Mutation of the Protein Kinase C Phosphorylation Site on Rat alpha 1 Na+,K+-ATPase Alters Regulation of Intracellular Na+ and pH and Influences Cell Shape and Adhesiveness*

(Received for publication, January 28, 1997, and in revised form, March 31, 1997)

Roger Belusa Dagger §, Zheng-Ming Wang Dagger §, Takako Matsubara Dagger , Bo Sahlgren Dagger , Irina Dulubova , Angus C. Nairn , Erkki Ruoslahti par **, Paul Greengard and Anita Aperia Dagger Dagger Dagger

From the Dagger  Department of Woman and Child Health, Karolinska Institute, S-112 81 Stockholm, Sweden, the  Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021, and the par  La Jolla Cancer Research Center, The Burnham Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The enzyme Na+,K+-ATPase creates the transmembrane Na+ gradient that is of vital importance for functioning of all eukaryotic cells. Na+,K+-ATPase can be phosphorylated by protein kinase A (PKA) and protein kinase C (PKC), and these sites of phosphorylation have been identified. In the present study, we have examined the physiological significance of PKC phosphorylation of rat Na+,K+-ATPase. In COS cells transfected with wild type rat Na+,K+-ATPase alpha 1, intracellular Na+ was higher and pH was lower than in cells transfected with rat Na+,K+-ATPase alpha 1 in which the PKC phosphorylation site, Ser-23, had been mutated into alanine. Phorbol dibutyrate inhibited Na+,K+-ATPase-dependent ATP hydrolysis and Rb+ uptake in cells expressing wild type Na+,K+-ATPase but not in cells expressing S23A Na+,K+-ATPase. Cells expressing the S23A mutant had a more rounded appearance and attached less well to fibronectin than did untransfected cells or cells transfected with wild type rat Na+,K+-ATPase alpha 1. These results indicate a functional role for PKC-mediated phosphorylation of rat Na+,K+-ATPase alpha 1 and suggest a connection between this enzyme and cell adhesion.


INTRODUCTION

A precise intracellular cation composition is of vital importance for cell homeostasis and function. The maintenance of high K+ and low Na+ concentrations, characteristic of the eukaryotic cell, is critically dependent on Na+,K+-ATPase (1, 2). The intracellular Na+ concentration is such that Na+,K+-ATPase is not saturated with regard to this substrate. Therefore, Na+,K+-ATPase activity is dependent on the activity of the Na+ influx (leak) pathway (3). For this reason the pump has often been considered to have a passive role in the regulation of cellular Na+ and K+ homeostasis (4). Recently it has been shown that Na+,K+-ATPase activity can be regulated by hormones, second messengers, protein kinase A (PKA)1 and protein kinase C (PKC) (5-8). Together, these various results suggest that the response of Na+,K+-ATPase to altered ion levels is hormonally modulated allowing for greater versatility in the regulation of cellular homeostasis.

Serine 23 has recently been identified as the major PKC phosphorylation site in rat Na+,K+-ATPase alpha 1 (9, 10). In this study Ser-23 was mutated to alanine and the functional consequences examined in intact cells.


EXPERIMENTAL PROCEDURES

Mutation

A pCMV/ouabain-resistant vector (Pharmingen, San Diego, CA) with the rat Na+,K+-ATPase alpha 1 subunit was used, and the S23A mutation was introduced using a polymerase chain reaction mutation strategy (Pfu DNA polymerase from Stratagene, La Jolla, CA). To produce the two fragments needed to create the mutation, polymerase chain reaction was carried out with the following primer pairs: pair 1, antisense primer 5'-CTCTTTCTAGTCTCCAGCCACAGG-3' and sense primer 5'-CCCATGTTCTGATACAGCTGC-3'; pair 2, antisense primer 5'-GACAAGAAGGCCAAGAAGGCGAA-3' and sense primer 5'-CAGATCACCAACGACGACATC-3'. The following polymerase chain reaction reaction cycles were used: 96 °C for 3 min followed by 30 cycles of 57 °C for 30 s, 72 °C for 1 min, and 95 °C for 20 s and finally 72 °C for 5 min. The two fragments were subjected to a polynucleotide kinase reaction (Boehringer Mannheim) for 30 min at 37 °C and then ligated with the ligase chain reaction (Pfu DNA ligase, Stratagene) with wild type rat Na+,K+-ATPase alpha 1 cDNA as a template. The following ligase chain reaction reaction cycles were used: 96 °C for 2 min and 60 °C for 2 min followed by 30 cycles of 96 °C for 20 s and 60 °C for 20 s.

