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J. Biol. Chem., Vol. 280, Issue 10, 8705-8713, March 11, 2005

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Parathyroid Hormone-mediated Regulation of Na⁺-K⁺-ATPase Requires ERK-dependent Translocation of Protein Kinase C^*

Syed J. Khundmiri, William L. Dean¶, Kenneth R. McLeish¶||, and Eleanor D. Lederer||

From the Departments of Medicine and ^¶Biochemistry and Molecular Biology, University of Louisville and ^||Veterans Affairs Medical Center, Louisville, Kentucky 40202

Received for publication, July 29, 2004 , and in revised form, December 14, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Parathyroid hormone (PTH) inhibits Na⁺-K⁺-ATPase activity by serine phosphorylation of the ₁ 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 PKC by 102 ± 16% and PKCI by 41 ± 7% but had no effect on PKCII and PKC. Both PKC and PKCI phosphorylated the Na⁺-K⁺-ATPase ₁ subunit in vitro. PTH increased the activity of PKC but not PKCI. Coimmunoprecipitation assays demonstrated that treatment with PTH enhanced the association between Na⁺-K⁺-ATPase ₁ subunit and PKC, whereas the association between Na⁺-K⁺-ATPase ₁ subunit and PKCI remained unchanged. A PKC inhibitory peptide blocked PTH-stimulated serine phosphorylation of the Na⁺-K⁺-ATPase ₁ subunit and inhibition of Na⁺-K⁺-ATPase activity. Pharmacologic inhibition of MEK-1 blocked PTH-stimulated translocation of PKC, whereas transfection of constitutively active MEK-1 cDNA induced translocation of PKC and increased phosphorylation of the Na⁺-K⁺-ATPase ₁ subunit. In contrast, PTH-stimulated ERK activation was not inhibited by pretreatment with the PKC inhibitory peptide. Inhibition of PKC expression by siRNA did not inhibit PTH-mediated ERK activation but significantly reduced PTH-mediated phosphorylation of the Na⁺-K⁺-ATPase ₁ subunit. Pharmacologic inhibition of phosphoinositide 3-kinase blocked PTH-stimulated ERK activation, translocation of PKC, and phosphorylation of the Na⁺-K⁺-ATPase ₁ subunit. We conclude that PTH stimulates Na⁺-K⁺-ATPase phosphorylation and decreases the activity of Na⁺-K⁺-ATPase by ERK-dependent activation of PKC.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Na⁺-K⁺-ATPase is an energy-dependent enzyme responsible for the maintenance of intracellular sodium and potassium balance (1). In the proximal renal tubule, the activity of this 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⁺-K⁺-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⁺-K⁺-ATPase activity have been implicated in the pathogenesis of some forms of hypertension associated with impaired salt excretion (2, 3). Not surprisingly, Na⁺-K⁺-ATPase activity is highly regulated by many hormones through multiple signaling pathways, including those relying on protein kinase C (PKC)¹ activation (4).

Multiple PKC isoforms have been identified, comprising a family of closely related serine/threonine kinases (5). Upon activation, PKC translocates from cytosol to membrane compartments (6–9). Three major classes of PKCs have been identified: conventional, Ca⁺-dependent PKCs (, I, II, and ); novel, Ca⁺-independent PKCs (, , , and ); and atypical PKCs ( and ) (10–12). Several laboratories have demonstrated differential regulation of PKC isoforms in kidney by hormones such as angiotensin II (11), dopamine (13), and norepinephrine (14). Parathyroid hormone (PTH), through interaction with the G protein G_q, activates protein kinase C through a phospholipase C-dependent pathway (15).

Several laboratories, including our own, reported that PTH-stimulated PKC activation is critical for inhibition of Na⁺-K⁺-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⁺-K⁺-ATPase activity in opossum kidney cells (OK), a model of renal proximal tubule (19). The PKC isoforms stimulated by PTH that regulate Na⁺-K⁺-ATPase have not been identified. We have previously demonstrated that PTH regulation of Na⁺-K⁺-ATPase activity is PKC- and ERK-dependent (17, 18). However, the mechanisms for PTH-mediated ERK and PKC activation involved in Na⁺-K⁺-ATPase regulation have not been determined. Based on our previous work showing that PKC activates the ERK pathway, we postulated that PTH regulates Na⁺-K⁺-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⁺-K⁺-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⁺-K⁺-ATPase in OK cells by ERK-dependent activation of PKC.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—PTH (1–34) was purchased from Bachem Biosciences Inc. (King of Prussia, PA). Polyclonal antibodies against the Na⁺-K⁺-ATPase ₁ subunit (for immunoprecipitation), recombinant active PKC, PKC1, 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⁺-K⁺-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 (CRQIKIWFQNRRMKWKK) (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.

