Phosphorylation of Na,K-ATPase by Protein Kinase C at Ser18 Occurs in Intact Cells but Does Not Result in Direct Inhibition of ATP Hydrolysis*

(Received for publication, January 23, 1997, and in revised form, April 16, 1997)

Marina S. Feschenko Dagger and Kathleen J. Sweadner Dagger §

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Na,K-ATPase activity has been demonstrated to be regulated by a variety of hormones in different tissues. It is known to be directly phosphorylated on its alpha -subunit, but the functional effects of protein kinases remain controversial. We have developed a sensitive, antibody-based assay for detection of the level of phosphorylation of the alpha 1-isoform of rat Na,K-ATPase at the serine residue that is most readily phosphorylated by protein kinase C (PKC) in vitro, Ser18. By stimulation of endogenous PKC and inhibition of phosphatase activity, it was possible to consistently obtain a very high stoichiometry of phosphorylation (close to 0.9) in several types of intact cells. This demonstrates the accessibility and competency of the site for endogenous phosphorylation. The cells used were derived from rat (NRK 52E, C6, L6, and primary cultures of cerebellar granule cells, representing epithelial cells, glia, muscle cells, and neurons). In the presence of the phosphatase inhibitor calyculin A, full phosphorylation was preserved during subsequent assays of enzyme activity in vitro. Assay of the hydrolysis of ATP in NRK and C6 cells, however, indicated that there was no significant effect of phosphorylation on the Vmax of the Na,K-ATPase or on the apparent affinity for Na+. Any regulatory effect of PKC on sodium pump activity thus must be lost upon disruption or permeabilization of the cells and is not a direct consequence of enzyme alteration by covalent phosphorylation of Ser18.


INTRODUCTION

The Na,K-ATPase is a membrane protein responsible for maintaining the electrochemical gradients of Na+ and K+ in almost all mammalian cells. Since many physiological events in the cells depend on the concentrations of these ions, the mechanisms regulating Na,K-ATPase activity are of great importance. Direct phosphorylation and dephosphorylation of Na,K-ATPase by protein kinases and protein phosphatases have been proposed to be mechanisms for modulation of sodium pump work. Since many hormones are known to affect activities of protein kinases or phosphatases, the reversible phosphorylation of the Na,K-ATPase molecule may present a missing link between hormone action and changes in enzyme activity. The alpha -subunit of Na,K-ATPase has been shown to be a substrate for Ca2+-, phospholipid-dependent protein kinase (PKC)1 both in vitro (1-6, 17) and in intact cells (5, 7-10), and two PKC phosphorylation sites have been identified for rat alpha 1 in vitro (6, 11). Both sites (Ser11 and Ser18) are located at the N terminus of the alpha -subunit. Using direct sequencing of phosphorylated ATPase, we showed that Ser18 was the major phosphorylation site with a commercially available mixture of alpha , beta , and gamma  isoforms of PKC.

A physiological role for protein kinase C regulation of Na,K-ATPase is controversial, however. Although there are reports of inhibition of enzyme activity as a direct result of phosphorylation in the dogfish shark, duck salt gland, and rat kidney enzymes (2, 6, 12), most of the literature deals with indirect evidence that PKC stimulation by phorbol esters can change Na,K-ATPase activity in intact cells or membrane preparations. Moreover, both activation (13, 14) and inactivation (2, 7, 9, 15, 16) of the Na,K-ATPase have been reported upon stimulation of PKC. Our attempts to detect an effect of PKC phosphorylation on purified rat kidney enzyme showed no change in ATPase activity (17).

In this study we developed a nonradioactive antibody-based assay to answer three central questions: 1) is the same major site used by endogenous PKC in intact cells as by exogenous PKC in vitro?; 2) what is the balance between PKC activity and activity of phosphatases responsible for alpha 1 dephosphorylation?; and 3) does activation of PKC and phosphorylation of Na,K-ATPase in cells result in any change in ATPase activity or Na+ affinity?


EXPERIMENTAL PROCEDURES

Na,K-ATPase and Protein Kinase C

Purified rat Na,K-ATPase was prepared by the method of Jørgensen (18), using the medulla from frozen kidneys obtained from PelFreez Biologicals (Rogers, AR). Enzyme preparations were stored in buffer containing 20 mM Tris (pH 7.2), 320 mM sucrose, 1 mM EDTA. The specific activity of Na,K-ATPase was 1200 µmol/h/mg. Protein concentration was determined by the method of Lowry using bovine serum albumin as a standard (19). PKC from rat brain was purchased from Boehringer Mannheim GmbH Biochemica (Germany). The PKC preparations used consist of alpha -, beta -, and gamma -isoforms (information provided by the supplier).

Phosphorylation of Na,K-ATPase by PKC

Purified Na,K-ATPase at a concentration of 10-50 µg/ml was preincubated with PKC (1.5 µg/ml) for 3 min at 30 °C in buffer containing 10 mM Tris phosphate (pH 7.4), 5 mM magnesium acetate, 500 µM CaCl2, 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma), and 80 µg/ml phosphatidyl serine (in a sonicated suspension) (Sigma, P7769). The reaction was started by the addition of [gamma -32P]ATP (1800 cpm/pmol) to a final concentration 60 µM. Phosphorylation proceeded for 30 min at 30 °C and was stopped with an equal volume of electrophoresis sample buffer containing 125 mM Tris, pH 6.8, 4% SDS, 20% glycerol, and bromphenol blue (Laemmli sample buffer). Control samples were prepared the same way except that instead of PKC, an equal volume of PKC storage buffer (50% glycerol, 0.02% Tween 20, 20 mM Tris-HCl, 0.5 mM EDTA, 0.5 mM EGTA, 5 mM dithiothreitol, pH 7.5) was added. Electrophoresis and transfer to nitrocellulose were performed as described below. Phosphoproteins were visualized by overnight autoradiography of the dried nitrocellulose on Kodak XAR-5 film. Densitometry was performed with a Molecular Dynamics 300A laser densitometer.