The mutated fragment was purified by agarose gel electrophoresis, digested with ApaI (Boehringer Mannheim) and BstBI (New England Biolabs Inc.) restriction enzymes and substituted for the wild type fragment. Each complete clone was sequenced with an Applied Biosystems Taq Dye Deoxy terminator cycle sequencing kit in a Perkin-Elmer DNA thermal cycler 9600 to confirm the mutation and exclude other mutations. The S23A mutation produced a BglI restriction site in the mutated clone.

Transfection

Wild type and mutant Na+,K+-ATPase alpha 1 were stably transfected into COS7 cells using the calcium phosphate method (11). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and 5% penicillin/streptomycin in 37 °C humidified air with 5.3% CO2. COS cells, which are derived from early embryonic monkey kidneys, express a highly ouabain-sensitive Na+,K+-ATPase. Selection of transfected clones was achieved as described (12-14). Briefly, cells were grown in 10 µM ouabain for 3-4 weeks, and the medium was changed every 3rd day. Untransfected COS cells died within 2 days in ouabain medium, while cells expressing the relatively ouabain-insensitive rat Na+,K+-ATPase alpha 1 subunit survived. Following this ouabain selection procedure, 200-300 single clones with approximately the same growth rate were pooled and replated in DMEM containing 10 µM ouabain. With the exception of the study shown in Fig. 3B, all experiments were performed on this mixture of clones.


Fig. 3. A, phase-contrast micrographs of subconfluent cells expressing wild type and S23A Na+,K+-ATPase grown on uncoated glass coverslips. A clear difference in cell phenotype is noted. The cells expressing wild type Na+,K+-ATPase resembled nontransfected COS cells and were well spread out with long extensions. In contrast, cells expressing S23A Na+,K+-ATPase were more rounded, and only few extensions were noted. B, relative proportion of well spread out cells and rounded cells in different clones. Single cell clones were divided into two categories; cells with long extensions and cells with short or no extensions. Number of cell clones expressing wild type Na+,K+-ATPase are shown with filled bars, and cell clones expressing S23A Na+,K+-ATPase are shown with unfilled bars. Data are mean from two independent experiments. Clonal selection and quantitation of spreading were performed as described under "Experimental Procedures."
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We selected clones of cells, expressing wild type and S23A Na+,K+-ATPase, that had similar levels of transfected Na+,K+-ATPase activity. The abundance of Na+,K+-ATPase, as determined by immunoblotting, was also within the same range in both cell types. In pilot studies we could clearly distinguish between ouabain-sensitive (endogenous) and ouabain-insensitive (transfected) Na+,K+-ATPase populations that were completely inhibited by ouabain concentrations of 10-5 M and 5 × 10-3 M, respectively. Approximately 60% of Na+,K+-ATPase was resistant to 10-5 M ouabain and was considered to be rat Na+,K+-ATPase. Furthermore, the expression of wild type and S23A Na+,K+-ATPase mRNA was in each protocol verified by reverse transcriptase-polymerase chain reaction with RNase-free DNase (Pharmacia Biotech Inc., Uppsala, Sweden)-treated total RNA. By measuring Rb+ flux (see below), we could verify the presence of functioning Na+,K+-ATPase in the plasma membrane. Rb+ flux sensitive to 5 × 10-3 M, but not to 10-5 M, ouabain was not significantly different in cells expressing wild type and S23A Na+,K+-ATPase.