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⁺-K⁺-ATPase, and regulation of Na⁺-K⁺-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% CO₂. 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^–7 M PTH (bovine 1–34) in the presence or absence of 100 nM PKC isoform-specific peptide inhibitor or 10^–5 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 µg/ml aprotinin, 20 µg/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 x 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 2x 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 –70 °C. To the pellet, 500 µl 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 x 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 2x Laemmli sample buffer was added, boiled for 5 min, and stored at –70 °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 µg/ml aprotinin, 1 µg/ml leupeptin, 100 µl/ml phosphatase inhibitor mixture, 1% Triton X-100, 0.5% Nonidet P-40, and 0.5% SDS were centrifuged at 70,000 x g for 1 h in a Beckman ultracentrifuge. 100 µg 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 (Spectrafuge; National Labnet Co.). The supernatant was incubated overnight at 4 °C with 50 ng of anti-PKC, PKC1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit polyclonal antibodies against the ₁ subunit of Na⁺-K⁺-ATPase (Upstate Biotechnology, Inc.). Protein G- (PKC, or PKC1) or A- (Na⁺-K⁺-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 2x 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⁺-K⁺-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^–7 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 µg 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 µl 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 CaCl₂), substrate mixture (500 µM PKC substrate), cAMP-dependent protein kinase/calmodulin kinase inhibitor mixture (2 µM protein kinase A inhibitor peptide PKI, and 20 µM R24571 [GenBank] 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 MgCl₂, 500 µM ATP and 100 µCi of [-³²P]ATP) were added to the protein G-Sepharose beads and incubated at 30 °C for 10 min. After 10 min a 25-µl 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^–7 M PTH (bovine, 1–34) 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 MgCl₂ 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 x g for 20 min. The pellet containing crude membrane material was resuspended in the homogenizing buffer.

In Vitro Phosphorylation of Na⁺-K⁺-ATPase ₁ Subunit by PKC and PKC1—Crude membranes were prepared from OK cells as described above. Crude membranes were solubilized in IP buffer, and Na⁺-K⁺-ATPase ₁ subunit was immunoprecipitated from 100 µg of membrane protein as described above. Protein A-Sepharose beads containing the immunoprecipitated Na⁺-K⁺-ATPase ₁ subunit were incubated in 40 µl of PKC phosphorylation buffer containing 100 mM HEPES, pH 7.4, 6.5 mM CaCl₂, 4 mM dithiothreitol, 50 mM MgCl₂, 5 mM ATP, 1 mM IP20 (cAMP-dependent protein kinase inhibitory peptide), PKC activator (1 mg/ml phosphatidyl serine, Promega), and 100 nCi of [-³²P]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 2x 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⁺-K⁺-ATPase ₁ subunit antibodies was performed.

Determination of Na⁺-K⁺-ATPase Activity as K⁺-stimulated pNPPase Activity—K⁺-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⁺-K⁺-ATPase activity as K⁺-stimulated pNPPase activity is measured by assessing its ability to cleave p-nitrophenyl phosphate to p-nitrophenol in the presence of high molar concentration of potassium. 20 µl of the membrane (25–30 µg of protein) was added to 0.88 ml of assay buffer (30 mM histidine, 150 mM KCl, 20 mM MgCl₂, 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 µl 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 x 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 µmoles of p-nitrophenol released/mg protein/h.