Gel Electrophoresis, Electrophoretic Transfer, and Immunostaining

Electrophoresis was performed as described previously (20), using 10% polyacrylamide and the Bio-Rad mini-ProteanII apparatus. Electrophoretic transfer of proteins was performed in buffer containing 20 mM Tris, 150 mM glycine, and 20% methanol at 0.15 A overnight.

For immunostaining, nitrocellulose membranes were first blocked with 20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl and 0.5% Tween 20 (TBS-Tween) for 30-40 min at room temperature. Incubations with the primary and horseradish peroxidase-conjugated secondary antibodies were performed in the same buffer for 1.5 and 1 h, respectively. Membranes were extensively washed with TBS-Tween after each step and finally incubated with luminol reagent (Pierce). Membranes were then exposed to Kodak XAR-5 film for 0.5-20 min. Three different Na,K-ATPase antibodies were used in this study: monoclonal antibody McK1, which binds near the N terminus of the alpha 1-subunit (21); monoclonal antibody 6F, which has an epitope approximately 40 amino acids away from the McK1 epitope (22); and polyclonal antiserum K1, which was raised against purified rat kidney Na,K-ATPase alpha 1-subunit and is known to recognize polypeptide fragments from different parts of the molecule (N-terminal and C-terminal halves) (21, 23). All three antibodies demonstrated similar sensitivity (luminescence per unit of antigen) when used at the following dilutions: 1:5,000 for McK1, 1:400 for 6F, and 1:2,000 for K1. For sequential staining of the same blot, the monoclonal antibodies were stripped off by 30-min incubation in 10% acetic acid, 40% methanol and extensively washed with TBS-Tween. To assess the retention of antigen, blots were restained with 0.1% Amido Black. There was no significant loss of Na,K-ATPase after the stripping procedure. Antibody 6F was obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA).

Cell Cultures

Three rat cell lines (NRK-52E (normal rat kidney), C6 glioma, and L6 skeletal muscle myoblasts) were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (100 IU/ml penicillin, 100 µg/ml streptomycin) in a 5% CO2, 95% air atmosphere. NRK and C6 cells were used in phosphorylation experiments immediately upon reaching 90-100% confluence. L6 cells were allowed to fuse and form myotubes in Dulbecco's modified Eagle's medium containing 2% fetal bovine serum for 7-10 days. It should be noted that the 52E subclone of NRK has an epithelial phenotype and is different from the NRK fibroblastic subclone commonly used in virology (24).

Primary neuronal cultures were prepared from the cerebellum of 7-day-old rats as described (25). In brief, 10 cerebelli were cleaned of meninges, chopped with razor blades, and then treated with 0.025% (w/v) trypsin for 10 min in Ca2+- and Mg2+-free Dulbecco's phosphate-buffered saline. The reaction was stopped by adding 2% fetal bovine serum. The cells were pelleted and resuspended in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mM L-glutamine, 30 mM glucose, and 24.5 mM KCl. The cell suspension was filtered through a mesh with a pore size of 75 µm (Falcon cell strainer) and seeded onto polylysine-coated 35-mm tissue culture dishes (Falcon). After 15 min at 37 °C, the medium containing unattached cells was removed, and fresh medium was added. To prevent proliferation of nonneuronal cells, cytosine arabinoside (10 µM) was added to the culture medium 24 h after seeding. Cells were incubated at 37 °C in a 5% CO2, 95% air atmosphere for 7-8 days prior to phosphorylation experiments.

In Vivo Phosphorylation

Monolayers of the cells grown in 35- or 60-mm tissue culture dishes were preincubated for 1.5-2 h at 37 °C in a 5% CO2, 95% air atmosphere in Puck's buffer (Sigma) supplemented with 0.25 mM CaCl2, 0.5 mM MgSO4, 1 mM NaH2PO4, 22 mM NaHCO3, and 10 mM Hepes. Then drugs (PMA, calyculin A) or their vehicle (ethanol) were added at the concentrations indicated in the figure legends, and the cells were incubated for 20-30 min at 37 °C. The buffer was discarded, and dishes with the attached cells were immediately placed on ice. 0.2-0.5 ml of 100 °C 10 mM Tris-HCl buffer (pH 7.4) containing 1% SDS was then added to each dish. The cells were scraped and clarified by centrifugation for 30 min at 40,000 rpm at 10 °C. Supernatants were collected, diluted 1:1 with Laemmli sample buffer, and loaded on gels.

Cell Membrane Preparations

Intact cells were incubated to enhance phosphorylation as described above. After the incubation buffer was discarded, 1-2 ml of ice-cold hypotonic buffer containing 10 mM Tris phosphate (pH 7.2), 5 mM magnesium acetate, 1 mM EGTA, 1 mM EDTA, and 100 nM calyculin A was added to each dish. Dishes were incubated on ice for 15-20 min and then scraped, homogenized, and centrifuged for 30 min at 40,000 rpm, 4 °C. The supernatants were discarded, and the pellets were washed once in the same hypotonic buffer. The final pellets were resuspended in buffer containing 20 mM Tris (pH 7.2), 320 mM sucrose, 1 mM EDTA, and 100 nM calyculin A.

