and analyzed by Western blotting for Na ϩ -K ϩ -ATPase ␣ 1 subunit, PKC␣, and PKC␤I. A shows representative blots from three independent experiments. B shows a graph of the quantitative data as the means Ϯ S.E. in arbitrary densitometry units from these three independent experiments. The figure shows that PTH stimulates increased association of PKC␣ with the Na ϩ -K ϩ -ATPase ␣ 1 subunit, whereas association of PKC␤1 is unchanged. *, p Ͻ 0.05. WB, Western blot. subunit. Pretreatment with the PKC␣ inhibitory peptide blocked PTH-mediated phosphorylation of Na ϩ -K ϩ -ATPase ␣ 1 subunit.
To determine whether PKC␣ played a role in PTH-mediated regulation of Na ϩ -K ϩ -ATPase activity, OK cells were treated with 10 Ϫ7 M PTH for 15 min in the presence or absence of 100 nM PKC␣ inhibitory peptide. As shown in Fig. 6, PTH inhibited Na ϩ -K ϩ -ATPase activity by about 25%. Pretreatment with PKC␣ inhibitory peptide, but not with Antennapedia carrier peptide, blocked PTH-mediated inhibition of Na ϩ -K ϩ -ATPase activity, as measured by K ϩ -dependent pNPPase assay (Fig.  6A) and 86 Rb uptake (Fig. 6B).
Role of ERK in PTH-mediated Phosphorylation of Na ϩ -K ϩ -ATPase ␣ 1 Subunit-As described previously (18), inhibition of ERK by the MEK-1 inhibitor U0126 (final concentration, 10 Ϫ5 M) inhibited PTH-mediated phosphorylation of Na ϩ -K ϩ -ATPase ␣ 1 subunit (Fig. 5). To determine whether activation of PKC␣ was upstream or downstream of ERK, we measured the translocation of PKC␣ and activation of ERK in OK cells treated with PTH for 15 min following 30 min of pretreatment with vehicle, PKC␣ inhibitory peptide, or U0126. Inhibition of either PKC␣ or ERK blocked PTH-mediated translocation of PKC␣ to the membrane (Fig. 7A). Inhibition of PKC␣ did not alter PTH-mediated activation of ERK (Fig. 7B), suggesting that PKC␣ activation is downstream of ERK activation in the PTH-mediated signaling cascade. To confirm that PTH-stimulated ERK activation is not dependent upon PKC␣ activity, we reduced the expression of PKC␣ by transfection with PKC␣ siRNA and treated the cells with 10 Ϫ7 M PTH for 15 min. As shown in Fig. 8A, PKC␣ siRNA reduced the expression of PKC␣ by about 70%, whereas it had no effect on the expression of PKC␤1. Western blot analysis of the Na ϩ -K ϩ -ATPase ␣ 1 subunit phosphorylation showed that PTH stimulated phosphoryl-ation in control and mock transfected cells. However, transfection with PKC␣ siRNA completely blocked PTH-stimulated Na ϩ -K ϩ -ATPase ␣ 1 subunit phosphorylation (Fig. 8B). PTH increased ERK phosphorylation in all groups, including cells transfected with PKC␣ siRNA (Fig. 8C).
To investigate the involvement of ERK in the translocation of PKC␣ and phosphorylation of the Na ϩ -K ϩ -ATPase ␣ 1 subunit, a cDNA for active MEK-1 was transfected into OK cells. Expression of active MEK-1 resulted in activation of ERK (Fig.  9A), stimulated translocation of PKC␣ (Fig. 9B), and enhanced Na ϩ -K ϩ -ATPase ␣ 1 subunit phosphorylation (Fig. 9C). Additionally, OK cells were incubated with 100 g/ml constitutively active recombinant GFP-linked MEK-1 using Bioporter transfection reagent (Gene Therapy System). Uptake of MEK-1 resulted in increased activation of endogenous ERK (Fig. 10A), translocation of PKC␣ to the membrane fraction (Fig. 10B), increased phosphorylation of the Na ϩ -K ϩ -ATPase FIG. 5. Effect of PKC␣ inhibitory peptide and U0126 on PTHmediated Na ؉ -K ؉ -ATPase ␣ 1 subunit phosphorylation. OK cells were treated with 10 Ϫ7 M PTH for 15 min in the continued presence or absence of 100 nM PKC␣ inhibitory peptide or U0126. The Na ϩ -K ϩ -ATPase ␣ 1 subunit was immunoprecipitated from crude membrane fractions and analyzed by Western blot using anti-phosphoserine antibodies. A representative Western blot from three independent experiments is shown. The graph presents quantitative data as the means Ϯ S.E. in arbitrary densitometry units from these three independent experiments. Both PKC␣ inhibitory peptide and inhibition of ERK activation with U0126 blocked Na ϩ -K ϩ -ATPase ␣ 1 subunit phosphorylation.*, p Ͻ 0.05. WB, Western blot.
