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Am J Physiol Cell Physiol 273: C1981-C1986, 1997;
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Vol. 273, Issue 6, C1981-C1986, December 1997

Regulation of rat Na+-K+-ATPase activity by PKC is modulated by state of phosphorylation of Ser-943 by PKA

Xian-Jun Cheng, Jan-Olov Höög, Angus C. Nairn, Paul Greengard, and Anita Aperia

Department of Woman and Child Health and Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-11281 Stockholm, Sweden; and Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously shown that the rat Na+-K+-ATPase alpha 1-isoform is phosphorylated at Ser-943 by protein kinase A (PKA) and at Ser-23 by protein kinase C (PKC), which in both cases results in inhibition of enzyme activity. We now present evidence that suggests that the phosphorylation of Ser-943 by PKA modulates the response of Na+-K+-ATPase to PKC. Rat Na+-K+-ATPase alpha 1 or a mutant in which Ser-943 was changed to Ala-943 was stably expressed in COS cells. The inhibition of enzyme activity measured in response to treatment with the phorbol ester, phorbol 12,13-dibutyrate (PDBu; 10-6 M), was significantly reduced in the cells expressing the Ala-943 mutant compared with that observed in cells expressing wild-type enzyme. In contrast, for cells expressing Na+-K+-ATPase alpha 1 in which Ser-943 was mutated to Asp-943, the effect of PDBu was slightly enhanced. The PDBu-induced inhibition was not mediated by activation of the adenosine 3',5'-cyclic monophosphate/PKA system and was not achieved via direct phosphorylation of Ser-943. Sp-5,6-DCl-cBIMPS, a specific PKA activator, increased the phosphorylation of Ser-943, and this was associated with an enhanced response to PDBu. Thus the effect of PKC on rat Na+-K+-ATPase alpha 1 is determined not only by the activity of PKC but also by the state of phosphorylation of Ser-943.

adenosine 3',5'-cyclic monophosphate-dependent protein kinase; phorbol ester; COS cells; site-directed mutagenesis; protein kinase C

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

SODIUM-POTASSIUM-ADENOSINETRIPHOSPHATASE is an integral plasma membrane protein responsible for generating and maintaining electrochemical gradients across the cell membrane (26, 34). It transduces energy from ATP hydrolysis to the active pumping of three Na+ out of and two K+ into the cell, thereby generating electrochemical gradients. These gradients are essential for numerous cellular activities and functions such as active transport of certain solutes, regulation of cell volume, and restoration of the membrane potential in electrically excitable tissues. The enzyme is composed of a large catalytic alpha -subunit and a smaller glycosylated beta -subunit, the function of which has not yet been well defined.

The activity of Na+-K+-ATPase can be regulated by a variety of hormones and neurotransmitters, the actions of which appear to be mediated directly or indirectly by protein phosphorylation (2, 7, 16, 20, 30). The catalytic alpha -subunit of rat renal Na+-K+-ATPase has well-characterized phosphorylation sites for adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC). In the rat alpha 1-isoform, PKA phosphorylates Ser-943, a residue at the carboxy terminus of the enzyme that is highly conserved between isoforms and species (4, 17, 19). The phosphorylation site(s) for PKC varies between isoforms and species but is generally present in the amino terminus. In the rat alpha 1-isoform Ser-23 is phosphorylated (18, 29); in the Bufo marinus alpha 1-isoform Thr-15 and Ser-16 are phosphorylated (4). Our previous studies have shown that phosphorylation of either site is associated with inhibition of enzyme activity (19, 29).

In a number of other cases in which a protein is phosphorylated on different sites by distinct protein kinases, it has been found that the phosphorylation of one site modulates the effects of phosphorylation or dephosphorylation at the other site (9, 14, 21, 28). Therefore, the aim of the present study was to examine whether the state of phosphorylation of the PKA site, Ser-943, in rat renal Na+-K+-ATPase alpha 1-subunit, might influence the effect of PKC on the activity of this ion pump.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Construction of mutant cDNAs. The method employed in this study for mutation has been described in detail elsewhere (12, 19). In brief, mutations were introduced into the rat Na+-K+-ATPase alpha 1 cDNA cloned in the Bluescript vector by oligonucleotide-directed site-specific mutagenesis. The oligonucleotides used in the present study were 5'-GCT GGA AGA CAG CAT TCC TTC TGG-3' and 5'-GCT GGA AGA CAT CAT TCC TTC TGG-3', which converted Ser-943 (starting the numbering from the initiation Met) of the rat Na+-K+-ATPase alpha 1-subunit to Ala-943 and Asp-943, respectively. To verify that the mutations were correct, nucleotide sequencing was performed using the dideoxynucleotide chain-termination method (32).