Na,K-ATPase Activity Measurements

First, the test tube assay for the hydrolysis of ATP was performed as described (17). The released 32Pi was quantified by the formation of a phosphomolybdate complex. The reaction was started by the addition of 20-40 µg of NRK or C6 membranes in 0.5 ml of buffer containing 50 mM Tris-HCl (pH 7.4), 3 mM MgCl2, 3 mM NaATP, 1 mM EGTA, 1 mM EDTA, 5 mM NaN3, 115 mM NaCl, 5 mM KCl, and 100 nM calyculin A, with or without 2 mM ouabain. The reaction proceeded for 15-20 min at 37 °C and then was stopped with 0.5 ml of quenching solution (1 N sulfuric acid, 0.5% ammonium molybdate). Then 1 ml of isobutanol was added, and the phosphomolybdate complex was extracted into the organic phase by 10 s of vortex mixing, followed by separation of phases by centrifugation in a table top centrifuge. Aliquots of the upper phase were counted with a scintillation counter. Second, a spectrophotometric assay in which the generation of ADP is coupled to the oxidation of NADH and monitored continuously by absorbance at 340 nm was performed as described (17), except that 3 mM sodium azide and 100 nM calyculin A were present in the assay medium. Third, determination of total Na,K-ATPase activity in perforated cultured cells was performed as described (26). After phosphorylation, monolayers of cells were washed briefly with 10 mM Tris-EGTA (pH 7.4) containing 100 nM calyculin A and placed in the same solution with the addition of 0.03 mg/ml of alamethicin. The cells were incubated for 10 min at room temperature, scraped, and homogenized briefly with a Dounce homogenizer (loose pestle) to disperse the cells, and 50-µl aliquots were used in activity assays (approximately 50-100 µg of total protein/aliquot). The assay was performed as described above for the test tube assay.


RESULTS

An Antibody-based Assay for PKC Phosphorylation of Rat alpha 1

Previously, we showed that phosphorylation of Na,K-ATPase by PKC occurs at two sites on the N terminus of the rat alpha 1-subunit: Ser11 and Ser18 (Fig. 1) (11). A total stoichiometry of 0.9 mol/mol was found for rat alpha 1 when purified enzyme was incubated with purified kinase. Direct sequencing of phosphorylated protein showed that 75-80% of the phosphate incorporated was bound to Ser18 and only 20-25% to Ser11. The conclusion was that Ser18 is the main PKC phosphorylation site in rat, and neither of the sites was fully phosphorylated, unless some preexisting, stable phosphorylation interfered with the incorporation of 32P. Interestingly, Ser18 is absent in Na,K-ATPase alpha -subunit sequences from many other species; that was shown to be the reason for much lower levels of phosphorylation (0.15 mol/mol) in pig and dog alpha 1-subunits.


Fig. 1. N-terminal sequences of Na,K-ATPase alpha -subunits in different species. All serines and threonines are underlined. For rat, dog, and pig alpha 1, the numbering starts at glycine, since the first five amino acids are known to be removed during biosynthesis. Note that not all investigators follow this numbering convention, and hence for rat alpha 1, Ser11 is the same residue as Ser16 in some papers, and Ser18 is the same residue as Ser23. Each boldface P above the sequences indicates a PKC phosphorylation site in rat alpha 1 (11). The horizontal bar shows the epitope for monoclonal antibody McK1 (DKKSKK) (21, 22).
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Earlier, monoclonal antibody McK1 was found to bind to the amino acid sequence DKKSKK, which includes Ser18 (Fig. 1) (21, 22). This antibody does not recognize Na,K-ATPase from pig and dog, which have the shorter sequence DKKK at that location. Since the McK1 epitope and the major site for PKC phosphorylation include the same amino acid residues, we hypothesized that phosphorylation of Ser18 might affect McK1 binding, making it possible for this antibody to probe the level of PKC phosphorylation of Na,K-ATPase in cell cultures and tissues. As shown in Fig. 2 below, this is successful under appropriate experimental conditions.


Fig. 2. Antibody-based detection of phosphorylation of Ser18 in vitro. Different amounts of purified rat kidney Na,K-ATPase (0.5 µg (lanes 1 and 4); 0.2 µg (lanes 2 and 5); 0.1 µg (lanes 3 and 6)) were incubated with (lanes 4-6) or without (lanes 1-3) 15 ng of PKC for 30 min at 30 °C in a final volume of 10 µl. A, 32P, autoradiogram of 32P-labeled proteins after SDS-gel electrophoresis and electrophoretic transfer to nitrocellulose; McK1, staining of the same blot with the monoclonal antibody McK1. The exposure times required for luminol detection are too short to detect any interference from the radioactivity of 32P. B, densitometry of McK1 binding. Data points for the nonphosphorylated samples of lanes 1-3 are plotted (squares). The arrow shows where the signal for the phosphorylated sample in lane 4 of panel A falls. The phosphorylated sample of lane 5 did not fall on the McK1 binding curve, and sample 6 was below the detection level. The dashed line shows the calculated linear correlation between the amount of Na,K-ATPase and the antibody binding signal.
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First, experiments were performed to assess the sensitivity and linearity of antibody signals from McK1 and two antibodies used as controls. Since Na,K-ATPase is not very abundant in cell cultures, a high sensitivity method is required to detect antigen-antibody binding. Horseradish peroxidase-luminol staining of Western blots is known to be several orders of magnitude more sensitive than traditional staining with colorigenic substrates, and it is possible to scan the images obtained to quantify and compare their optical densities. Caution is needed in using this method for quantifying the amount of antigen in different samples. The limitations of the assay are imposed both by the luminescent method itself and by the binding characteristics of the antibody. At higher concentrations of antigen the luminescent signal becomes nonlinear and finally reaches a plateau (due either to exhaustion of luminol reagent or x-ray film saturation), while at lower antigen concentrations it may drop abruptly below the detection level. There is a linear zone that can be shifted by adjusting the time of exposure of the blot to x-ray film. We found that if the amounts of antigen in samples differ more than 3-fold, accurate quantitation is not possible unless calibration curves are made in each experiment. Since cell cultures contain only small amounts of Na,K-ATPase, such samples are more likely to face the problem of having the signal below detection level rather than reaching saturation.