⁸⁶Rb Uptake—Ouabain-sensitive ⁸⁶Rb uptake was measured as an index of Na⁺-K^+-ATPase-mediated ion transport as described previously (18) following the method of Okafor et al. (29). OK cells were pretreated with 5 µM 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 ⁸⁶Rb (1 µCi/ml ⁸⁶RbCl) in Dulbecco's modified Eagle's without serum. Half the cells received ouabain (final concentration, 1 mM) 15 min prior to the start of ⁸⁶Rb uptake. ⁸⁶Rb 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 µl) of the lysate was used to measure radioactivity. The difference between the ⁸⁶Rb uptake measured in the presence of 1 mM ouabain and the absence of ouabain was used as a measure of Na⁺-K⁺-ATPase-mediated transport activity. The uptake data are expressed as nmol of rubidium (⁸⁶Rb) 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 µl of Genesilencer reagent was diluted with 25 µl of serum-free EMEM. In another tube 25 µl of siRNA diluent was diluted with 15 µl 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% CO₂ 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% CO₂.

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% CO₂, 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 µg/ml GFP-linked constitutively active recombinant MEK-1 was mixed with 5 µl 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% CO₂ incubator. After 4 h, the cells were washed thrice with 1x 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 ± S.E. All of the experiments were repeated at least three times unless otherwise stated to document reproducibility. The p values are calculated using SigmaStat software utilizing a paired t test. A p value less than 0.05 was a priori considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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^–7 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 PKC1 to the membrane fraction, whereas no translocation of PKCII 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 ± 16%, whereas PKC1 was increased by 41 ± 7%. To determine whether translocation of PKC and PKC1 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 PKC1 activity.

View larger version (24K): [in this window] [in a new window]   FIG. 1. Translocation of PKC isoforms to the membrane fraction. OK cells were treated with 10–7 M PTH for 15 min. The membrane fractions were subjected to 10% SDS-PAGE, the proteins were transferred to nitrocellulose membrane, and Western blotting for PKC isoforms , I, II, and was performed. Representative Western blots for the four PKC isoforms are shown from three independent experiments. The bar graph presents quantitative analysis of the blots as the means ± S.E. in arbitrary densitometry units from three independent experiments. The results demonstrate that PTH induces translocation of PKC and PKC1 to the membrane. *, p < 0.05. WB, Western blot.

View larger version (18K): [in this window] [in a new window]   FIG. 2. Effect of PTH on activation of PKC and PKCI. OK cells were treated with 10–7 M PTH for 15 min. PKC and PKCI were immunoprecipitated from whole cell lysates, and PKC activity was measured as described under "Experimental Procedures." Each bar represents the mean activity ± S.E. (pmol of 32Pi incorporated into PKC substrate peptide/mg protein/min) from three independent experiments. The results demonstrate that PTH activates PKC. *, p < 0.05.

View larger version (30K): [in this window] [in a new window]   FIG. 3. In vitro phosphorylation of Na+-K+-ATPase 1 subunit by PKC or PKCI. The Na+-K+-ATPase 1 subunit was immunoprecipitated from OK cell crude membrane fractions and subjected to in vitro phosphorylation by recombinant active PKC or PKCI. A representative autoradiograph of Na+-K+-ATPase 1 subunit phosphorylation from three independent experiments is shown (upper panel). The lower panel is a Western blot for the Na+-K+-ATPase 1 subunit showing equal loading in each condition. The graph presents quantitative data as the means ± S.E. in arbitrary densitometry units from three independent phosphorylation experiments. The figure shows that PKC associates with Na+-K+-ATPase 1 subunit upon activation by PTH. *, p < 0.05. WB, Western blot.

View larger version (26K): [in this window] [in a new window]   FIG. 4. Association between Na+-K+-ATPase 1 subunit, PKC, and PKCI. OK cells were treated with 10–7 M PTH for 15 min. The Na+-K+-ATPase 1 subunit, PKC, or PKCI was immunoprecipitated from crude membrane fractions and analyzed by Western blotting for Na+-K+-ATPase 1 subunit, PKC, and PKCI. A shows representative blots from three independent experiments. B shows a graph of the quantitative data as the means ± S.E. in arbitrary densitometry units from these three independent experiments. The figure shows that PTH stimulates increased association of PKC with the Na+-K+-ATPase 1 subunit, whereas association of PKC1 is unchanged. *, p < 0.05. WB, Western blot.