FIG. 6. Effect of PKC␣ inhibitory peptides on PTH-mediated inhibition of Na ؉ -K ؉ -ATPase activity. OK cells were treated with 10 Ϫ7 M PTH for 15 min in the presence or absence of 100 nM PKC␣ inhibitory peptide. A shows Na ϩ -K ϩ -ATPase activity measured in crude membranes expressed as percentages of control K ϩ -dependent pN-PPase activity. The results demonstrate that PTH increases ouabain sensitive K ϩ -dependent pNPPase activity. Each bar represents the mean (ϮS.E.) percentage of respective control from four independent experiments performed in triplicate. B shows ouabain sensitive 86 Rb uptake stimulated by 10 Ϫ7 M PTH in the presence and the absence of 100 nM PKC␣ inhibitory peptide. Each bar represents the mean (Ϯ S.E.) percentage of respective control from four independent experiments performed in triplicate. The results show that PKC␣ inhibitory peptide significantly inhibits PTH-mediated inhibition of K ϩ -dependent pN-PPase activity and ouabain-sensitive 86 Rb uptake. *, p Ͻ 0.05. FIG. 8. Effect of PKC␣ siRNA on PKC␣ expression, PTH-mediated ERK activation, and Na ؉ -K ؉ -ATPase ␣ 1 subunit phosphorylation. OK cells were transfected with PKC␣ siRNA or control siRNA or were cultivated with reagent or vehicle alone, followed by incubation with or without 10 Ϫ7 M PTH for 15 min. A shows an immunoblot for PKC␣ and PKC␤1 of lysates from control cells and cells transfected with PKC␣ siRNA and control siRNA. The figure demonstrates that PKC␣ siRNA specifically reduces PKC␣, but not PKC␤1, expression. B shows a representative Western blot of Na ϩ -K ϩ -ATPase ␣ 1 subunit immunoprecipitated from crude membrane fractions from the same groups of cells using anti-phosphoserine antibodies. The figure shows that PTH induces phosphorylation of Na ϩ -K ϩ -ATPase ␣ 1 subunit in control cells, whereas down-regulation of PKC␣ expression inhibits PTH-stimulated phosphorylation. C shows a representative Western blot of the same groups of cells from two independent experiments for phospho-ERK (upper panel) and ERK1 (lower panel). The results show that down-regulation of PKC␣ expression by siRNA has no effect of PTH-stimulated ERK phosphorylation. WB, Western blot. The blot is representative of three independent experiments. C is a Western blot of Na ϩ -K ϩ -ATPase ␣ 1 subunit immunoprecipitated from the crude membrane fractions from the same groups of cells using anti-phosphoserine antibodies (upper panel) or anti-Na ϩ -K ϩ -ATPase ␣ 1 subunit (lower panel). The results show that transfection with active MEK-1 stimulated phosphorylation of the Na ϩ -K ϩ -ATPase ␣ 1 subunit, and equal amounts of the subunit were immunoprecipitated. The blots are representative of three independent experiments. WB, Western blot.

FIG. 7. Effect of PKC␣ inhibitory peptide and U0126 on PTH-mediated PKC␣ translocation and ERK activation.
OK cells were treated with 10 Ϫ7 M PTH for 15 min in the continued presence or absence of 100 nM PKC␣ inhibitory peptide or U0126. A presents a Western blot of membrane fractions for PKC␣ showing that both PKC␣ inhibitory peptide and ERK inhibition with U0126 block PTH-stimulated translocation of PKC␣. The graph presents densitometric analysis of three independent experiments as mean Ϯ S.E. B presents a representative Western blot of cytosolic proteins for phospho-ERK (upper panel) and ERK2 (lower panel) showing that PTH-stimulated ERK activation is blocked by U0126 but not PKC␣ inhibitory peptide. The graph shows densitometric quantitation of phospho-ERK in three independent experiments expressed as the means Ϯ S.E. in arbitrary densitometry units, confirming the inhibition of ERK phosphorylation by U0126 but not by PKC␣ inhibitory peptide. *, p Ͻ 0.05. WB, Western blot. ␣ 1 subunit (Fig. 10C), and inhibition of Na ϩ -K ϩ -ATPase activity (Fig. 10D).
To investigate whether phosphorylation of Na ϩ -K ϩ -ATPase ␣ 1 subunit was mediated by ERK-activated PKC␣, constitutively active MEK-1 was transiently transfected into OK cells in the presence or absence of PKC␣ inhibitory peptide. As shown in Fig. 11, PKC␣ inhibition had no effect on activation of ERK by transfection of active MEK-1 (Fig. 11A). Inhibition of PKC␣, however, blocked translocation of PKC␣ (Fig. 11B) and phosphorylation of Na ϩ -K ϩ -ATPase ␣ 1 subunit by expression of constitutively active MEK-1 (Fig. 11C).