To test whether the McK1 antibody can be used to detect PKC phosphorylation, we compared binding of the antibody to phosphorylated and nonphosphorylated Na,K-ATPase (Fig. 2A). Three different concentrations of purified rat kidney Na,K-ATPase were used. Na,K-ATPase was incubated in phosphorylation buffer with (lanes 4-6) or without (lanes 1-3) exogenous PKC and [gamma -32P]ATP. Samples were subjected to SDS-gel electrophoresis and transferred to nitrocellulose membrane. Phosphoproteins were visualized by autoradiography and then immunostained with monoclonal antibody McK1 using the horseradish peroxidase-luminol method. Phosphorylation of Na,K-ATPase by PKC dramatically decreased the binding of McK1 antibody. Thus, Ser18 is an important amino acid residue in the McK1 epitope, and covalent modification by phosphorylation prevents antibody binding. The assay is consequently based on detecting how much unmodified Ser18 is left after PKC phosphorylation. Incorporation of 32P as detected by scintillation counting was linear at all concentrations of Na,K-ATPase used (not shown), while 32P autoradiography (not shown) and McK1 binding (Fig. 2B) deviated at higher antigen concentration. Since the signal for the phosphorylated sample in lane 4 of Fig. 2A ("unknown") fell in the linear zone of the McK1 binding curve, we were able to calculate the amount of unmodified Na,K-ATPase in this sample. The result was 0.13 µg, which is 26% of the 0.5 µg used in the phosphorylation experiment. These data correlate very well with our previous finding that approximately 75% of Ser18 becomes modified after PKC phosphorylation in vitro (11).

PKC Phosphorylation in Intact Cells---To assess the generality of our findings, four different types of rat cells were used in this study: 1) NRK (clone 52E); 2) C6 glioma; 3) L6 skeletal muscle myotubes; and 4) primary cultures of cerebellar granule neurons. All of these cells express the alpha 1-isoform of Na,K-ATPase. PKC phosphorylation was determined by quantifying McK1 binding relative to control antibody. Intact cells were incubated with or without protein kinase and phosphatase modulators. Fig. 3A shows the result of an experiment with NRK cells. Treatment of the cells with PMA significantly decreased McK1 binding, while the combination of PMA and the protein phosphatase inhibitor calyculin A practically abolished it, meaning that most of the Na,K-ATPase became phosphorylated at Ser18. Calyculin A alone was ineffective, suggesting that in the absence of stimuli, the activity of PKC in the NRK cell line is low. Prolonged treatment with PMA (24 h), which is known to down-regulate PKC, did not change the basal level of Na,K-ATPase phosphorylation (results not shown). 6F binding was unaffected by any of the treatments, as expected from the observation that its epitope is approximately 40 amino acids away (22). Fig. 3B summarizes the results of phosphorylation experiments with all four cell culture types. Binding of McK1 antibody was normalized to binding of 6F or K1 antibody. PMA treatment alone increased phosphorylation of Na,K-ATPase in NRK and C6 cells (as detected by the loss of McK1 binding). It was less effective in cerebellar neurons and practically ineffective in L6 cells. The combination of PMA and calyculin A caused a dramatic increase in the phosphorylation level of Na,K-ATPase in all four types of cell culture.


Fig. 3. Antibody-based detection of phosphorylation of Ser18 after PKC stimulation in intact cells. Intact cells were incubated with or without 1 µM PMA or 100 nM calyculin A for 30 min at 37 °C. The cell lysates were loaded on an SDS gel, transferred to nitrocellulose, and stained with McK1 antibody. McK1 antibody was then stripped off by a 30-min incubation in 10% acetic acid, 40% methanol, and the blot was restained with either monoclonal antibody 6F (shown in A) or polyclonal antibody K1 (used in some experiments summarized in B). A, a representative experiment with NRK cells. B, densitometry for n = 3-8 (for each of four different types of cell cultures), expressed as percentage of maximal McK1 binding in samples without PMA or calyculin A. Each data point was first normalized against the binding of 6F or K1 to correct for any variation in loading. NRK, normal rat kidney 52E; C6, C6 glioma; L6, L6 skeletal myotubes; CN, primary cultures of cerebellar granule neurons.
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Protein Phosphatase Activity in Cell Cultures

The effect of calyculin A in potentiating the effect of PMA on the level of phosphorylation of Ser18 showed that protein phosphatases are very active in the cell cultures tested. Treatment of intact cells with 1 µM PMA alone was able to bring about 50% phosphorylation of Na,K-ATPase at most. Attempts to increase phosphorylation by raising the concentration of PMA up to 10 µM and/or prolonging the incubation time were unsuccessful (results not shown). The combination of 1 µM PMA with 100 nM calyculin A caused 70-90% PKC phosphorylation of Ser18 in all experiments. The level of phosphorylation was unaffected by a 10-fold decrease in PMA concentration, indicating that PKC was fully activated at the lower PMA concentration (0.1 µM), while the same decrease in calyculin A concentration (final concentration, 10 nM) resulted in 15-20% loss of phosphorylation (data not shown). Thus, to reach the highest level of phosphorylation, the activity of protein phosphatases must be controlled with an appropriate concentration of phosphatase inhibitor.