Expression of the cDNA in COS cells. The entire cDNAs coding for wild-type and mutated alpha 1 were excised from the Bluescript vector and subcloned into the eukaryotic expression vector pXM (24) as described (12, 19). The vector pXM contains the adenovirus major late promoter and the simian virus 40 origin, early gene enhancer, and polyadenylation sequence. To obtain cell lines with the Na+-K+-ATPase cDNA stably integrated into chromosomes, plasmid DNA was linearized with Nde I and transfected into COS-7 cells (12, 19) using the calcium phosphate/DNA precipitation method (11). Due to a difference in ouabain binding affinity between monkey and rat alpha 1-subunits, rat cells survive concentrations of ouabain that kill monkey cells (COS cells). Therefore, we used ouabain sensitivity to select those COS cells in which the cDNA encoding the rat alpha 1-subunit had been transfected, expressed in a stable way, and assembled with the monkey beta -subunit to form a functional Na+-K+-ATPase in the plasma membrane. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum under conditions described elsewhere (12, 19). Sixty hours after cell transfection, ouabain was added to the medium at a final concentration of 10-5 M. After 10 days, hundreds of individual ouabain-resistant colonies appeared. These colonies were pooled and propagated.

Determination of Na+-K+-ATPase activity. Na+-K+-ATPase activity, in membranes isolated from the transfected cell lines, was measured by determination of Pi production (12, 19). After preincubation of cells with drugs, cells were lysed and cell membranes were prepared, washed, and resuspended in TME buffer [75 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.5, 12.5 mM MgCl2, 1.5 mM EDTA]. Crude cell membranes were quickly frozen on dry ice and thawed at room temperature to open vesicles formed during the membrane preparation. Aliquots of membrane fragments were incubated for 15 min at 37°C in 100 µl of a solution containing (in mM) 10 NaCl, 20 KCl, 120 choline chloride, 5 MgCl2, 1 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 30 Tris · HCl, pH 7.4, 3 Tris-ATP, and tracer amounts of [gamma -32P]ATP. In experiments in which 5 or 70 mM NaCl was used, the concentration of choline chloride was varied to keep the ionic strength constant. To prevent dephosphorylation of Na+-K+-ATPase, the buffer was supplemented with the protein phosphatase inhibitors okadaic acid (2.5 × 10-7 M) and FK-506 (2.5 × 10-8 M). All studies were performed in the presence of 10-5 M ouabain to inhibit endogenous COS cell Na+-K+-ATPase activity. An amount of enzyme was selected so that total ATP hydrolysis did not exceed 20%, and ATP hydrolysis was linear with time. The reaction was stopped by the addition of 700 µl of activated charcoal. The [32P]Pi liberated was determined in the supernatant after centrifugation. For the determination of ouabain-insensitive ATPase activity, NaCl and KCl were omitted, and 5 × 10-3 M ouabain was added. Protein content of cell membranes was determined by the method of Bradford (10) using a kit from Bio-Rad and using bovine serum albumin (BSA) as a standard.

The dose dependence of the effect of ouabain on Na+-K+-ATPase activity in the transfected cells revealed the presence of two populations of Na+-K+-ATPase, the endogenous enzyme sensitive to 10-5 M ouabain and the transfected enzyme insensitive to 10-5 M ouabain but completely inhibited by 5 × 10-3 M ouabain (data not shown). Transfected Na+-K+-ATPase was estimated (15) to be ~50-60% of total Na+-K+-ATPase (12, 19). All subsequent experiments were carried out in the presence of 10-5 M ouabain.

