HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 275, Issue 44, 34693-34700, November 3, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/44/34693    most recent M005869200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Feschenko, M. S. Articles by Sweadner, K. J. Search for Related Content PubMed PubMed Citation Articles by Feschenko, M. S. Articles by Sweadner, K. J. Social Bookmarking                      What's this?

Interaction of Protein Kinase C and cAMP-dependent Pathways in the Phosphorylation of the Na,K-ATPase^*

Marina S. Feschenko, Elizabeth Stevenson, and Kathleen J. Sweadner

From the Laboratory of Membrane Biology, Neuroscience Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129

Received for publication, July 5, 2000, and in revised form, August 3, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To test the hypothesis that there is cross-talk between the protein kinase C (PKC) and protein kinase A (PKA) pathways in the regulation of the Na,K-ATPase, we measured its phosphorylation in mammalian cell cultures. Phosphorylation of the PKC site, Ser-18, appeared to be due to the activation of the  isoform of the kinase. In NRK-52E and L6 cells, this phosphorylation was reduced by prior activation of a cAMP-dependent signaling pathway with forskolin. In principle this would be consistent with direct interaction between the two phosphorylation sites, but further investigation suggested a more indirect mechanism. First, phosphorylation of Ser-938, the PKA site, could not be detected despite the presence of active PKA. Second, there was a major reduction in the phosphorylation of unrelated phosphoproteins as a consequence of elevation of cAMP, suggesting generalized reduction of kinase activity or activation of phosphatase activity. In NRK-52E and L6, phosphorylation of the Na,K-ATPase at Ser-18 paralleled this global change. In C6 cells, in contrast, there was no cAMP effect on Na,K-ATPase phosphorylation at Ser-18 and no global cAMP effect on other phosphoproteins. The cross-talk is evidently mediated by events occurring at the cellular level.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Na,K-ATPase¹ is a membrane protein that has two major subunits,  and . Phosphorylation of the  subunit by protein kinases has been proposed to mediate the regulation of the enzyme. The phosphorylation sites for protein kinase A (cAMP-activated) (1-3) and protein kinase C (2, 4, 5) have been identified. Rat Na,K-ATPase 1 subunit is phosphorylated in vitro by protein kinase C (PKC) predominantly at Ser-18 and by protein kinase A (PKA) at Ser-938.² Site-directed mutagenesis studies have supported the participation of these sites in Na,K-ATPase regulation (1, 6-10). The molecular mechanism of regulation is an open question, however, because seemingly inconsistent functional effects have been reported with different experimental systems, including either inhibition or activation ostensibly through the same kinase. A variety of mechanisms have been proposed, including direct inhibition or stimulation by kinases (11-13), regulation of enzyme activity through pathways that include lipid metabolites (8, 14), and regulation by recruitment to, and internalization from, the plasma membrane (10, 15). Only in some cases has it been shown whether (and which) sites on the Na,K-ATPase are actually used by protein kinases in intact cells, however (5, 16-20). Even less is known about the phosphorylation stoichiometry reached in intact cells in experimental conditions (16, 21).

Interactions between PKC and cAMP-dependent pathways in regulation of kidney Na,K-ATPase have been suspected for some time (22). Recently it has been reported that phosphorylation of the 1 subunit by protein kinase C is modulated by a cAMP-stimulated pathway (23) and possibly by the state of phosphorylation of the protein kinase A site, Ser-938 (17). Such cross-talk between cAMP-dependent and Ca²⁺, phospholipid-dependent signaling pathways might affect the final state of the enzyme and account for some of the complexity in the literature. Cross-talk could occur at several levels: regulation of kinase activities (24), regulation of phosphatase activities (18), or direct conformational changes of Na,K-ATPase affecting the accessibility of phosphorylation sites.

The possibility that kinases interact through affecting the conformation of the Na,K-ATPase is supported by certain evidence. The  subunit is the catalytic subunit, and it undergoes conformational transitions upon binding its ligands during the working cycle. The Na⁺ binding conformation is known as E1, and the K⁺ binding conformation is known as E2. In the test tube, the enzyme can be arrested in various specific conformations by the choice of ligands added, and the conformational state can be probed by such means as fluorescence microscopy and protease sensitivity (25). We observed that ligands that stabilize the Na,K-ATPase in different conformations affected phosphorylation by protein kinase C and protein kinase A in vitro in a reciprocal way (3). Phosphorylation by protein kinase A was maximal in the presence of ligands that promote the E1 conformation, whereas phosphorylation by protein kinase C was maximal in ligands that promote the E2 conformation. In related observations, Logvinenko et al. (12) report that protein kinase C shifts the conformation from E2 to E1, as measured with eosin fluorescence. Furthermore, phosphorylation by protein kinase A in vitro requires the presence of Triton X-100 (3, 26), whereas phosphorylation by protein kinase C is prevented in the same conditions (3). Triton X-100 inactivates enzyme activity in these conditions, and kinetic effects on enzyme partial reactions suggests that the inactive enzyme is in the E1 conformation (27). These observations indicate that accessibility of the two phosphorylation sites depends on the conditions, making it plausible that phosphorylation of the sites could interact in a complementary manner.

Using an antibody-based method for quantifying phosphorylation at Ser-18 by protein kinase C, we were able to demonstrate very high levels of phorbol ester-stimulated phosphorylation in intact cells from three different rat cell lines: NRK-52E (normal rat kidney), L6 (muscle), and C6 (glioma). With these, we investigated the effect of activation of the cAMP pathway on Ser-18 phosphorylation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- Calyculin A, forskolin, 3-isobutyl-1-methylxanthine (IBMX), and phorbol myristoyl acetate (PMA) were from RBI/Sigma (Natick, MA), and cyclosporin A was from Alexis Biochemicals (San Diego, CA). Protein kinase A catalytic subunit (catalog #P8289), anti-mouse IgG-agarose, and BAPTA-AM were obtained from Sigma.