Another major concern is retention of the phosphate introduced by PKC. To study the functional consequences of phosphorylation it is important both to obtain the highest level of phosphorylation possible and to preserve the phosphorylation during subsequent experimental manipulations. The stability of PKC phosphorylation in perforated C6 cells and membrane preparations isolated from NRK and C6 cells was investigated in the conditions of ATPase activity assays. We found that in ATPase activity assay medium in the absence of calyculin A, almost complete dephosphorylation of the Ser18 site occurred after 15-30 min of incubation at 37 °C (Fig. 4A). The presence of 100 nM of calyculin A throughout the assay prevented dephosphorylation (Fig. 4B). The result indicates that activity of protein phosphatase in the cells tested is very high and is not removed during cell permeabilization or membrane isolation. It can be seen that there was little change in the phosphorylation level of the control samples; this was true of both cell lines, further supporting the conclusion that both have similar, low levels of basal phosphorylation.


Fig. 4. Inhibition of phosphatase activities in cell membrane preparations. Intact C6 cells in the presence of 100 nM calyculin A were incubated with (lanes 2 and 4) or without (lanes 1 and 3) 1 µM PMA for 30 min at 37 °C. Membrane fractions were isolated as described under "Experimental Procedures." Samples were either loaded on an SDS-gel directly (lanes 1 and 2) or incubated in ATPase assay medium for 20 min at 37 °C followed by precipitation with 3 volumes of chloroform/methanol (2/1, v/v) (lanes 3 and 4). After electrophoresis and electrophoretic transfer, the blots were stained with McK1 antibody first, stripped with 10% acetic acid, 40% methanol, and restained with 6F antibody. A, calyculin A was present during the initial treatment of intact cells (lanes 1 and 2) but was omitted from the subsequent incubation in assay medium (lanes 3 and 4), and phosphorylation was lost. B, 100 nM calyculin A was included in all buffers used prior to the precipitation step, and phosphorylation was preserved.
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Functional Consequences of PKC Phosphorylation at Ser18

Two different kinds of preparations were used in activity experiments: C6 cells perforated with alamethicin by the method of Askari and co-workers (26) and membrane preparations isolated from cultured C6 and NRK cells. Intact cells were incubated with 1 µM PMA and 100 nM calyculin A (phosphorylated samples) or with calyculin A only (controls), and all of the subsequent steps were performed in the presence of 100 nM calyculin A to preserve phosphorylation of Ser18. The level of phosphorylation obtained was checked in every experiment. Almost stoichiometric phosphorylation was obtained in the PMA-treated cells (Fig. 5). To test whether the difference between control and phosphorylated sample was exaggerated because the McK1 signal dropped below the detection level, we loaded two different amounts (20 and 40 µg) of NRK membranes on the gel. McK1 staining was normalized against K1 staining, and the percentage of unmodified Ser18 was calculated in phosphorylated samples (lanes 2 and 4) relative to that in control samples (lanes 1 and 3). The results were, respectively, 10 and 12%, confirming that the difference in the signals was not exaggerated.


Fig. 5. Estimation of the stoichiometry of phosphorylation at Ser18 in NRK membranes. This figure shows that the apparent high stoichiometry of phosphorylation of Ser18 in NRK cells is not an artifact of insufficient gel loading. 20 µg (lanes 1 and 2) or 40 µg (lanes 3 and 4) of control (lanes 1 and 3) or phosphorylated (lanes 2 and 4) NRK membranes were loaded on an SDS-gel. To generate standard curves, different amounts of purified Na,K-ATPase were loaded on the same gel: 0.02 µg (lane 5); 0.05 µg (lane 6); 0.10 µg (lane 7); 0.20 µg (lane 8). Phosphorylation was evidently nearly complete, since doubling the amount loaded did not increase the McK1 signal in lane 4 (compare with Fig. 2A).
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Activities of control and phosphorylated samples were compared in maximal turnover conditions using hydrolysis of [gamma -32P]ATP in test tubes or, in a few experiments, the coupled ATPase assay (Table I). No significant effect (p > 0.05) of phosphorylation was found for membrane preparations of NRK or C6 cells or for preparations of permeabilized C6 cells, despite approximately 90% phosphorylation of Ser18. Table I lists the means and S.D. values, the p value for each group, and the 95% confidence interval. Given the observed S.D. values, statistical analysis showed that we would have 80% power to detect increases or decreases in activity of 13, 9, or 24%, respectively, in the groups given in Table I. This estimates the accuracy of the assay and puts a quantitative limit on the conclusion that there was effectively no change in Na,K-ATPase activity.

Table I. Na,K-ATPase activity after phosphorylation

Intact NRK or C6 cells were incubated in the presence of 100 nM calyculin A with (phosphorylated) or without (control) 1 µM PMA for 30 min at 37 °C. Cells were either permeabilized with alamethicin or membrane fractions were isolated as described under "Experimental Procedures." Na,K-ATPase activity was measured in maximal turnover conditions using hydrolysis of [gamma -32P]ATP or the coupled assay. 100 nM calyculin A was present in all buffers. Na,K-ATPase activities of phosphorylated samples are expressed as percentages of activities of corresponding control samples. All experiments were done in the presence of 1 mM EGTA to inhibit Ca-ATPase and 5 mM NaN3 to inhibit mitochondrial ATPase. Under these conditions, ouabain-sensitive activity varied from 30 to 45% of total ATPase activity seen in the preparations of permeabilized C6 cells and from 44 to 70% in membrane preparations of C6 and NRK cells. The actual specific Na,K-ATPase activities were 0.87-3.22 µmol/mg of protein/h in permeabilized cells and 2.4-4.8 µmol/mg of protein/h in membranes.
n Mean and S.D. p value 95% confidence interval (low-high)

% of control
C6 cells 5 96.40  + 13.96 0.548 79.07 -113.73
C6 membranes 8 102.88  + 7.75 0.336 96.4 -109.36
NRK membranes 7 90.70  + 10.00 0.054 81.43 -99.97

Alteration of Na+ affinity has been hypothesized to mediate hormonal effects on Na,K-ATPase activity (14, 27, 28), and effects of PKC have been observed on Na,K-ATPase conformational equilibria (6). We used the coupled assay to measure Na,K-ATPase activity at different concentrations of Na+ to determine whether phosphorylation of Ser18 resulted in a change in Na+ affinity (Fig. 6). Again, there was no significant effect as detected with control and phosphorylated preparations of NRK membranes. Calyculin A by itself did not affect the activity of Na,K-ATPase in any of the assays used.