Role of PI 3-Kinase in Phosphorylation of Na ϩ -K ϩ -ATPase ␣ 1 Subunit, PKC␣ Translocation, and ERK Activation-To determine whether PI 3-kinase played a role in the activation and translocation of PKC␣ and eventual phosphorylation of Na ϩ -K ϩ -ATPase ␣ 1 subunit, we pretreated OK cells with either 10 Ϫ5 M wortmannin or 5 ϫ 10 Ϫ5 M LY294002 for 30 min at 37°C followed by treatment with 10 Ϫ7 M PTH. As shown in Fig. 12, wortmannin and LY294002 blocked PTH-mediated activation of ERK (Fig. 12A), translocation of PKC␣ to membrane fraction (Fig. 12B), and phosphorylation of the Na ϩ -K ϩ -ATPase ␣ 1 subunit (Fig. 12C).

DISCUSSION
Protein kinase C is one of the essential signaling elements through which PTH regulates Na ϩ -K ϩ -ATPase activity in the renal proximal tubule. The present study and previously published work shows that PKC can directly phosphorylate the Na ϩ -K ϩ -ATPase ␣ 1 subunit (32). In this report, we demonstrate that PTH selectively and uniquely stimulates the translocation of one PKC isoform, PKC␣, to associate with the ␣ 1 subunit of Na ϩ -K ϩ -ATPase in the membrane. This association is necessary for phosphorylation of the ␣ 1 subunit and inhibition of sodium pump activity. A role for PKC␣ in regulation of Na ϩ -K ϩ -ATPase is consistent with previous studies. Middleton et al. (31) demonstrated that treatment of OK cells with a phorbol ester resulted in membrane translocation of the PKC␣ isoform, inhibition of Na ϩ -K ϩ -ATPase activity, and phosphorylation of the Na ϩ -K ϩ -ATPase ␣ 1 subunit. Liang and Knox (19) demonstrated involvement of PKC␣ in inhibition of Na ϩ -K ϩ -ATPase activity by nitric oxide and phorbol 12-myristate 13-acetate.
We also identified PKC␤1, PKC␤II, and PKC isoforms in OK cells, but none appear to have a significant role in PTH regulation of Na ϩ -K ϩ -ATPase. Although PKC␤1 coimmunoprecipitated with the Na ϩ -K ϩ -ATPase ␣ 1 subunit and phosphorylated the ␣ 1 subunit in an in vitro phosphorylation assay, treat- The data indicate that transfection with MEK-1 stimulated membrane translocation of PKC␣. C shows a Western blot of immunoprecipitated Na ϩ -K ϩ -ATPase ␣ 1 subunit using anti-phosphoserine representative of two independent experiments. The results show that MEK-1 transfection stimulated phosphorylation of Na ϩ -K ϩ -ATPase ␣ 1 subunit. WB, Western blot. ment with PTH did not stimulate PKC␤1 activity or increase the association of PKC␤1 with Na ϩ -K ϩ -ATPase. These findings are also consistent with the report of Middleton et al. (31), which detected PKC␤1 in OK cells but failed to identify its activation with phorbol esters. Studies on regulation of the sodium pump through PKC activation in renal proximal tubules demonstrate variable involvement of other PKC isoforms. Budu et al. (13) showed that treatment of OK cells expressing rodent wild type Na ϩ -K ϩ -ATPase ␣ 1 subunit with a serotonin agonist increased sodium pump activity in a PKC␤-dependent manner. Efendiev et al. (33) demonstrated a role for PKC and PKC␤ (I or II not specified) in the regulation of rat proximal tubule sodium pump activity by phorbol ester and dopamine.
The other novel finding emerging from this study is that the major role for PTH-stimulated ERK activity in regulation of Na ϩ -K ϩ -ATPase activity is to activate PKC␣. Our data demonstrate that inhibition of ERK blocked PTH-mediated translocation of PKC␣, suggesting that activation of PKC␣ is dependent upon ERK activation. Previous studies demonstrated that PKC␣ is phosphorylated at Thr 497 , Thr 638 , and Ser 657 residues (34). Computer-based phosphorylation motif screening of PKC␣ identified a potential ERK1/2 phosphorylation site at Thr 638 (scansite.mit.edu) (35). Therefore, it is likely that PKC␣ is regulated through direct phosphorylation by ERK1/2. This observation is consistent with the studies of Adayev et al. (36), which showed that 8-hydroxy-2-(di-n-propylamino)tetralin-mediated activation of PKC␣ is dependent upon activation of ERK in a hippocampal cell model. Our present demonstration that PKC␣ activation is ERK-dependent is also consistent with our previously published findings that 1) the initial activation of ERK is PKC-independent (37) and 2) Na ϩ -K ϩ -ATPase regulation by PKC is ERK-dependent (17).