ATP Hydrolysis

Cells expressing wild type and S23A Na+,K+-ATPase were seeded (3.0 × 105 cells/well) in 30-mm diameter wells and grown to confluence. The cells were incubated for 20 min at 37 °C in DMEM with or without 1 µM PDBu. Cells were lysed by treatment with 1 mM Tris-HCl, pH 7.5, on ice for 15 min, and a crude membrane fraction was prepared as described (15). The membrane fraction was frozen in aliquots and stored overnight at -70 °C. Na+,K+-ATPase-dependent ATP hydrolysis was measured in triplicate as described (12). Transfected Na+,K+-ATPase activity was determined as the difference between ATPase activity at 10-5 M and 5 × 10-3 M ouabain.

Cellular Uptake of K+ as Assessed by Determination of 86Rb+ Flux

All experiments were performed in phosphate-buffered saline (PBS), which contained (in mM) 130 NaCl, 4 KCl, 1.5 CaCl2, 1 MgCl2, 4 Trizma phosphate, 10 D-glucose. The osmolality of this solution was adjusted to 315 mosM/liter by choline chloride addition, and the pH was adjusted to 7.4 at 37 °C. Experiments were performed at 37 °C in a water bath under a humidified atmosphere with 5% CO2, 95% air. After removal of the culture medium, cells were washed twice and preincubated for 40 min in the presence of ouabain (10-5 M) to inhibit the activity of endogenous Na+,K+-ATPase or 5 × 10-3 M to inhibit both endogenous and transfected Na+,K+-ATPase. PDBu, when used, was added after 20 min of preincubation. 86Rb+ flux measurements were initiated by adding to each well 0.1 ml of PBS containing 0.25 µCi/ml 86Rb+. 86Rb+ uptake was linear for 5 min (data not shown), and recordings were made after 2 min. Reactions were stopped by rinsing cells three times with ice-cold PBS, containing 5 mM BaCl2. Cells were then lysed by the addition of 0.6 ml of 1 mM NaOH. Radioactivity in cell lysates were measured using a scintillation counter (LKB, Wallac, Turku, Finland). Protein was determined using a Bio-Rad assay with bovine serum albumin as standard.

Rat wild type and S23A Na+,K+-ATPase-dependent 86Rb+ uptake was determined as the difference between the accumulated 86Rb+ uptake rate in the presence of 10-5 M and 5 × 10-3 M ouabain.

Measurement of Intracellular Na+

Cells grown on 25-mm coverslips and placed in Petri dishes were incubated in DMEM culture medium containing a membrane-permeable acetoxymethyl (AM) ester of the Na+-sensitive fluorescent indicator, Na+-binding benzofuran isophthalate (SBFI/AM) 10-5 M (16, 17) for 3 h at 37 °C and equilibrated with 5% CO2, in air. Probenecid (0.6 mM) was added to block the transport of SBFI out of the cells. The coverslips were mounted on a microscope stage. The DMEM was replaced, and the cells were superperfused with physiological salt solution containing (in mM): 110 NaCl, 5 KCl, 1 CaCl2, 1.2 MgCl2, 1 Na2HPO4, 25 NaHCO3, 20 HEPES, 5 glucose, 1 sodium butyrate, pH 7.4, at 37 °C. In K+-free solutions, the KCl in physiological salt solution was omitted.

Fluorescence was measured using a SPEX dual-beam excitation spectrofluorometer (18). The excitation monochromators were set at 345 and 380 nm, the emission at 510 nm, and fluorescence was recorded as the 345/380 nm ratio. After loading the cells, Petri dishes were placed on the stage of a Zeiss Neoflurar X40, 1.75 objective, at 37 °C and UV measurements selected using a computerized spectrophotometer system (DM3000 cm, SPEX Inc., New York). Emitted light was measured by photon counting (Hamamatsu R928). All experimental solutions were kept at 37 °C and equilibrated with 5% CO2 in air.