Fig. 6. Na,K-ATPase activity at different Na+ concentrations with and without phosphorylation. Phosphorylated and control NRK membranes were prepared as described in Table I and under "Experimental Procedures." Ouabain-sensitive ATPase activities of the samples were measured in the coupled assay with three different Na+ concentrations and in the presence of 100 nM calyculin A. The Na,K-ATPase activities of the phosphorylated samples are expressed as percentages of the activities of the corresponding control samples. Open bars, control NRK sample; hatched bars, phosphorylated sample. n = 3.
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DISCUSSION

PKC-mediated Phosphorylation of Ser18 Occurs in Vivo

The Na,K-ATPases of various animal species differ both in the sites where PKC phosphorylates the alpha -subunit and in the recorded maximal stoichiometries of phosphorylation. We have used the rat alpha 1 Na,K-ATPase because it is possible to obtain a stoichiometry of 32P incorporation close to 1.0 at a defined site near the N terminus. Under these conditions, we hoped to have the best chance of finding a rigorous correlation between phosphorylation and any alteration in the activity of the enzyme. No effect of phosphorylation was seen in our prior work using purified Na,K-ATPase and purified PKC (17). That left open the possibility that the site phosphorylated in vitro was not used physiologically, however. Ser18 is unique to rat alpha 1, and it is reasonable to question whether it is a legitimate phosphorylation site in vivo, or whether it becomes accessible only upon some disruption of protein structure that occurs during enzyme purification. Here we have demonstrated with four different types of rat cell cultures that, in intact cells stimulated with phorbol esters, the site is robustly phosphorylated when protein phosphatase activity is inhibited with calyculin A.

There is now little doubt that PKC phosphorylation of Na,K-ATPase alpha -subunit can take place in intact cells and tissues. Direct PKC phosphorylation of Na,K-ATPase alpha -subunit was demonstrated in OK (opossum kidney) cells (7); in normal and diabetic rat sciatic nerve (8); in COS-7 cells expressing Bufo marinus alpha -subunit (5, 10); in rat choroid plexus (9); and in NRK, C6, and L6 cell lines and primary cultures of rat cerebellar granule neurons (present study). In the case of the rat choroid plexus stimulated with phorbol 12, 13-dibutyrate or serotonin, phosphopeptide maps indicated that the same sites were used as with purified enzyme (9), consistent with the results presented here. The mapping technique used could not resolve Ser11 from Ser18, however, leaving open the possibility that only Ser11 was phosphorylated in vivo. The identity of the sites phosphorylated in intact cells or tissues has been even less clear in other work. In transfected COS-7 cells expressing B. marinus alpha -subunit, a substantial background of unidentified basal phosphorylation activity was stimulated by phorbol ester even in mutations of the Thr15 and Ser16 PKC phosphorylation sites, suggesting that there may be additional sites (5). In similar cultures, activation of alpha -adrenergic or muscarinic cholinergic receptors that normally increase PKC activity through Ca2+ entry and diacylglycerol release instead increased Na,K-ATPase phosphorylation at the protein kinase A site (Ser943), in a process inhibitable by H-89, a protein kinase A inhibitor (10). In other cases the site(s) of endogenous PKC action on the Na,K-ATPase alpha -subunit have not been identified (7, 8). With the potential for complex regulatory interactions, the interpretation of experimental results should be easier when the phosphorylation of specific sites can be explicitly measured. An assay that makes it possible to assess phosphorylation directly in every experiment should make quantitative assessment of cause and effect more feasible.

Advantages and Limitations of an Antibody-based Assay

The binding of McK1 requires unphosphorylated Na,K-ATPase, and it thus provides a good assay to demonstrate near complete phosphorylation, since it sensitively detects the fraction that has not reacted with the kinase. It is more difficult to make conclusions about the basal level of phosphorylation if it is low, however, since a small reduction in antibody binding could be hard to detect. The available indirect evidence suggests that the basal phosphorylation of Na,K-ATPase in the cell lines tested was low. Down-regulation of PKC by prolonged incubation with phorbol ester did not increase binding of McK1 antibody, and the phosphatase inhibitor calyculin A alone failed to significantly decrease it. In addition, there was little increase in McK1 binding when incubation of membranes at 37 °C allowed phosphatase action. For example, in Fig. 4A, if there had been significant basal phosphorylation in the control membranes (lane 1), then after permitting phosphatase action during prolonged incubation in assay medium (lane 3) there would have been an increase in McK1 binding. PKC activity appears to contribute very little to the resting level of Na,K-ATPase phosphorylation at Ser18. In rat choroid plexus, the basal level of phosphorylation was found to be 6-7% of that obtained after stimulation of PKC with phorbol 12,13-dibutyrate (9). Assuming that stoichiometric phosphorylation was obtained with phorbol 12,13-dibutyrate, a basal level on the order of 6-7% (or less) would be unlikely to be detected in our assay.