Cell Cultures-- Three rat cell lines, NRK (normal rat kidney, clone 52E), C6 glioma, and L6, were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (100 IU of penicillin/ml, 100 µg of streptomycin/ml) in a 5% CO₂, 95% air atmosphere. NRK and C6 cells used in phosphorylation experiments were near confluence. L6 cells were allowed to fuse and form myotubes in DMEM containing 2% fetal bovine serum for 7-10 days. The Na,K-ATPase isoforms expressed by these cultures are: NRK-52E, 1 and 1; C6, 1 and 3; L6, 1 and 3, with a small amount of 1 (28).³

Cell Treatment-- Monolayers of cells in 35-mm tissue culture dishes were preincubated for 1.5-2 h at 37 °C in 5% CO₂ in DMEM without serum. Then drugs (forskolin, IBMX, calyculin A, PMA, cyclosporin A) or their vehicle (Me₂SO or ethanol) were added at concentrations and for times indicated in the figure legends, and the cells were incubated at 37 °C. The medium was discarded, dishes were placed on ice, and 0.2 ml of 1% SDS at 100 °C in 10 mM Tris-HCl buffer, pH 7.4, was added to each dish. The dishes were scraped, and the detergent extract was clarified by centrifugation for 30 min at 40,000 rpm. Supernatants were diluted 1:1 with Laemmli sample buffer, and aliquots (30 µl/well, approximately 15 µg of protein) were loaded on SDS gels.

Gel Electrophoresis, Electrophoretic Transfer, and Immunostaining-- Electrophoresis and Western blotting were performed as described previously (16) using 10% polyacrylamide gels. Monoclonal antibody McK1 (29, 30) was used as a phosphorylation-blocked antibody to detect PKC phosphorylation of Na,K-ATPase at Ser-18 as described previously (16). A dialyzed ammonium sulfate concentrate was prepared and used at 1:20,000. Details on procedures for obtaining quantitative data are described elsewhere (31). Polyclonal antibody 470 (a generous gift of Dr. A. C. Nairn, Rockefeller University), raised against a phosphorylated peptide derived from the PKA site of Na,K-ATPase, was used to detect phosphorylation at Ser-938 at 1:20,000 (1). This same antibody detected many other phosphoproteins. Polyclonal antibody to phosphoserine from Zymed Laboratories Inc. (San Francisco, CA) was used to detect phosphorylated proteins in cell lysates at 1:700. Horseradish peroxidase-conjugated secondary antibodies were obtained from Sigma, and luminol reagents were from Pierce.

Polyclonal antibody K1 (1:5,000) (32) and monoclonal antibody 6F (1:500) (30) (Developmental Studies Hybridoma Bank, Iowa City, IA) were routinely used for Na,K-ATPase  subunit detection independent of the state of phosphorylation. Although not shown in most figures, this control for uniform protein recovery and gel loading was always performed. Blots were also routinely stained with Amido Black to check protein loading.

Immunofluorescence Localization of PKC Isoforms-- Protein kinase C isoforms were detected with antibodies from a kit (anti-PKC isozyme sampler set 10267-001) from Life Technologies, Inc. NRK-52E cells growing on multi-well glass slides (Falcon) were fixed for 6 min with 20 °C 100% methanol after incubation either with control medium or medium containing 1 µM PMA for 15 min. After fixation, the cells were washed with phosphate-buffered saline and stored at 4 °C overnight. Then they were permeabilized with 0.3% Triton X-100 in phosphate-buffered saline and 5% goat serum and incubated for 1 h with primary antibody diluted (1:200) in phosphate-buffered saline plus 5% goat serum, 0.1% Triton X-100 overnight at 4 °C. After three washes, they were incubated with rhodamine-conjugated goat-anti-rabbit IgG (1:50), washed three times, and photographed with a Zeiss IM35 fluorescence microscope.

Intracellular cAMP Measurement-- For cAMP measurement, cells were seeded in culture medium in 96-well plates at 10⁵ cells/well and incubated overnight at 37 °C. The next day the medium was replaced with fresh DMEM without serum, and cells were incubated for 2 h. Then forskolin and IBMX were added to final concentrations of 50 and 500 µM, respectively. Control cells were treated with the equivalent volume of Me₂SO. After 15 min of incubation at 37 °C, the medium was aspirated, and intracellular cAMP was assayed by the non-acetylation method with kit RPN 225 from Amersham Pharmacia Biotech. This method employs competitive enzyme-linked immunosorbent assay with antibody against cAMP.

Immunoprecipitation-- C6 or NRK cells in 100-mm tissue culture dishes were incubated with drugs as described in the text. The incubation medium was aspirated, and cells were immediately placed on ice. After a brief wash with ice-cold TBS buffer (10 mM Tris HCl, pH 7.5, 150 mM NaCl), 1 ml of ice-cold TBS buffer containing three detergents, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium deoxycholate, and protease and protein phosphatase inhibitor mixture (0.2 mM phenylmethylsulfonylfluoride, 10 µg/ml aprotinin, 5 µg/ml pepstatin A, 1 mM benzamidine, 0.1 mM Na₃VO₄, and 10 mM NaF) (TIPA buffer) was put in each dish and incubated on ice for 10-15 min. When cells had been treated with calyculin A, the protein phosphatase inhibitor was added to the dissociation buffer at the same final concentration to ensure continued inhibition. Cell extracts were disrupted by repeated aspiration through 21-gauge needles, and the detergent extracts were centrifuged at 10,000 rpm for 5 min at 4 °C. Supernatants containing 500 µg of total cellular protein were incubated (with rotation) overnight at 4 °C with 2-4 µg of monoclonal antibody 6H against Na,K-ATPase  subunit (a generous gift of Dr. M. J. Caplan, Yale University Medical School). The next day, 40 µl of anti-mouse IgG agarose (pre-washed and resuspended in TIPA buffer) was added and incubated for two more hours. Immunoprecipitates were collected by centrifugation at 10,000 rpm for 10 min at 4 °C and washed 2 times with 1 ml of TIPA buffer per sample. After the final wash, the pellet was resuspended in 40 µl of 1× electrophoresis sample buffer. Samples were incubated for 20 min at room temperature and centrifuged at 10,000 rpm for 10 min. Supernatants were saved. Pellets were washed with an additional 20 µl of 1× electrophoresis sample buffer and centrifuged again. The supernatants were combined and heated for 10 min at 65 °C before loading on the gel.