At the end of each experiment, the following calibration procedure was carried out. The SBFI-loaded cells were superperfused with a solution containing (in mM): 5 KCl, 1 CaCl2, 1.2 MgCl2, 1 KH2PO4, 20 HEPES, 5 glucose, pH 7.4, at 37 °C. The Na+ concentration ranged from 0 to 137 mM. Osmolality was kept constant by the addition of choline chloride. The solution also contained ouabain (5 × 10-3 M) and the Na+ ionophores, gramicidin (10-5 M), monensin (10-5 M), and nigericin (10-5 M) (16, 19). The intracellular Na+ (Na+i) calibration curve was found to be almost linear from about 1 to 20 mM, the physiological intracellular range, and a change of less than 0.5 mM Na+i could be detected (data not shown).

Measurement of Intracellular pH

Cells were cultured in DMEM and incubated with the membrane permeable AM ester of fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF/AM), (10-5 M) for 30-60 min at 37 °C. The coverslips were mounted on a microscope stage. The DMEM was replaced, and the cells were superfused with standard physiological salt solution.

Fluorescence was measured in a SPEX dual-beam excitation spectrofluorometer as described (18). The excitation monochromators were set at 485 and 445 nm, the emission at 520 nm, and cell fluorescence was recorded as the 485/445 nm ratio. Calibration was performed at the end of each experiment as described (18). Cells were superfused with a solution containing (in mM): 120 KCl, 20 NaCl, 20 HEPES, and 0.025 nigericin. The pH of the extracellular medium was varied between 6.0 and 8.0.

Cell Morphology

After transfection, dilution series were made on the cell population. The cell suspension was aliquoted in microtiter wells (Costar) and grown in medium containing 10-5 M ouabain. After 2-4 weeks single cell clones were visible. Their morphology was characterized using light microscopy. They could be divided into two categories, cells with long cellular extensions and cells with short or no cellular extensions. This difference in shape was most obvious when the cells were subconfluent. In two independent experiments 150 clones of subconfluent cells expressing wild type Na+,K+-ATPase and 120 clones of cells expressing S23A Na+,K+-ATPase were characterized in each experiment.

Cell Attachment

Cell attachment on fibronectin-coated substrates was assayed in microtiter wells (Costar). All wells were preincubated overnight at 4 °C with varying concentrations of fibronectin. Before adding cells, each well was preincubated with 1% bovine serum albumin for 30 min in 37 °C and later washed with serum-free DMEM. Two alternative methods were used to assess the earliest stages of cell attachment. 1) Cells expressing wild type or S23A Na+,K+-ATPase were grown in a 3 µCi/ml [3H]thymidine containing medium supplemented with 10% fetal calf serum and harvested after approximately 72 h at 60-80% confluence. 2.5 × 104 cells/well were seeded in serum-free DMEM. Cells were allowed to attach to fibronectin (5 µg/ml)-coated substrates during 40 min of incubation in 37 °C and later washed with serum-free DMEM. Radioactivity was measured in a Wallac scintillation counter. Results are presented as percent remaining radioactivity of total. 2) Cells expressing wild type or S23A Na+,K+-ATPase were harvested at 60-80% confluence, and 2.5 × 104 cells/well were seeded in serum-free DMEM. After different incubation times the wells were washed with serum-free DMEM and fixed in PBS containing 4% paraformaldehyde and stained with a 20% methanol cresyl-violet solution. Cells were counted in three randomly selected areas (each area containing >60 cells) within the center of the well. Two types of cells were recognized: cells that had started to spread and had a flattened appearance and cells that were rounded and appeared loosely attached. The ratio between cells that had a flattened appearance and the total amount of cells was plotted against the concentration of the surface coating or time of incubation.