The lability of the phosphate attached to Ser18 in medium without phosphatase inhibitors makes it unlikely that there is much phosphorylation left in conventional membrane preparations, and thus it is unlikely that prior studies employing the McK1 antibody to detect Na,K-ATPase on Western blots are compromised by the new observation that phosphorylation prevents antibody binding. Whether or not any phosphate on Ser18 would be preserved during tissue fixation is a separate question, however. It is conceivable that McK1 staining of tissue sections could be altered by the phosphorylation state at the time of fixation, a possibility that remains to be tested.

Phosphorylation by PKC at Other Sites

Most recent reports have demonstrated the existence of PKC phosphorylation sites close to the N terminus of the Na,K-ATPase alpha -subunit. There is marked sequence divergence at this location, making it necessary to identify phosphorylation sites in each species individually. The identified sites include Ser11 and Ser18 for rat alpha 1 (6, 11); Ser11 for sheep (5), pig, and dog alpha 1 (11) (deduced because it is the only serine or threonine in the short phosphorylated N-terminal segment that was removed by trypsinolysis); and Thr15 and Ser16 in Bufo alpha 1 (5). Bufo Ser16 is the homolog of mammalian Ser11 (Fig. 1). It is curious that the stoichiometry of phosphorylation of Ser11 and its homolog seldom exceeds 0.15 (Refs. 5 and 11; by inference in Ref. 1), while that of Ser18 reaches 0.7-0.9 (Ref. 11 and present study) or 0.5-0.65 (6). A higher level of phosphorylation of Na,K-ATPase by PKC has been reported for only one other Na,K-ATPase, the dogfish shark rectal gland enzyme, where a stoichiometry of 2.0 was found (2). Unfortunately, the sequence of that Na,K-ATPase is not known, nor is the location of the sites (both serine and threonine) of phosphorylation.

The species difference in maximum observed phosphorylation stoichiometry for Ser11 and Ser18 has a correlate in the structure of the phosphorylation site. The high stoichiometry in rat Ser18 corresponds with a surrounding sequence that resembles a typical consensus sequence for PKC: basic residues at one or two positions on either side of the candidate Ser/Thr (29). The low stoichiometry typical of mammalian Ser11 or Bufo Ser16 corresponds with a surrounding sequence with no Lys or Arg residues (Fig. 1). PKCbeta II is autophosphorylated at sites that also are not typical PKC consensus sites (30). The autophosphorylation sites do not have any clear homology to the Ser11 site on Na,K-ATPase, but they have in common with it several nearby proline, glycine, and glutamate residues. It remains to be determined whether Ser11 can be phosphorylated to a higher level by endogenous kinases in cells. If it cannot, then any functional consequences expected from its phosphorylation must be quantitatively limited compared with the consequences of phosphorylation at sites that can be fully phosphorylated.

An antibody-protein binding site typically encompasses a 600-Å2 surface area and involves contacts with 20 or more residues, not necessarily adjacent in the linear sequence (31). The mapping of the epitope for McK1 makes it clear that Ser18 is a crucial contact; even the single substitution of Ala18 in mouse and human greatly decreases antibody affinity, and the deletion of Lys17 and Ser18 abolishes binding (21). Whether Ser11 is also contacted by the antibody cannot be determined without co-crystallization and an atomic structure, but the failure of McK1 to bind to a chimera containing amino acids 1-13 but lacking amino acids 14-18 suggests that Ser11 is not an important part of the antibody binding site (22). Consequently, we cannot say anything about the phosphorylation state of Ser11 in these experiments. It is plausible that phosphate incorporation occurs at both serines after stimulation of PKC in intact cells.

There is some possibility of PKC phosphorylation at other sites, since there are more than 30 potential consensus sequences for PKC phosphorylation. It has been reported that PKC produces phosphothreonine as well as phosphoserine in dog kidney enzyme (1), although the dog alpha 1 sequence does not have threonine near the N terminus. The maximum observed stoichiometry in that study was 0.15. A phosphorylation site was mapped to an internal location in duck salt gland enzyme by selective proteolysis (4); previous work by the same group had shown phosphate incorporation into both serine and threonine, implying that there were at least two sites (3). The maximum observed stoichiometry was 0.3. For rat alpha 1 phosphorylated in vitro, no phosphorylation was detected at any location other than the N terminus, as determined by proteolytic fingerprinting (11). In the present study we of course would not have detected phosphorylation at alternative sites even if it occurred as a consequence of activation of specific PKC isoforms in intact cells. In any event, any such phosphorylation did not have detectable consequences on enzyme activity.

The Balance between Kinase and Phosphatase Activities

The observed physiological level of phosphorylation represents the balance between kinase and phosphatase activity, and in fact the second messenger-mediated regulation of phosphatase activity has been implicated in the physiological regulation of Na,K-ATPase (28, 32). In our experiments with four different kinds of cells, we found that PMA alone was able to increase phosphorylation at Ser18 by 50% at most, while in combination with calyculin A phosphorylation reached 70-90%. The result suggests strong phosphatase activity in all four cell cultures. Interestingly, in L6 myotubes PMA alone was practically ineffective in increasing phosphorylation (similar to observations in normal rat sciatic nerve (8)), but the application of the phosphatase inhibitor calyculin A alone changed the phosphorylation of Na,K-ATPase by 20-30%. Thus, it is possible that in L6 cells there was some basal phosphorylation at Ser18 that is continuously removed by phosphatase.

It is not obvious why PKC activities in the cell cultures tested appeared so low, while protein phosphatases were highly active. It is conceivable that there is little PKC activation because of relatively low hormone levels in culture medium with 10% fetal serum or because some pathways are inactive due to the absence of contacts with other cell types present in normal tissues. A similar imbalance between protein kinase and protein phosphatase activities was found by others in isolated rat adipocytes where protein kinase activities were measured directly (33).