Phosphorylation of Na,K-ATPase by PKA-- For phosphorylation by protein kinase A in vitro and in cell extracts, purified Na,K-ATPase from rat renal medulla was obtained by the procedure of Jørgensen (33), as described previously (16).

In vitro phosphorylation with purified Na,K-ATPase and purified protein kinase A was performed as described previously (3), except that ³²P was used previously, and here phosphorylation was detected with a Ser-938 phosphoprotein-specific antibody. Triton X-100 at 0.05% was included because without it the Na,K-ATPase is not phosphorylated by this kinase. We previously showed that its addition did not alter the ability of protein kinase A to phosphorylate kemptide as an artificial substrate (3).

Phosphorylation in Cell Extracts-- Cell extracts of NRK and C6 cells were made as follows. NRK cells grown in 100 mM tissue culture dishes were washed two times with warm DMEM without serum and once with ice-cold TBS-buffer (10 ml/dish). The buffer was aspirated, and 1 ml/dish of ice-cold extraction buffer was added containing 50 mM Tris HCl, pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 10% (v/v) glycerol, 0.1% (v/v) 2-mercaptoethanol, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 25 µg/ml leupeptin, and 25 µg/ml aprotinin. The dishes were incubated on ice for 10 min, and the cell extracts were scraped and homogenized with a Polytron homogenizer in two bursts of 10-15 s. Extracts were clarified by centrifugation at 1000 rpm for 5 min, then supernatants were separated into aliquot and frozen at -80 °C.

It is our experience that protein phosphatase remains active in disrupted cells when calyculin A is not present, and so the extracts should have had relatively low phosphoprotein levels, as suggested by the faint stain of background bands by antibody 470 seen in Fig. 8, compared with Fig. 5. The inhibitor was added only during the assay. For the phosphorylation assays, 30-µg samples of extract protein were supplemented with 0.5 mM cAMP, 1 mM ATP, 2 mM MgCl₂, 100 nM calyculin A, and 0.05% Triton X-100 from concentrated stocks (100-fold or more). 1 µg of purified Na,K-ATPase and 100 ng of PKA catalytic subunit were added where indicated, and incubations were for 30 min at 30 °C. Laemmli sample buffer was added from a 4× concentrate, and the entire sample was loaded on gels.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phosphorylation and Dephosphorylation of the PKC Site of Na,K-ATPase in Intact Cells-- Previously we developed a semi-quantitative, antibody-based assay for the detection of PKC phosphorylation of Na,K-ATPase in intact cells (16). The method has several advantages as follows. There is no need to starve cells of inorganic phosphate or load ³²P, there is no need to perform immunoprecipitation, and with this method we were able to demonstrate close to stoichiometric levels of phosphorylation. Furthermore, the method is insensitive to phosphorylation turnover rates, unlike ³²P. The epitope of monoclonal antibody McK1 includes Ser-18 (29, 30). Incorporation of phosphate at Ser-18, the major PKC phosphorylation site in rat 1, blocks binding of McK1 on Western blots. Thus, McK1 binding can be used to estimate the amounts of nonphosphorylated Ser-18 in a sample, and the loss of binding represents the amount of phosphorylated Ser-18. Fig. 1A shows a representative phosphorylation experiment with two different cell lines: NRK-52E and C6. Activation of PKC with PMA alone caused some phosphorylation (some reduction in McK1 binding), but much more was seen (70-90%) when PMA was added with protein phosphatase inhibitors calyculin A (shown) or okadaic acid (not shown). Calyculin A alone had no effect, suggesting that in the absence of stimuli the activity of PKC in these cell lines was low. Since calyculin A and okadaic acid are known to inhibit protein phosphatase types 1 and 2A, one or both of these phosphatases (or related phosphatases) is likely to be responsible for the dephosphorylation of Ser-18. Cyclosporin A, an inhibitor of protein phosphatase 2B (calcineurin), failed to increase Ser-18 phosphorylation levels in the presence or absence of PMA in either cell line (Fig. 1A).

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Detection of PKC phosphorylation at Ser-18 in intact cells. After treatments with drugs, NRK or C6 cells were lysed and subjected to SDS-gel electrophoresis, transferred to nitrocellulose, and stained with McK1 antibody. A reduction in binding reflects an increase in phosphorylation at Ser-18. Blots were later stripped and stained with 6F antibody, which is unaffected by phorphorylation, as a control for uniform gel loading (not shown). A, cells were incubated with or without 1 µM PMA or 100 nM calyculin A for 30 min at 37 °C. When cyclosporin A (CsA) was used, cells were preincubated with 80 µM cyclosporin A for 1 h at 37 °C. Activation of PKC with PMA produced some phosphorylation, which was enhanced by calyculin A but not by cyclosporin A. B, cells were preincubated with 5 µM PMA for 24 h to down-regulated PMA-sensitive isoforms of PKC. After a brief washing they were incubated with 100 nM calyculin A or a combination of calyculin A and 1 µM PMA for 30 min at 37 °C to stimulate phosphorylation at Ser-18. No phosphorylation was detected. C, cells were incubated in regular or calcium-free medium in the presence or absence of 100 µM BAPTA-AM. Then PKC phosphorylation was stimulated by PMA treatment with or without calyculin A as described above. 1, control; 2, PMA; 3, calyculin A; 4, calyculin A and PMA. All data are representative examples of replicate experiments.