We and others recently demonstrated that ERK might directly phosphorylate the Na ϩ -K ϩ -ATPase ␣ 1 subunit (18,38). In our studies, inhibition of MEK-1 by U0126 blocked PTHmediated phosphorylation of the Na ϩ -K ϩ -ATPase ␣ 1 subunit and inhibition of Na ϩ -K ϩ -ATPase activity. Further, we showed that the Na ϩ -K ϩ -ATPase ␣ 1 subunit immunoprecipitated from OK cells transfected with exogenous rat ␣ 1 was phosphorylated when incubated with constitutively active recombinant GFP-ERK1 in an in vitro phosphorylation assay. Interestingly, phosphorylation occurred in wild type and Ser 18 to alanine mutant rat ␣ 1 but not in the Ser 11 to alanine mutant (18). Feschenko and Sweadner (32) demonstrated that Ser 11 is a PKC phosphorylation site. Ser 11 is not followed by proline and therefore is not expected to be a substrate of mitogen-activated protein kinases, suggesting that in vitro phosphorylation by ERK may not be physiological. In the present work, we tested whether transfection with constitutively active MEK-1 into native OK cells could phosphorylate the Na ϩ -K ϩ -ATPase ␣ 1 subunit. The results showed that phosphorylation occurred in these cells but was completely blocked by an inhibitor of PKC␣. Similar results were obtained when constitutively active recombinant GFP-MEK-1 was introduced into native OK cells. We cannot exclude the possibility that PTH-stimulated ERK can directly phosphorylate the Na ϩ -K ϩ -ATPase ␣ 1 subunit. However, the data presented here show that PTH-stimulated activation of PKC␣ by an ERK-dependent pathway is required for phospho-FIG. 12. Effect of wortmannin and LY294002 on PTH-mediated ERK activation, PKC␣ translocation, and N ؉ -K ؉ -ATPase ␣ 1 subunit phosphorylation. OK cells were treated with 10 Ϫ7 M PTH for 15 min in the presence or absence of 10 Ϫ5 M wortmannin or 5 ϫ 10 Ϫ5 M LY294002. A shows a Western blot of cytosolic proteins for phosphorylated and total ERK representative of three independent experiments and the corresponding densitometric analysis. The data show that wortmannin and LY294002 block PTH-stimulated ERK phosphorylation. *, p Ͻ 0.05. B shows a Western blot of membrane proteins using anti-PKC␣ antibodies representative of three independent experiments, and the corresponding densitometric analysis. The data show that wortmannin and LY294002 block translocation of PKC␣. *, p Ͻ 0.05. C shows a Western blot of immunoprecipitated Na ϩ -K ϩ -ATPase ␣ 1 subunit using anti-phosphoserine antibodies representative of three independent experiments and the corresponding densitometric analysis, presented as the means Ϯ S.E. in arbitrary densitometry units. The data indicate that inhibition of PI3 kinase with wortmannin or LY294002 block PTHstimulated phosphorylation of the Na ϩ -K ϩ -ATPase ␣ 1 subunit. *, p Ͻ 0.05. WB, Western blot. rylation of the Na ϩ -K ϩ -ATPase ␣ 1 subunit.
The present study showed that inhibition of PI 3-kinase blocked PTH regulation of sodium pump phosphorylation and activity. These data are consistent with dependence of ERK activation on PI 3-kinase activity (37) and the dependence of PTH regulation of Na ϩ -K ϩ -ATPase activity on ERK activation (17,18). Recently, Al Khalili et al. (38) demonstrated that inhibition of PI 3-kinase by wortmannin and inhibition of PKC by GF109203X blocked ERK activation and phosphorylation of the Na ϩ -K ϩ -ATPase ␣ 1 subunit in primary human skeletal muscle cells. Similarly, Isenovic et al. (39) also showed in vascular smooth muscle cells that angiotensin II increased sodium pump activity through activation of PI 3-kinase and ERK.
In conclusion, the results of this study define a novel signal transduction pathway leading from PTH receptor ligation to inhibition of Na ϩ -K ϩ -ATPase activity in one model of proximal tubular cells, the opossum kidney cell line. The results demonstrate that PTH stimulates ERK by a PI 3-kinase-dependent mechanism. ERK activation is required for activation and translocation of PKC␣, leading to enhanced association with the Na ϩ -K ϩ -ATPase ␣ 1 subunit. This association facilitates subsequent phosphorylation of the Na ϩ -K ϩ -ATPase ␣ 1 subunit and inhibition of sodium pump activity.