View larger version (21K): [in this window] [in a new window]   FIG. 5. Effect of PKC1 inhibitory peptide and U0126 on PTH-mediated Na+-K+-ATPase subunit phosphorylation. OK cells were treated with 10–7 M PTH for 15 min in the continued presence or absence of 100 nM PKC inhibitory peptide or U0126. The Na+-K+-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 ± S.E. in arbitrary densitometry units from these three independent experiments. Both PKC inhibitory peptide and inhibition of ERK activation with U0126 blocked Na+-K+-ATPase 1 subunit phosphorylation.*, p < 0.05. WB, Western blot.

View larger version (16K): [in this window] [in a new window]   FIG. 6. Effect of PKC inhibitory peptides on PTH-mediated inhibition of Na+-K+-ATPase activity. OK cells were treated with 10–7 M PTH for 15 min in the presence or absence of 100 nM PKC inhibitory peptide. A shows Na+-K+-ATPase activity measured in crude membranes expressed as percentages of control K+-dependent pNPPase activity. The results demonstrate that PTH increases ouabain sensitive K+-dependent pNPPase activity. Each bar represents the mean (±S.E.) percentage of respective control from four independent experiments performed in triplicate. B shows ouabain sensitive 86Rb uptake stimulated by 10–7 M PTH in the presence and the absence of 100 nM PKC inhibitory peptide. Each bar represents the mean (± 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+-dependent pNPPase activity and ouabain-sensitive 86Rb uptake. *, p < 0.05.

View larger version (27K): [in this window] [in a new window]   FIG. 7. Effect of PKC inhibitory peptide and U0126 on PTH-mediated PKC translocation and ERK activation. OK cells were treated with 10–7 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 ± 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 ± S.E. in arbitrary densitometry units, confirming the inhibition of ERK phosphorylation by U0126 but not by PKC inhibitory peptide. *, p < 0.05. WB, Western blot.

View larger version (34K): [in this window] [in a new window]   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 10–7 M PTH for 15 min. A shows an immunoblot for PKC and PKC1 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 PKC1, expression. B shows a representative Western blot of Na+-K+-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+-K+-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.

View larger version (38K): [in this window] [in a new window]   FIG. 9. Effect of transient transfection of active MEK-1 on ERK activation, PKC translocation, and Na+-K+-ATPase 1 subunit phosphorylation. OK cells were transiently transfected with constitutively active MEK-1 cDNA, transfected with plasmid alone, or exposed only to vehicle. A shows a Western blot for phosphorylated and total ERK representative of three independent experiments. The blots show that transfection with active MEK-1 induces phosphorylation of ERK. B shows a Western blot of membrane proteins from the same groups of cells for PKC. The results show that PKC translocated to the membrane in cells transfected with active MEK-1 but not in mock transfected cells. The blot is representative of three independent experiments. C is a Western blot of Na+-K+-ATPase 1 subunit immunoprecipitated from the crude membrane fractions from the same groups of cells using anti-phosphoserine antibodies (upper panel) or anti-Na+-K+-ATPase 1 subunit (lower panel). The results show that transfection with active MEK-1 stimulated phosphorylation of the Na+-K+-ATPase 1 subunit, and equal amounts of the subunit were immunoprecipitated. The blots are representative of three independent experiments. WB, Western blot.

View larger version (23K): [in this window] [in a new window]   FIG. 10. Effect of uptake of constitutively active recombinant MEK-1 on ERK activation, PKC translocation, Na+-K+-ATPase 1 subunit phosphorylation, and Na+-K+-ATPase activity. Constitutively active recombinant MEK-1 was introduced into OK cells using Bioporter reagent. A shows a Western blot for phosphorylated ERK (upper panel) and ERK2 representative of three independent experiments. The results show that introduction of active MEK-1-induced phosphorylation of ERK. B shows a Western blot of membrane proteins using anti-PKC antibodies representative of three independent experiments. The results show that introduction of active MEK-1 stimulated translocation of PKC to the membrane. C shows a Western blot of immunoprecipitated Na+-K+-ATPase 1 subunit using anti-phosphoserine antibodies representative of three independent experiments. The results show that introduction of active MEK-1 stimulated phosphorylation of Na+-K+-ATPase 1 subunit. D shows the effect of MEK-1 transduction on ouabain-sensitive K+-dependent pNPPase activity in crude membrane fractions. The results are presented as the mean (± S.E.) percentage of control from four independent experiments performed in triplicate. The data show that introduction of MEK-1 significantly inhibits basal activity. *, p < 0.05. WB, Western blot.