Sensitivity to phorbol ester and to calcium narrowed down the isoforms of PKC that are likely to be phosphorylating the Na,K-ATPase in these cells. Down-regulation of PKC by prolonged incubation with PMA completely abolished PKC phosphorylation in both cell lines (Fig. 1B). Preincubation of the cells in calcium-free DMEM and in the presence of the calcium chelator BAPTA-AM abolished PKC phosphorylation of Na,K-ATPase in both NRK and C6 cells (Fig. 1C). Taken together the results suggest that phosphorylation of Ser-18 is carried out by a phorbol ester-sensitive, calcium-dependent PKC isoform, such as , , or . NRK-52E grows as an epithelial layer with tight junctions between cells and has organized apical and basolateral surfaces (34). Using PKC isoform-specific antibodies and immunocytochemistry, we observed that , , and  isoforms were present in NRK-52E cells but  showed the most robust redistribution to the plasma membrane when treated with 1 µM PMA. Redistribution of  to the basolateral plasma membrane, where the Na,K-ATPase is found, could be seen as a sharp line of stain between cells (Fig. 2) compared with the cytoplasmic location in resting cells. PKC isoforms  and  had a perinuclear distribution in resting cells (Fig. 2) and showed less robust relocalization to the plasma membrane. The  isoform was found at the basolateral membrane in resting and stimulated cells (Fig. 2), but since it is not calcium-sensitive, it cannot be the kinase that acts on Na,K-ATPase. PKC  and  isoforms were also detected in NRK-52E cells with isoform-specific antibodies but did not show any detectable stain at the basolateral membrane (data not shown). The results were supported by Western blot analysis of membrane and cytosolic fractions of control and PMA-treated NRK-52E and C6 cells (data not shown). In aggregate, the data suggest that the PKC  isoform is the most likely candidate for Na,K-ATPase phosphorylation.

View larger version (98K): [in this window] [in a new window]   Fig. 2.   Recruitment of PKC isoforms to the basolateral membrane in NRK-52E. Immunofluorescence microscopy was used to detect PKC isoforms and assess whether they were recruited to the membrane surface where the Na,K-ATPase is found. For each antibody shown, cells were either untreated or treated with 1 µM PMA for 15 min in DMEM culture medium without serum, then fixed, permeabilized, and stained. Cells stained with secondary antibody alone showed almost no detectable stain (not shown). The PKC isoforms are indicated on the figure. In control cells stained for , , and , the large central nuclei can be seen as ghosts, and with  and  antibodies there is distinctive perinuclear stain. The  isoform showed the best recruitment from a diffuse distribution in the cytoplasm to the lateral membrane where the Na,K-ATPase also resides. The  isoform shares that basolateral membrane location with or without PMA treatment, but it does not have the calcium dependence required to catalyze phosphorylation of the Na,K-ATPase.

Elevation of Intracellular cAMP and PKC Phosphorylation-- McK1 antibody was employed to investigate whether activation of the PKA-dependent pathway through the elevation of cAMP concentrations would cause changes in PKC phosphorylation levels of Na,K-ATPase Ser-18 in intact cells (Fig. 3). Interestingly, a cell-specific response was observed. In NRK cells, preincubation with forskolin (an adenylate cyclase activator) and IBMX (a phosphodiesterase inhibitor) before calyculin A and PMA treatment resulted in a 60-80% decrease in PKC phosphorylation of Ser-18. 1,9-Dideoxyforskolin, an analog that does not stimulate adenylate cyclase, had no effect (data not shown). In contrast, in C6 cells, forskolin/IBMX failed to alter the phosphorylation of Na,K-ATPase at Ser-18. In a third cell line tested, L6 myogenic cells, the response to forskolin/IBMX pretreatment was similar to that observed in NRK cells; a significant decrease in PKC phosphorylation of Ser-18 was seen. Fig. 3A shows representative results, and Fig. 3B shows the averages of densitometric values for four experiments, with estimates of the level of phosphorylation derived from the loss of McK1 antibody binding. Forskolin/IBMX failed to decrease phosphorylation at Ser-18 when cells were preincubated with calyculin A and PMA first (Fig. 3B). When all four drugs were added together, an intermediate level of phosphorylation was seen (not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Activation of a cAMP-dependent pathway decreases PKC phosphorylation at Ser-18. NRK, L6, or C6 cells were preincubated for 15 min at 37 °C with forskolin (50 µM) and IBMX (500 µM) or vehicle. Then 100 nM calyculin A was added with or without 1 µM PMA, and cells were further incubated for 20 min. Detection of phosphorylation at Ser-18 was performed with McK1 antibody. A, a representative experiment in which NRK and L6 cells responded with a reduction in phosphorylation. B, densitometry from n = 4 experiments, expressed as percentage of McK1 stain in control samples. Panel B also includes results from experiments in which the sequence of treatments was changed so that cells were preincubated with calyculin A and PMA for 15 min, and then forskolin/IBMX (F+I) was added and incubated for 20 min more. In this case, activation of the cAMP-dependent pathway did not decrease phosphorylation at Ser-18 in any of the three cell lines.

The difference between C6 and the other two cell lines was related to the amount of cytoplasmic cAMP accumulated: there was a 1000-fold increase in cAMP in NRK cells, a 400-fold increase in L6 cells, but just a 5-fold increase in C6 cells (Table I). Thus intracellular cAMP levels negatively correlated with the levels of PKC phosphorylation of Na,K-ATPase seen in Fig. 3B. Calyculin A had no effect on cAMP levels (data not shown). Treatment of NRK cells with a cell-permeable cAMP analog, 1 mM dibutyryl cyclic AMP, brought about a 6-fold increase in intracellular cAMP-immunoreactive signal as measured with the enzyme-linked immunosorbent assay method. The treatment did not affect the PKC phosphorylation of Ser-18, suggesting that the final cAMP levels obtained, like those in C6 cells treated with forskolin/IBMX, were not enough to activate the pathway (data not shown).