Chemicals

Ouabain was obtained from Merck (Darmstadt, Germany), 86Rb+ from Amersham Life Science, Inc., bovine serum albumin from Boehringer Mannheim, [gamma -32P]ATP from NEN Life Science Products, and Dulbecco's modified Eagle's medium from Life Technologies, Inc. Human fibronectin was from the Finnish Red Cross Blood Service. SBFI and BCECF were obtained from Molecular Probes (Eugene, OR). Nigericin, gramicidin, monensin, probenecid, PDBu, and vitronectin were from Sigma. All other reagents were of analytic grade or of highest available purity.

Statistical Analysis

Values are given as means ± S.E. Data were analyzed by Student's t test and analysis of variance test. p values < 0.05 are considered significant.


RESULTS

Na+,K+-ATPase Activity

Na+,K+-ATPase activity was determined in microsomal preparations from transfected cells as described (12). Determinations were made both at saturating (70 mM) and nonsaturating Na+ (10 mM) concentrations (Table I). As with the other protocols, ouabain (10-5 M) was present in all media to inhibit endogenous Na+,K+-ATPase. In cells expressing wild type Na+,K+-ATPase, the PKC activator, PDBu, inhibited enzyme activity by 24 ± 7% at 70 mM Na+ and 27 ± 5% at 10 mM Na+. In cells expressing S23A Na+,K+-ATPase, PDBu had no effect at either 10 mM or 70 mM Na+.

Table I. Effect of PDBu on Na+,K+-ATPase activity measured as ouabain-sensitive ATP hydrolysis in COS cells stably transfected with wild type or S23A rat Na+,K+-ATPase alpha 1

The studies were performed either at saturating (70 mM) or nonsaturating (10 mM) Na+ concentration. n refers to the number of experiments. All ATPase determinations were made in triplicate. Values are mean ± S.E.

Cell type Sodium concentration Na+,K+-ATPase activity
 -PDBu +PDBu

mM nmol/Pi/mg protein/h
Wild typea 70 3185  ± 261 2383  ± 209b
(n = 8)
S23A 70 2529  ± 222 2679  ± 217
(n = 12)
Wild type 10 1454  ± 106 1096  ± 59b
(n = 6)
S23A 10 1321  ± 99 1398  ± 86
(n = 9)

a Cells incubated with and without PDBu were studied in parallel.
b Indicates significant, p < 0.05, difference between nontreated and PDBu-treated cells.

86Rb+ Uptake

It is generally accepted that 86Rb+ can be used as a tracer for K+. Na+,K+-ATPase-dependent K+ uptake was therefore assessed by determination of the difference in cellular uptake of 86Rb+ in the presence and absence of 5 × 10-3 M ouabain. PDBu (5 × 10-6 M) significantly reduced Na+,K+-ATPase-dependent 86Rb+ uptake to 77 ± 6% (n = 8) of the control value in cells expressing wild type Na+,K+-ATPase (p < 0.01 by paired t test). PDBu stimulated Na+,K+-ATPase-dependent 86Rb+ uptake in cells expressing S23A Na+,K+-ATPase to 140 ± 10% of the control value (n = 6), p < 0.02 (see "Discussion").

Intracellular Na+ and pH

Under basal conditions, Na+i was significantly lower in cells expressing S23A Na+,K+-ATPase than in cells expressing wild type Na+,K+-ATPase (Fig. 1A). Values of Na+i are generally lower, when determined with fluorescent probes, which measure free Na+i, than with indirect methods that use, for example, isotopes or an electron probe to measure total Na+i (16, 17, 19). Intracellular pH was slightly but significantly higher in cells expressing S23A Na+,K+-ATPase compared with those expressing wild type Na+,K+-ATPase.