In different cells and tissues, PKC and phosphatase activities may be regulated independently, affecting the observed level of Na,K-ATPase phosphorylation. A significant level of basal phosphorylation of Na,K-ATPase has been seen in normal rat sciatic nerve (8). Since okadaic acid also increased phosphorylation of Na,K-ATPase approximately 3-fold, the authors concluded that the phosphorylation state of the alpha -subunit results from a balance between a strong phosphatase activity and the activity of PKC. Basal phosphorylation in primary cultures of cerebellar granule neurons appears to be low, however (Fig. 3). This contradicts a prediction that dephosphorylation of a high basal level could explain the increase in Na,K-ATPase activity seen when such cultures are treated with glutamate (34). The mechanism of the effect of glutamate on Na,K-ATPase activity remains to be determined.

Functional Consequences of Phosphorylation of Na,K-ATPase

Despite a high stoichiometry of phosphorylation of Ser18 in vitro, in our prior study we did not see any effect on Na,K-ATPase activity (11, 17). This appears to contradict several other reports. First, there was 50% inhibition of purified dogfish shark enzyme reported at a stoichiometry of 2.0 (2), although species differences or phosphorylation at the second site could obviously be responsible. More importantly, 30% inhibition was reported in purified rat renal cortex Na,K-ATPase at a stoichiometry of 0.5, accompanying an effect on conformation affecting apparent affinity for K+ (6). There have also been reports of PKC-mediated inhibition of Na,K-ATPase activity correlated with phosphorylation of alpha  in cells or tissues: 28% in rat choroid plexus (9) and 30% in OK cells (but not in LLC-PK1 cells) (7). Modulatory effects on Na+ affinity have been suggested in other work to be important for Na,K-ATPase regulation (27, 28, 35). Nevertheless, we failed to see any statistically significant effect on ATP hydrolytic rate in C6 or NRK membranes or in permeabilized C6 cells in maximal velocity conditions or on Na+ affinity.

We consider it likely that the observation of no effect under conditions where the level of phosphorylation is carefully monitored is a more significant result than the observation of inhibition, because there are alternative ways to obtain inhibition. These alternatives include activation of phospholipase A2 and the release of arachidonic acid metabolites, activation of cross-talking pathways that result in Na,K-ATPase phosphorylation at other sites, and modulation of phosphatase activity, to name a few that can be documented. In rat kidney-proximal tubules, Na,K-ATPase inhibition caused by activation of PKC can be inhibited by ethoxyresorufin, an inhibitor of the cytochrome P450-dependent monooxygenase pathway, and mepacrine, an inhibitor of phospholipase A2 (36, 37). A phospholipase A2-mediated source of lipid-derived inhibitors was also implicated in a study showing that PKC inhibited proximal tubule Na,K-ATPase in customary experimental conditions (somewhat hypoxic) but stimulated it when solutions were oxygenated (14). Cross-talking pathways include not only the activation of protein kinase A seen in COS-7 cells (10), but also Na,K-ATPase inhibition mediated by increases in NO and cGMP seen in rat renal medullary tissue slices (38). The kinase-mediated regulation of calcineurin, a protein phosphatase, has been proposed to impact on the regulation of rat proximal tubule Na,K-ATPase by either protein kinase A or PKC (28). Although it may be argued that inhibition has been seen with purified rat renal Na,K-ATPase (6), in that case enzyme was purified from renal cortex, from which it is difficult to obtain very high specific activities. The enzyme purified from rat renal medulla in our prior work had a 2-3 times higher specific activity and thus may have had lower levels of contaminants that mediate Na,K-ATPase inhibition. The absence of inhibition mediated by other pathways in the cell cultures tested here may reflect their quiescent physiological status or their adaptation to continuous cell division.

The lack of a direct effect of phosphorylation on Na,K-ATPase activity leaves a central question unanswered: why does PKC phosphorylate the enzyme, and what are the real consequences for the cell? The existence of alternative pathways for Na,K-ATPase inhibition or activation does not mean that the documented phosphorylation has no physiological significance, only that a more complex mechanism may be involved. Phosphorylation may affect interaction of Na,K-ATPase with other intracellular modulators. Examples of such mechanisms are known. Phosphorylation of the calmodulin-binding domain of the plasma membrane Ca2+-pump by PKC reduced its interaction with calmodulin (39). Phosphorylation of Ser38 of the Ca2+-ATPase of cardiac sarcoplasmic reticulum (SERCA2a) by Ca2+/calmodulin-dependent kinase was originally thought to enhance the Vmax of Ca2+ uptake up to 60% (40). A recent report from the same laboratory did not confirm the conclusion, however, finding instead that phosphorylation of the SERCA2a inhibitor phospholamban increased the apparent affinity of SERCA2a for Ca2+, presumably through dissociation of phosphorylated phospholamban from Ca2+-ATPase (41). Meanwhile, the physiological role of phosphorylation of SERCA2a at Ser38 as well as of phosphorylation of Na,K-ATPase at Ser18 remains to be elucidated.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant NS 27653 (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: 149-6118, Massachusetts General Hospital, 149 13th St., Charlestown, MA 02129. Tel.: 617-726-8579; Fax: 617-726-7526; sweadner{at}helix.mgh.harvard.edu.
1   The abbreviations used are: PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SERCA2a, Ca2+-ATPase of cardiac sarcoplasmic reticulum.

ACKNOWLEDGEMENTS

We thank Elkan Halpern (Department of Radiology, Massachusetts General Hospital) for the statistical analysis of Na,K-ATPase activity.


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