Is PKC Phosphorylation of Ser-18 Modulated by Phosphorylation at the Na,K-ATPase PKA Site?-- To detect phosphorylation at the PKA site of Na,K-ATPase, Ser-938, we used polyclonal antibody 470 raised against a PKA-phosphorylated synthetic peptide corresponding to amino acids ICKTRRNS*VFQQG (the asterisk indicates the phosphoserine) between the H8 and H9 transmembrane segments of the 1 subunit (1). Fig. 4A shows that antibody 470 strongly bound purified rat Na,K-ATPase phosphorylated by purified PKA catalytic subunit in vitro and did not recognize non-phosphorylated  subunit, as reported originally (1). Antibody 6F was used on identical blots to illustrate that protein loading was the same.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Lack of PKA phosphorylation at Ser-938 and immunoprecipitation of Na,K-ATPase from cells. A, 0.5 µg of purified rat kidney Na,K-ATPase was incubated with or without 100 ng of PKA catalytic subunit in the presence of 0.05% Triton X-100 for 10 min at 30 °C. After SDS-gel electrophoresis and electrophoretic transfer, phosphorylation detection was performed with antibody 470 raised against a phosphorylated peptide derived from the PKA site of Na,K-ATPase. An identical blot was stained with monoclonal antibody 6F to document the loading of Na,K-ATPase. B, Na,K-ATPase was immunoprecipitated with monoclonal antibody 6H from C6 cells treated for 30 min at 37 °C with vehicle (1), forskolin/IBMX (2), forskolin/IBMX in combination with calyculin A (3); calyculin A alone (4) at the concentrations described in Fig. 3. In lane 5, no cell lysate was added, so the immunoreactivity represents added IgG. Immunostaining with antibody 470 was used to detect PKA phosphorylation at Ser-938, but none was seen. The same blot was restained with polyclonal antibody K1 against Na,K-ATPase  subunit to document successful immunoprecipitation. Prestained Mr markers from Bio-Rad were used, as indicated on the right. C, purified rat kidney Na,K-ATPase was phosphorylated by PKA in vitro as above. Different amounts of phosphorylated enzyme were loaded on the gel, transferred to nitrocellulose, and then stained either with antibody 470 or with K1. Lane 1, 1 µg; lane 2, 0.5 µg; lane 3, 0.25 µg; lane 4, 0.12 µg; lane 5, 0.06 µg. Both antibodies were able to detect similar amounts of protein, demonstrating that the absence of stain in B was not due to lack of sensitivity.

The Na,K-ATPase  subunit was next immunoprecipitated from C6 cells (Fig. 4B) and NRK-52E cells (not shown) that had been treated with or without forskolin/IBMX to activate PKA and with or without calyculin A to inhibit endogenous phosphatase activity. The precipitates were stained with antibody 470 to detect phosphorylation and with antibody K1 to document the recovery and loading of the  subunit. No phosphorylation at the PKA site was detected in either cell line. To illustrate the sensitivity of the antibody, Fig. 4C shows a control in which purified Na,K-ATPase, phosphorylated in vitro with PKA catalytic subunit, was serially diluted and stained in parallel with the antibody 470 and K1 antibodies.

We considered the possibility that phosphorylation of Ser-938 occurred but was lost during immunoprecipitation. The phosphatase inhibitor calyculin A, which inhibits PP1, PP2A, and related phosphatases, was present throughout the incubation and immunoprecipitation procedure for the samples in lanes 3 and 4 of Fig. 4B. PP2B and PP2C were unlikely to have been active because of the absence of cofactors needed for their activation. Further evidence that the lack of phosphorylation was unlikely to be due to phosphatase activity was obtained by incubating crude cell extracts, which contain endogenous phosphoproteins, under identical buffer conditions (with and without calyculin A). An anti-phosphoserine antibody was then used as a probe for the endogenous phosphoproteins on blots. The phosphorylation of other phosphoproteins was unchanged by the incubation (data not shown).

Elevated cAMP Affects the Phosphorylation of Many Proteins-- Antibody 470 was then used to attempt to detect phosphorylation of Ser-938 on blots of crude cell preparations. Instead of specifically staining the Na,K-ATPase, however, the antibody reacted with many different proteins in all three cell lines (Fig. 5). This is not uncommon with phosphorylated peptide-directed antibodies because phosphoserine tends to be immunodominant. The Na,K-ATPase, if phosphorylated, could not be detected in the background of stain of other proteins. The fact that the stained proteins were phosphoproteins was supported by the dramatic increase in staining when cells were treated with the phosphatase inhibitor calyculin A with or without addition of PMA. That calyculin A alone increased the phosphorylation levels of many proteins suggests that there are kinases that function in basal conditions in these cell lines and that their targets are normally dephosphorylated again by the high PP1/PP2A-like phosphatase activity. This contrasts with protein kinase C phosphorylation of Na,K-ATPase Ser-18, which required stimulation by PMA before the enhancing effect of phosphatase inhibition was seen in the same cell lines (Fig. 1A). The 470 antibody thus samples a subset of the "total" population of phosphoproteins in the cell. The proteins detected are presumably abundant, and the high molecular weights of many of them suggest that some could be structural proteins.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Detection of multiple phosphorylated proteins in extracts of intact cells with antibody 470. Cells were treated with drugs at the concentrations and for the times described for Fig. 3, and blots were stained with antibody 470 instead of McK1. Calyculin A alone greatly increased staining. The addition of PMA with calyculin A increased the phosphorylation of some additional proteins, most visible in C6 and L6. Forskolin/IBMX alone had little effect, but pretreatment with forskolin/IBMX reduced the accumulation of phosphoproteins in NRK and L6 cells treated with calyculin A and PMA, paralleling the effect seen on Ser-18 in Fig. 3. No effect of forskolin/IBMX pretreatment was seen in C6 cells. A representative set of experiments is shown. The same Mr markers were used in each panel.