View larger version (49K): [in this window] [in a new window]   FIG. 11. Effect of PKC inhibition on ERK activation, PKC translocation, and Na+-K+-ATPase 1 subunit phosphorylation induced by transient transfection of active MEK-1. OK cells were transiently transfected with active MEK-1 cDNA in the presence or absence of PKC inhibitory peptide. A shows a Western blot of cytosolic proteins for phosphorylated ERK and total ERK. A representative Western blot from two independent experiments is shown. The results show that transfection of MEK-1 induced activation of ERK. B shows a Western blot of membrane proteins using anti-PKC antibodies. A representative Western blot from two independent experiments is shown. The data indicate that transfection with MEK-1 stimulated membrane translocation of PKC. C shows a Western blot of immunoprecipitated Na+-K+-ATPase 1 subunit using anti-phosphoserine representative of two independent experiments. The results show that MEK-1 transfection stimulated phosphorylation of Na+-K+-ATPase 1 subunit. WB, Western blot.

View larger version (29K): [in this window] [in a new window]   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 10–7 M PTH for 15 min in the presence or absence of 10–5 M wortmannin or 5 x 10–5 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 < 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 < 0.05. C shows a Western blot of immunoprecipitated Na+-K+-ATPase 1 subunit using anti-phosphoserine antibodies representative of three independent experiments and the corresponding densitometric analysis, presented as the means ± S.E. in arbitrary densitometry units. The data indicate that inhibition of PI3 kinase with wortmannin or LY294002 block PTH-stimulated phosphorylation of the Na+-K+-ATPase 1 subunit. *, p < 0.05. WB, Western blot.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We also identified PKC1, PKCII, and PKC isoforms in OK cells, but none appear to have a significant role in PTH regulation of Na⁺-K⁺-ATPase. Although PKC1 coimmunoprecipitated with the Na⁺-K⁺-ATPase ₁ subunit and phosphorylated the ₁ subunit in an in vitro phosphorylation assay, treatment with PTH did not stimulate PKC1 activity or increase the association of PKC1 with Na⁺-K⁺-ATPase. These findings are also consistent with the report of Middleton et al. (31), which detected PKC1 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⁺-K⁺-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⁺-K⁺-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 Thr⁴⁹⁷, Thr⁶³⁸, and Ser⁶⁵⁷ residues (34). Computer-based phosphorylation motif screening of PKC identified a potential ERK1/2 phosphorylation site at Thr⁶³⁸ (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⁺-K⁺-ATPase regulation by PKC is ERK-dependent (17).

We and others recently demonstrated that ERK might directly phosphorylate the Na⁺-K⁺-ATPase ₁ subunit (18, 38). In our studies, inhibition of MEK-1 by U0126 blocked PTH-mediated phosphorylation of the Na⁺-K⁺-ATPase ₁ subunit and inhibition of Na⁺-K⁺-ATPase activity. Further, we showed that the Na⁺-K⁺-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 Ser¹⁸ to alanine mutant rat ₁ but not in the Ser¹¹ to alanine mutant (18). Feschenko and Sweadner (32) demonstrated that Ser¹¹ is a PKC phosphorylation site. Ser¹¹ 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⁺-K⁺-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⁺-K⁺-ATPase ₁ subunit. However, the data presented here show that PTH-stimulated activation of PKC by an ERK-dependent pathway is required for phosphorylation of the Na⁺-K⁺-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⁺-K⁺-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⁺-K⁺-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⁺-K⁺-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⁺-K⁺-ATPase ₁ subunit. This association facilitates subsequent phosphorylation of the Na⁺-K⁺-ATPase ₁ subunit and inhibition of sodium pump activity.