Fig. 1. Basal levels of Na+i (A) and pH (B) in transfected cells. Na+i was determined in 18 cells each expressing the wild type or S23A Na+,K+-ATPase. pH was determined in 62 cells expressing the wild type and 44 cells expressing the S23A Na+,K+-ATPase. Values are means ± S.E. *, p < 0.01.
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Exposure of cells to ouabain or K+-free medium caused similar increases in Na+i in cells expressing wild type or S23A Na+,K+-ATPase (data not shown). In both cell types, Na+i increased linearly with incubation time for at least 15 min. Addition of PDBu caused an increase in Na+i in cells expressing wild type Na+,K+-ATPase after a lag period of a few minutes (Fig. 2A). After 15 min of PDBu incubation, the increase in Na+i averaged 7.6 ± 0.4 mM (p < 0.01). PDBu had no effect on Na+i in cells expressing S23A Na+,K+-ATPase.


Fig. 2. A, the effect of PDBu on Na+i in cells expressing wild type or S23A Na+,K+-ATPase. The left panel shows a tracing from an individual cell; the right panel summarizes results from five experiments in each group. B, the effect of PDBu on pH in cells expressing wild type and S23A Na+,K+-ATPase. The left panel shows tracings from individual cells; the right panel summarizes results from five experiments in wild type and seven experiments in S23A cells. A PBS solution containing PDBu (10-6 M) was added at the time indicated by the arrows. All values are means ± S.E.
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Na+i is critically dependent on the relationship between Na+ entry into the cell, the leak, and Na+,K+-ATPase, the pump. Activation of PKC by phorbols may stimulate one of the most important Na+ entry pathways, the amiloride-sensitive Na+/H+ exchanger (NHE). To test whether PDBu had dual and opposite effects in this system on the NHE and Na+,K+-ATPase, recordings were also made of intracellular pH, and the studies were repeated in the presence of amiloride, 10-5 M (20). PDBu increased intracellular pH in both types of cells, but the increase was of longer duration in cells expressing S23A Na+,K+-ATPase (Fig. 2B). Three min after PDBu addition, the pH started to fall in cells expressing wild type Na+,K+-ATPase but not in cells expressing S23A Na+,K+-ATPase. Amiloride abolished the pH response in both types of cell (data not shown). Preincubation with amiloride (10-5 M) abolished the PDBu-induced rise in Na+i in cells expressing wild type Na+,K+-ATPase (data not shown). Amiloride alone had no significant effect on basal Na+i in either type of cells.

Cell Morphology and Adhesiveness

COS cells normally start to spread out and appeared flattened within 30 min after they have been seeded to fibronectin coated plates. After 60 min all cells have started to spread. During the next 24-48 h they continued to spread and form long extensions. Most COS cells expressing wild type Na+,K+-ATPase also develop extensions within 48 h, while most cells expressing S23A Na+,K+-ATPase fail to do so. Fig. 3A illustrates the difference in shape between subconfluent cells expressing wild type and S23A Na+,K+-ATPase. Analysis of 100-150 individual clones indicated that more than 80% of the wild type cells had long extensions, while more than 85% of the S23A cells had a round appearance (Fig. 3B). To semiquantitatively compare the capacity of cells expressing wild type and S23A Na+,K+-ATPase to attach, [3H]thymidine-loaded cells were plated on nonadhesive plastic dishes coated with fibronectin. Forty min after plating, 48 ± 4.9% cells expressing wild type Na+,K+-ATPase, but only 18 ± 2.7% of cells expressing S23A Na+,K+-ATPase, were attached to fibronectin (Fig. 4A) (p < 0.01). The rate of cell spreading and flattening was visually estimated in cells that had been fixed and stained at various time points after plating. Sixty min after plating, approximately 80-90% of cells expressing wild type Na+,K+-ATPase, but only 40-50% of the cells expressing S23A Na+,K+-ATPase, appeared spread and flattened. After approximately 120 min, both cell types appeared spread and flattened to a similar extent (Fig. 4B). The difference in spreading between cells expressing wild type and S23A Na+,K+-ATPase at 60 min was similar when the fibronectin concentration varied between 2.5 and 25 µg/ml (Fig. 4C).