The most intriguing observations were made when forskolin/IBMX was added before calyculin A. As seen in Fig. 5, in NRK-52E and L6 cells this resulted in greatly reduced phosphorylation of the total pool sample of phosphoproteins. In C6 cells, it did not. This parallels what was seen in Fig. 3 above, where only the phosphorylation of Ser-18 was being probed. Fig. 6 shows the quantitation of the effect on total phosphorylation. Fig. 6 also demonstrates that forskolin/IBMX did not have the same effect if it was added after PMA and calyculin A. Since the effect of forskolin on PKC phosphorylation of Na,K-ATPase always correlated with total phosphorylation caused by other kinases, it suggested that the apparent cross-talk seen in Fig. 3 was actually secondary to more global cAMP-dependent regulation events.

View larger version (41K): [in this window] [in a new window]   Fig. 6.   Forskolin/IBMX reduces total phosphorylation. Densitometry is shown for n = 4 experiments similar to that in Fig. 5, expressed as percentage of maximal phosphorylation detected in the cells treated with PMA and calyculin A for 20 min at 37 °C. Different results were obtained depending on whether forskolin/IBMX was added before PMA and calyculin A or after, as indicated. Concentrations were as in Fig. 3, and times were as follows: for calyculin A alone, 15 or 20 min (20 min gave a close to maximal effect); for PMA plus calyculin, 20 min; for experiments with forskolin/IBMX (F+I), 15 min for the first additive and 20 min for the second.

To determine whether these observations could be reproduced with another probe of phosphoproteins, lysates prepared from NRK cells treated with the different drugs were stained on Western blots with a commercially available anti-phosphoserine antibody (Fig. 7). Although the anti-phosphoserine antibody (lanes marked 1) and antibody 470 (lanes marked 2) recognized different pools of phosphoproteins with only modest overlap, they both revealed the same major changes in total phosphorylation levels when the balance between protein phosphatase and protein kinase activities was shifted. Elevation of cAMP prevented the increase in phosphorylation that was seen when calyculin A was added to block phosphatase activity. Proteolysis was ruled out as a cause of the loss of signal by staining each blot with Amido Black at the end of every experiment.

View larger version (59K): [in this window] [in a new window]   Fig. 7.   Comparison of phosphorylation detection in intact cells with antibody 470 and anti-phosphoserine antibody. NRK cells were treated with forskolin/IBMX and calyculin A and PMA as described for Fig. 5. Lanes 1, antibody 470; lanes 2, anti-phosphoserine antibody from Zymed Laboratories Inc. Staining of lanes side by side with different antibodies was achieved by running the samples from fairly wide wells on the gel and then mounting the blot in a slot-blot apparatus with slots positioned over the wide lanes in pairs.

Protein Kinase A Is Present but Cannot Phosphorylate Na,K-ATPase-- Because the observed effects of forskolin/IBMX were quite different from the expectation that it should increase protein phosphorylation (at PKA sites) and because no PKA-mediated phosphorylation of Ser-938 was detected, the question was raised as to whether the cells somehow lacked active protein kinase A. This was tested by assaying the phosphorylation of the Na,K-ATPase in extracts of untreated NRK-52E cells with 500 µM exogenous cAMP to activate the endogenous kinase, 1 mM ATP, 2 mM MgCl₂, 100 nM calyculin A, and 0.05% Triton X-100. The results are shown in Fig. 8. No phosphorylation of endogenous Na,K-ATPase was detected (lane 1), but there was phosphorylation of exogenously added purified Na,K-ATPase (lane 2). When exogenous PKA catalytic subunit was added too, the level of phosphorylation increased. Considering that endogenous kinase and endogenous Na,K-ATPase were diluted in the preparation of the cell extract, the presence of any phosphorylation upon addition of cAMP suggests that there was no intrinsic problem with the endogenous PKA. The addition of Triton X-100 was required (data not shown). As discussed below, this could have implications for the biological significance of direct PKA-Na,K-ATPase interaction.

View larger version (64K): [in this window] [in a new window]   Fig. 8.   Detection of active PKA in cell extracts. Homogenized NRK cell extracts were prepared as described under "Experimental Procedures." Aliquots containing 30 µg of total protein were incubated for 30 min at 30 °C after the addition of cAMP, ATP, MgCl2, calyculin A, and 0.5% Triton X-100 (lane 1), plus 1 µg of purified Na,K-ATPase (lane 2), and plus both purified Na,K-ATPase and 100 ng of exogenous PKA catalytic subunit (lane 3). Detection of phosphorylated Ser-938 was performed on blots with antibody 470. The endogenous PKA activity was sufficient to phosphorylate a large fraction of the added Na,K-ATPase even though the extract was quite dilute compared with kinase in intact cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Investigation of mechanisms of Na,K-ATPase regulation has been quite controversial, but it is now clear that phosphorylation of its  subunit is not sufficient to cause direct inhibition of enzyme activity as a stereotypical response in all cell types. Instead, functional responses to activation of phosphorylation can range from stimulation to inhibition and are influenced by cellular factors such as oxygenation (35) and Ca²⁺ concentration (20). Kinases may regulate Na,K-ATPase by pathways that do not entail phosphorylation of the Na,K-ATPase (36), and kinase activation may regulate whether the Na,K-ATPase is transported between the plasma membrane and intracellular vesicular compartments with (10, 37) or without (38, 39) direct phosphorylation of the  subunit. Here we have tested the hypothesis that some of this variation is due to cross-talk between regulatory pathways.

Phosphorylation of Na,K-ATPase by Protein Kinase C-- One important factor in PKC-mediated regulation is that different effects may result from the activation of different isoforms of the kinase, as suggested by species-specific effects of kinases injected into oocytes (7). There are at least 12 PKC isoforms (40). PKC-, -, -, and - have been implicated in modulating Na,K-ATPase activity in various systems (41-46), however in none of these cases was there a direct connection to the phosphorylation state of a particular PKC site. We found that in both NRK and C6 cells, phosphorylation of Ser-18 was carried out by a phorbol ester- and Ca²⁺-sensitive PKC isoform. Only three isoforms (the "conventional" isoforms, , , and ) answer this description. Only the  isoform showed PMA-induced translocation to the basolateral membrane where the Na,K-ATPase is located. This strongly suggests that PKC- is responsible for Ser-18 phosphorylation.