	FOOTNOTES

 
^* This work was supported by Veterans Affairs Merit Reviews (to E. D. L. and K. R. M.); Scientist Development Grant 0435153N from the American Heart Association; American Heart Association, Ohio Valley Affiliate, Postdoctoral Fellowship Grant 0120318B (to S. J. K.); NIDDK, National Institutes of Health Grant DK62389 (to K. R. M.); and American Heart Association, Ohio Valley Affiliate, Grant-in-aid 0355350 (to W. L. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Kidney Disease Program, University of Louisville, 570 S Preston St., Louisville, KY 40202. Tel.: 502-852-0014; Fax: 502-852-4384; E-mail: syed.khundmiri{at}louisville.edu.

¹ The abbreviations used are: PKC, protein kinase C; PTH, parathyroid hormone; ERK, extracellular signal-regulated kinase; OK, opossum kidney; siRNA, small inhibitory RNA; PBS, phosphate-buffered saline; EMEM, minimal essential medium with Earl's salts; IP, immunoprecipitation; MOPS, 4-morpholinepropanesulfonic acid; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; GFP, green fluorescent protein; PI, phosphatidylinositol; pNPPase, p-nitrophenylphosphatase.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. D. Mochly-Rosen (Stanford University School of Medicine, Stanford, CA) for the gift of PKC isoform-specific inhibitory peptides. We thank Nina Lesousky for expert technical assistance.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Feraille, E., and Doucet, A. (2001) Physiol. Rev. 81, 345–418[Abstract/Free Full Text]
2. Zhang, Y., Norian, J. M., Magyar, C. E., Holstein-Rathlou, N. H., Mircheff, A. K., and McDonough, A. A. (1999) Am. J. Physiol. 276, F711–F719[Medline] [Order article via Infotrieve]
3. Hussain, T., and Lokhandwala, M. F. (2003) Exp. Biol. Med. 228, 134–142[Abstract/Free Full Text]
4. Mendez, C. F., Leibiger, I. B., Leibiger, B., Hoy, M., Gromada, J., Berggren, P. O., and Bertorello, A. M. (2003) J. Biol. Chem. 278, 44753–44757[Abstract/Free Full Text]
5. Nishizuka, Y. (1995) FASEB J. 9, 484–496[Abstract]
6. Kraft, A. S., and Anderson, W. B. (1983) Nature 301, 621–623[CrossRef][Medline] [Order article via Infotrieve]
7. Mochly-Rosen, D., Henrich, C. J., Cheever, L., Khaner, H., and Simpson, P. C. (1990) Cell Regul. 1, 693–706[Medline] [Order article via Infotrieve]
8. Cambier, J. C., Newell, M. K., Justement, L. B., McGuire, J. C., Leach, K. L., and Chen, Z. Z. (1987) Nature 327, 629–932[CrossRef][Medline] [Order article via Infotrieve]
9. Rotem, R., Paz, G. F., Homonnai, Z. T., Kalina, H., and Naor, Z. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7305–7308[Abstract/Free Full Text]
10. Aristimuno, P. C., and Good, D. W. (1997) Am. J. Physiol. 272, F624–F631[Medline] [Order article via Infotrieve]
11. Karim, Z., Defontaine, N., Paillard, M., and Poggioli, J. (1995) Am. J. Physiol. 269, C134–C140[Medline] [Order article via Infotrieve]
12. Boesch, D. M., and Garvin, J. L. (2001) Am. J. Physiol. 281, R861–R867
13. Budu, C. E., Efendiev, R., Cinelli, A. M., Bertorello, A. M., and Pedemonte, C. H. (2002) (2002) Br. J. Pharmacol. 137, 1380–1386[CrossRef][Medline] [Order article via Infotrieve]
14. Wang, X. D., Kiang, J. G., Atwa, M. A., and Smallridge, R. C. (1996) J. Investig. Med. 44, 566–574[Medline] [Order article via Infotrieve]
15. Dunlay, R., and Hruska, K. (1990) Am. J. Physiol. 27, F223–F231