Fig. 4. A, quantitation of cell attachment. [3H]thymidine-loaded cells expressing wild type Na+,K+-ATPase (filled bar) and cells expressing S23A Na+,K+-ATPase (unfilled bar) were seeded onto plates coated with 5 µg/ml concentration of fibronectin. After allowing cells to attach for 40 min at 37 °C, the wells were washed, and the remaining cells were measured. A significant (p < 0.01) difference in the capacity to attach was noted between the two cell types. Data are presented as mean ± S.E. (n = 6 in triplicates). The extent of cell adhesion was quantified as described under "Experimental Procedures." B and C, a time course study of the earliest stage of cell spreading. Wild type (black-square) and mutant (bullet ) cells were seeded onto plates coated with 5 µg/ml fibronectin (B) and various concentrations of fibronectin (C). Thirty to 120 min after plating, the cells were fixed and stained. All data points in B (30-120 min after plating) and C (60 min after plating) are the average of duplicate samples. Triplicate determination was made in each sample, and the variation was less than 10%.
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DISCUSSION

All mammalian cells are characterized by low Na+i and a high Na+ gradient across the plasma membrane that is generated by vectorial transport of Na+ mediated by Na+,K+-ATPase. Since the coupling ratio for outward Na+ transport and inward K+ transport is 3:2, Na+,K+-ATPase activity also generates a difference in membrane potential. The Na+,K+-ATPase-dependent electrochemical gradient is necessary for many vital cell functions, and total inhibition of Na+,K+-ATPase results in cell death (2, 21).

Na+i is generally too low to saturate Na+,K+-ATPase (Refs. 21 and 22 and this study), and the activity of the Na+,K+-ATPase is dependent on Na+i. Since the affinity for Na+ of the various subunits appears to vary, Na+i concentration is also expected to be dependent on which isoform(s) a given cell type expresses (21-23). The results of the present study indicate that other mechanisms are also involved in the regulation of Na+i. Thus we have shown using a variety of methods that cells transfected with S23A Na+,K+-ATPase alpha 1 subunit were no longer able to respond to PKC activation with the inhibition of ATP hydrolysis, decrease in Rb+ uptake, and increase in Na+i exhibited by cells expressing wild type rat Na+,K+-ATPase. These results highlight the significance of phosphorylation of Ser-23 of rat Na+,K+-ATPase alpha 1 subunit in response to activation of PKC in intact cells. Furthermore, our results suggest that this regulatory phosphorylation site may play a direct role in modulating basal Na+i, as well as an indirect role in modulating intracellular pH and other cellular phenotypes.

Many pumps and other ion transporters are involved in the regulation of Na+i and pH. The results of the present study illustrate that it is important not only to consider the regulation of Na+,K+-ATPase, but also its impact on other Na+-coupled transporters such as the NHE (Fig. 5). Activation of PKC stimulates NHE, resulting in an increase of intracellular pH (24, 25) and presumably an increase of Na+ influx (Fig. 5, A-D). Our results suggest that in cells expressing wild type rat Na+,K+-ATPase alpha 1, activation of PKC also inhibits the activity of Na+,K+-ATPase (Fig. 5, A and B). At early time points (<5 min, Fig. 5A), it appears that any increase in Na+ influx mediated by stimulation of NHE is compensated for by increased Na+i-dependent Na+,K+-ATPase activity. However, at later time points (>5 min, Fig. 5B) the combined effects of PKC on stimulation of Na+ entry and inhibition of Na+ exit pathways results in a measurable increase in Na+i (Fig. 5B). This increase in Na+i appears ultimately to create such an unfavorable Na+ gradient that down-regulates NHE activity, resulting in the transient nature of the pH increase observed. In cells expressing S23A Na+,K+-ATPase, PKC activation will lead only to activation of NHE (Fig. 5, C and D). In these cells, as observed at early time points in cells expressing wild type Na+,K+-ATPase, any increase in Na+i mediated by stimulation of NHE is compensated for by increased Na+,K+-ATPase activity (Fig. 5C). The observation that PDBu stimulated 86Rb+ uptake in cells expressing S23A Na+,K+-ATPase, which was measured in intact cells with low, nonsaturating Na+ concentration, is consistent with this conclusion. Moreover, in cells expressing S23A Na+,K+-ATPase, the increased pH induced by PDBu does not return to the basal level, because Na+i does not increase to a level sufficient to inhibit NHE (Fig. 5D). The regulation of Na+,K+-ATPase activity by phosphorylation of Ser-23 also appears to influence the basal ionic status of cells. Presumably because of a basal PKC activity, basal Na+i is higher in cells expressing wild type rat Na+,K+-ATPase alpha 1. Furthermore, most likely because of the relationship between the NHE leak and the Na+,K+-ATPase pump activity, the basal intracellular pH is lower in cells expressing wild type enzyme.