There are four major classes of protein phosphatases that could act to dephosphorylate the Na,K-ATPase, each with comparable specificity in regulation (47). Based on sensitivity to protein phosphatase inhibitors, we were able to conclude that dephosphorylation of Ser-18 is performed by a PP1/PP2A-like phosphatase (which could include PP4, PP5, or PP6 (48)). This is in agreement with studies in renal cortex and choroid plexus (18, 21).

Phosphorylation of Na,K-ATPase by Protein Kinase A-- Although the PKC site is in the first cytoplasmic segment near the N terminus, the PKA site is in what is proposed to be the third cytoplasmic loop, between the H8 and H9 transmembrane segments. Based on the expected homology to the structure of sarcoplasmic reticulum Ca²⁺-ATPase (49), the sites are widely separated, the PKC site on an exposed surface of the A domain (the "beak") and the PKA site on a loop that is sandwiched between the M6-M7 intracellular loop and the cluster of transmembrane helices, under the P-domain. This difference in the predicted disposition of the sites may account for an observation that is reinforced by the data presented here: that the PKC site is always accessible, whereas phosphorylation of the PKA site occurs only in certain conditions.

What evidence is there that Ser-938 is ever phosphorylated in living cells? The phosphorylation of endogenous Na,K-ATPase on Ser-938 by endogenously activated PKA has been demonstrated in only one paper to our knowledge. In this case, untransfected COS cells stimulated with isoproterenol showed increased phosphorylation (a 70% increase over a substantial background) of the monkey 1 subunit detected with the 471 antibody (6). In several other cases, however, attempts to demonstrate Ser-938 phosphorylation of endogenous Na,K-ATPase have been negative: rat sciatic nerve (23), choroid plexus (18), lung cells (50), and striatal neurons (19). In view of the broad ability of the related 470 antibody to stain other phosphoproteins, we would view the results in COS cell extracts with caution.

In several cases, activation of the PKA pathway has resulted in an increase in Na,K-ATPase phosphorylation, but the site of phosphorylation has not been defined (35, 51). Fisone et al. (18) clearly show that activation of PKA in choroid plexus resulted in the phosphorylation of sites distinct from the PKA site. Phosphorylation of Ser-938 (or its equivalent residue in Bufo enzyme) has been detected in transfectants (6, 20, 52), but proof is lacking that all of the exogenously expressed  subunit was folded correctly. The best evidence for a functional role for this residue comes from oocytes or transfectants expressing mutations of the site: Ser-938  Ala or Ser-938  Asp (6, 17, 20), but the weight of evidence now suggests that the regulation is indirect.

Here, endogenous phosphorylation of Na,K-ATPase Ser-938 by protein kinase A could not be detected. It could be argued that this was not because phosphorylation failed to occur but because forskolin/IBMX resulted in a secondary reduction of phosphorylation, for example by activating a protein phosphatase (53), and the phosphorylation was removed before immunoprecipitation. This argument would not hold for C6 cells, however, where forskolin/IBMX treatment did not alter the level of total phosphorylation or the level of Ser-18 phosphorylation and where forskolin/IBMX stimulation of phosphatase activity is consequently not a credible hypothesis. When excess exogenous, purified Na,K-ATPase was added to cell extracts, Ser-938 was phosphorylated by the endogenous PKA activity, but only when Triton X-100 was added, as reported before for both cell extracts and purified Na,K-ATPase (3, 26).

The possibility should be considered that Ser-938 is not actually used for phosphorylation in living cells under conditions that most people would regard as standard for activating the cAMP-dependent pathway. Instead, Ser-938 may become accessible to the kinase only when the Na,K-ATPase is structurally disturbed, either by Triton X-100 or other detergents (13), by heat denaturation (54), and hypothetically, by the misfolding of some fraction of  subunits when exogenously overexpressed. Epitope additions near this site disrupt folding to the extent that the site appears on the extracellular surface (55, 56) or result in poor cell growth and cloning efficiency (57), consistent with the partially buried location of the analogous segment in the Ca²⁺-ATPase. These observations do not rule out the possibility, however, that there could be physiological conditions in which access to the site could be actively induced.

Paradoxical Effects of Elevation of cAMP-- The molecular mechanism of the effect of elevated cAMP on phosphoprotein metabolism remains to be determined. Although at first glance it may seem paradoxical to see a widespread reduction in phosphorylation with agents that are universally used to increase phosphorylation at protein kinase A sites, PKA phosphorylates only some proteins, and global stimulatory effects are not expected. The traditional detection of phosphoproteins with ³²P also emphasizes rapidly turning over sites, which are more likely to be responsive to acute regulatory pathways, whereas antibody-based detection is independent of turnover rates. There are also special circumstances to bear in mind. First, the global effect and the effect on Ser-18 phosphorylation were not seen when protein phosphatases were inhibited first, i.e. when calyculin A addition preceded forskolin/IBMX addition. This suggests that the forskolin/IBMX effect requires dephosphorylation of some critical component of its pathway, identity unknown. Second, the effect was seen only in the two cell lines that responded to forskolin/IBMX with quite high cytoplasmic cAMP concentrations. We can speculate that the phenomenon is part of a homeostatic response to sustained or excessive cAMP elevation: a way to terminate the activity of the normal cAMP-activated pathway, independent of the simple hydrolysis of cAMP. Three possible mechanisms could be used: reduction of kinase activities (perhaps even a reduction in the supply of ATP); increase of phosphatase activity; or, most speculatively, restoration of phosphatase activity by the antagonism of calyculin A binding.