16. Ominato, M., Sato, T., and Katz, A. I. (1996) J. Membr. Biol. 152, 235–243[CrossRef][Medline] [Order article via Infotrieve]
17. Khundmiri, S. J., and Lederer, E. D. (2002) (2002) Am. J. Physiol. 282, F512–F522
18. Khundmiri, S. J., Bertorello, A. M., Delamere, N. A., and Lederer, E. D. (2004) J. Biol. Chem. 279, 17418–17427[Abstract/Free Full Text]
19. Liang, M., and Knox, F. G. (1999) Am. J. Physiol. 277, F859–F865[Medline] [Order article via Infotrieve]
20. Souroujon, M. C., and Mochly-Rosen, D. (1998) Nat. Biotechnol. 16, 919–924[CrossRef][Medline] [Order article via Infotrieve]
21. Disatnik, M. H., Boutet, S. C., Lee, C. H., Mochly-Rosen, D., and Rando, T. A. (2002) J. Cell Sci. 115, 2151–2163[Abstract/Free Full Text]
22. Schechtman, D., and Mochly-Rosen, D. (2002) Methods Enzymol. 345, 470–489[Medline] [Order article via Infotrieve]
23. Ridge, K. M., Dada, L., Lecuona, E., Bertorello, A. M., Katz, A. I., Mochly-Rosen, D., and Sznajder, J. I. (2002) Mol. Biol. Cell 13, 1381–1389[Abstract/Free Full Text]
24. Braun, M. U., and Mochly-Rosen, D. (2003) J. Mol. Cell Cardiol. 35, 895–903[CrossRef][Medline] [Order article via Infotrieve]
25. Clarke, H., Ginanni, N., Peralta, S., and Mullin, J. M. (2000) Kidney Int. 58, 1004–1015[CrossRef][Medline] [Order article via Infotrieve]
26. Lederer, E. D., Khundmiri, S. J., and Weinman, E. J. (2003) J. Am. Soc. Nephrol. 14, 1711–1719[Abstract/Free Full Text]
27. Hird, R. B., Wakefield, T. W., Mukherjee, R., Jones, B. U., Crawford, F. A., Andrews, P. C., Stanley, J. C., and Spinale, F. G. (1995) Circulation 92, 433–446[Abstract/Free Full Text]
28. Tran, C. M., and Farley, R. (1996) Biochemistry 35, 47–55[CrossRef][Medline] [Order article via Infotrieve]
29. Okafor, M. C., Mukhopadhyay, P., and Delamere, N. A. (2002) Investig. Ophthalmol. Vis. Sci. 43, 790–796[Abstract/Free Full Text]
30. Coulson, R., Proch, P. S., Olsson, R. A., Chalfant, C. E., and Cooper, D. R. (1996) Am. J. Physiol. 270, F263–F274[Medline] [Order article via Infotrieve]
31. Middleton, J. P., Khan, W. A., Collinsworth, G., Hannun, Y. A., and Medford, R. M. (1993) J. Biol. Chem. 268, 15958–15964[Abstract/Free Full Text]
32. Feschenko, M. S., and Sweadner, K. J. (1995) J. Biol. Chem. 270, 14072–14077[Abstract/Free Full Text]
33. Efendiev, R., Bertorello, A. M., and Pedemonte, C. H. (1999) FEBS Lett. 45, 45–48
34. Hansara, G., Garcia-Paramio, P., Prevostel, C., Whelan, R. D. H., Bornancin, F., and Parker, P. (1999) Biochem. J. 342, 337–344[CrossRef][Medline] [Order article via Infotrieve]
35. Yaffe, M. B., Leparc, G. G., Lai, J., Obata, T., Volinia, S., and Cantley, L. C. (2001) Nat. Biotechnol. 19, 348–353[CrossRef][Medline] [Order article via Infotrieve]
36. Adayev, T., Ray, I., Sondhi, R., Sobocki, T., and Banerjee, P. (2003) Biochim. Biophys. Acta 1640, 85–96[Medline] [Order article via Infotrieve]
37. Lederer, E. D., Sohi, S. S., and McLeish, K. R. (2000) J. Am. Soc. Nephrol. 9, 975–985
38. Al-Khalili, L., Kotova, O., Tsuchida, H., Ehren, I., Feraille, E., Krook, A., and Chibalin, A. V. (2004) J. Biol. Chem. 279, 25211–25218[Abstract/Free Full Text]
39. Isenovic, E. R., Jacobs, D. B., Kedees, M. H., Sha, Q., Milivojevic, N., Kawakami, K., Gick, G., and Sowers, J. R. (2004) Endocrinology 145, 1151–1160[Abstract/Free Full Text]

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