Fig. 5. A scheme illustrating the dual effects of PDBu on the Na+/H+ exchanger and on Na+,K+-ATPase in cells expressing wild type and S23A Na+,K+-ATPase. Since PDBu influences only the Na+/H+ exchanger in the cells expressing S23A Na+,K+-ATPase, the net effects on Na+i and pH would be expected to differ in the two cell types.
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Since mutation of S23A of rat Na+,K+-ATPase alpha 1 altered such important parameters as the concentration of Na+i and pH, it might also influence the phenotype of the cell in other ways. Such an effect was indeed observed, in that the shape of the cells expressing S23A Na+,K+-ATPase indicated that the attachment to substrate was perturbed. Furthermore, the ability of these cells to attach and spread on fibronectin was impaired. As cell attachment to this protein is mediated by integrins (27, 28), this raises the possibility of a change in the expression and/or function of integrins. The relationship between this phenomenon and the resetting of Na+i and pH will be a topic for future studies. Notably, an increase in intracellular pH following overexpression of a H+-ATPase has previously been shown to lead to a change in cell shape (29).

There is compelling evidence indicating that the activity of Na+,K+-ATPase can be modulated by hormones and that regulation of the enzyme is likely to be mediated by phosphorylation/dephosphorylation (6, 8, 12, 26). Recently, it was shown in rat choroid plexus that serotonin stimulated the phosphorylation of the alpha 1 subunit (at a site subsequently identified as Ser-23 (10)) and that this was accompanied by inhibition of Na+,K+-ATPase activity (30). In the rat renal tubule, Na+,K+-ATPase is inhibited by several natriuretic hormones, the effect of which may be mediated via PKC (31). By inhibiting the activity of Na+,K+-ATPase they should increase Na+i and thereby decrease the transcellular driving force for Na+. This may explain why these hormones inhibit Na+ reabsorption in the renal tubules. In conclusion, the results of the present study demonstrate the importance of PKC in the regulation of rat alpha 1 Na+,K+-ATPase and suggest that regulation of Na+,K+-ATPase by phosphorylation/dephosphorylation plays an important role in cell and body Na+ homeostasis.


FOOTNOTES

*   This work was supported by grants from the Swedish Medical Research Council (Project number 03644) and the Swedish Heart Lung Foundation (to A. A.) and by United States Public Health Service Grant MH-40899 (to P. G.).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.
§   The first two authors contributed equally to this work.
**   Participation to this work was initiated during stay at the Karolinska Institute as a Nobel Fellow.
Dagger Dagger    To whom correspondence should be addressed. Tel.: 46-8-672-2222; Fax: 46-8-672-1941.
1   The abbreviations used are: PKA, cAMP-dependent protein kinase; PKC, protein kinase C; AM, acetoxymethyl; BCECF, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; DMEM, Dulbecco's modified Eagle's medium; PDBu, phorbol 12,13-dibutyrate; PBS, phosphate-buffered saline; SBFI, Na+-binding benzofuran isophthalate; Na+i, intracellular Na+ concentration; NHE, Na+/H+ exchanger.

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