There are a few related observations in the Na,K-ATPase literature. In rat sciatic nerve, no increase in total phosphorylation (detected with ³²P) was seen after forskolin treatment, whereas Na,K-ATPase phosphorylation was decreased (23). In renal medullary tubules, dibutyryl cAMP and 8-bromo-cyclic AMP treatment resulted in a higher level of phosphorylation of Na,K-ATPase than forskolin/IBMX, although arguably the forskolin/IBMX should have resulted in higher intracellular cAMP levels (35). Finally, puzzling reductions of phosphorylation were reported some time ago in cAMP-treated liver membranes (58).

One thing that is clear is that much was learned by using anti-phosphoserine antibodies to detect the behavior of multiple phosphoproteins instead of investigating just one protein at a time. The use of forskolin/IBMX is so widely accepted as a way to activate PKA that controls that would have detected the phenomena described here are often omitted. Instead of evidence for conformation-mediated cross-talk between two sites on the Na,K-ATPase, we found evidence for cAMP modulation of phosphoprotein metabolism.

	FOOTNOTES

^* The work was supported by American Cancer Association Grant IRG-173-J (to M. S. F.) and National Institutes of Health Grant RO1NS27653 (to K. J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 617-726-8579; Fax: 617-726-7526; E-mail: sweadner@helix.mgh.harvard.edu.

Published, JBC Papers in Press, August 11, 2000, DOI 10.1074/jbc.M005869200

² Residues are numbered from the mature N terminus after biosynthetic cleavage of the first five amino acids of the nascent polypeptide chain.

³ E. Arystarkhova and K. J., Sweadner, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: Na, K-ATPase, sodium- and potassium-exchanging adenosine triphosphatase; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PP1 and PP2A, protein phosphatases 1 and 2A, respectively; PMA, phorbol-12-myristate-13-acetate; IBMX, 3-isobutyl-1-methylxanthine; DMEM, Dulbecco's modified Eagle's medium.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. C. Reidling, V. S. Subramanian, T. Dahhan, M. Sadat, and H. M. Said Mechanisms and regulation of vitamin C uptake: studies of the hSVCT systems in human liver epithelial cells Am J Physiol Gastrointest Liver Physiol, December 1, 2008; 295(6): G1217 - G1227. [Abstract] [Full Text] [PDF]

				B. Ashokkumar, N. D. Vaziri, and H. M. Said Thiamin uptake by the human-derived renal epithelial (HEK-293) cells: cellular and molecular mechanisms Am J Physiol Renal Physiol, October 1, 2006; 291(4): F796 - F805. [Abstract] [Full Text] [PDF]

				C. J. Ramnanan and K. B. Storey Suppression of Na+/K+-ATPase activity during estivation in the land snail Otala lactea J. Exp. Biol., February 15, 2006; 209(4): 677 - 688. [Abstract] [Full Text] [PDF]

				K. Balamurugan, N. D. Vaziri, and H. M. Said Biotin uptake by human proximal tubular epithelial cells: cellular and molecular aspects Am J Physiol Renal Physiol, April 1, 2005; 288(4): F823 - F831. [Abstract] [Full Text] [PDF]

				V. Sidhaye, J. D. Hoffert, and L. S. King cAMP Has Distinct Acute and Chronic Effects on Aquaporin-5 in Lung Epithelial Cells J. Biol. Chem., February 4, 2005; 280(5): 3590 - 3596. [Abstract] [Full Text] [PDF]

				B. d.Z. Silverman, W. Fuller, P. Eaton, J. Deng, J. R. Moorman, J. Y. Cheung, A. F. James, and M. J. Shattock Serine 68 phosphorylation of phospholemman: acute isoform-specific activation of cardiac Na/K ATPase Cardiovasc Res, January 1, 2005; 65(1): 93 - 103. [Abstract] [Full Text] [PDF]

				S. Soodvilai, V. Chatsudthipong, K. K. Evans, S. H. Wright, and W. H. Dantzler Acute regulation of OAT3-mediated estrone sulfate transport in isolated rabbit renal proximal tubules Am J Physiol Renal Physiol, November 1, 2004; 287(5): F1021 - F1029. [Abstract] [Full Text] [PDF]

				M. Einicker-Lamas, L. D. Wenceslau, R. R. Bernardo, L. Nogaroli, A. Guilherme, M. M. Oliveira, and A. Vieyra Sphingosine-1-Phosphate Formation Activates Phosphatidylinositol-4 Kinase in Basolateral Membranes from Kidney Cells: Crosstalk in Cell Signaling through Sphingolipids and Phospholipids J. Biochem., October 1, 2003; 134(4): 529 - 536. [Abstract] [Full Text] [PDF]

				W. Fuller, V. Parmar, P. Eaton, J. R Bell, and M. J Shattock Cardiac ischemia causes inhibition of the Na/K ATPase by a labile cytosolic compound whose production is linked to oxidant stress Cardiovasc Res, March 15, 2003; 57(4): 1044 - 1051. [Abstract] [Full Text] [PDF]

				M. S. Feschenko, E. Stevenson, A. C. Nairn, and K. J. Sweadner A Novel cAMP-Stimulated Pathway in Protein Phosphatase 2A Activation J. Pharmacol. Exp. Ther., July 1, 2002; 302(1): 111 - 118. [Abstract] [Full Text] [PDF]

				K. J. Sweadner and M. S. Feschenko Predicted location and limited accessibility of protein kinase A phosphorylation site on Na-K-ATPase Am J Physiol Cell Physiol, April 1, 2001; 280(4): C1017 - C1026. [Abstract] [Full Text] [PDF]

				K. M. Ridge, L. Dada, E. Lecuona, A. M. Bertorello, A. I. Katz, D. Mochly-Rosen, and J. I. Sznajder Dopamine-induced Exocytosis of Na,K-ATPase Is Dependent on Activation of Protein Kinase C-epsilon and -delta Mol. Biol. Cell, April 1, 2002; 13(4): 1381 - 1389. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 275/44/34693    most recent M005869200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Feschenko, M. S. Articles by Sweadner, K. J. Search for Related Content PubMed PubMed Citation Articles by Feschenko, M. S. Articles by Sweadner, K. J. Social Bookmarking                      What's this?