Determination of Na+-K+-ATPase phosphorylation. The state of phosphorylation of Na+-K+-ATPase at Ser-943 of the alpha -subunit was evaluated with an antibody that selectively detects the Ser-943-phosphorylated, but not dephosphorylated, form of Na+-K+-ATPase alpha -subunit (12, 19). This rabbit polyclonal antibody was raised against a PKA-phosphorylated synthetic peptide corresponding to amino acids 936-948 of rat Na+-K+-ATPase alpha 1-isoform. The characterization and application of this site-directed phosphorylation state-specific antibody have been described elsewhere (12, 19). To prevent degradation and dephosphorylation of Na+-K+-ATPase, cell lysates were prepared at 4°C in a buffer supplemented with protease and phosphatase inhibitors phenylmethylsulfonyl fluoride (0.1 mM), benzamidine (25 mM), leupeptin (20 µg/ml), antipain (20 µg/ml), pepstatin A (5 µg/ml), chymostatin (5 µg/ml), and sodium fluoride (50 mM). Proteins in cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to nitrocellulose membranes. Nitrocellulose membranes were probed with the phospho-Ser-943 antibody, and immunoreactivity was detected using an enhanced chemiluminescence technique (Amersham).

cAMP measurement. Confluent cultures of COS cells in 24-well plates were washed with 0.5 ml of prewarmed serum-free DMEM and preincubated under standard culture conditions for 10 min in 0.5 ml of DMEM containing 5 × 10-4 M 3-isobutyl-1-methylxanthine. After they were washed briefly with DMEM, cells were incubated for 15 min in 0.5 ml of DMEM in the absence or presence of 10-5 M forskolin or 10-6 M phorbol 12,13-dibutyrate (PDBu). Incubation was terminated by aspiration of the media followed by cooling on ice water. Six percent trichloroacetic acid (0.5 ml) was then added to each well, and the cells were scraped off, collected, and briefly sonicated. The cytosolic cAMP was acetylated before it was measured. cAMP was measured by radioimmunoassay according to the instructions from the manufacturer (Bio-Rad).

PKA activity measurement. PKA activity was determined using a kit (Promega, Madison, WI) that measured the transfer of 32P to a biotinylated PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) (27). Confluent monolayers of cells, grown in 24-well culture plates and pretreated with the indicated drugs or vehicle, were washed twice with phosphate-buffered saline and incubated on ice for 15 min in 100 µl of extraction buffer containing (in mM) 25 Tris · HCl, pH 7.4, 0.5 EDTA, 0.5 EGTA, 10 beta -mercaptoethanol, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. Cell lysates were collected. Aliquots of cell lysates (~10 µg of cell lysate protein) were incubated for 10 min at 30°C in 50 µl of a solution containing (in mM) 40 Tris · HCl, pH 7.4, 20 MgCl2, 0.1 mg/ml BSA, 0.1 biotinylated Kemptide, 0.1 ATP, and a tracer amount of [gamma -32P]ATP (sp act 3,000 Ci/mmol; Amersham International). Reactions were stopped by the addition of 25 µl of 7.5 M guanidine-HCl. Twenty-five microliters of the reaction mix were removed and spotted on a streptavidin-coated disk that specifically binds the biotinylated Kemptide. The excess free [gamma -32P]ATP and any nonbiotinylated proteins in the cell extract were removed by successive washing. The 32P incorporated into the PKA biotinylated peptide substrate on the disks was subsequently determined by liquid scintillation counting. Protein content in cell extracts was determined by the method of Bradford (10) using BSA as a standard. PKA activity was expressed as picomoles 32P incorporated per minute per milligram protein.

Chemicals. PDBu, okadaic acid, and FK-506 were purchased from Sigma (St. Louis, MO), 1-oleoyl-2-acetoyl-sn-glycerol (OAG) was from Avanti Polar Lipids, and H-89 and bisindolylmaleimide were from Calbiochem (San Diego, CA). All these drugs were stored as stock solutions in dimethyl sulfoxide (DMSO) at -20°C. The DMSO final concentration in the assay solutions was 0.1% (vol/vol), which was always added as vehicle to each control solution. (Sp)-5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphorothioate (Sp-5,6-DCl-cBIMPS) was purchased from BIOLOG Life Science Institute (Bremen, Germany), ouabain was from Merck (Darmstadt, Germany), [gamma -32P]ATP was from New England Nuclear (Boston, MA), and DMEM and fetal calf serum were from Life Technologies (Gaithersburg, MD).

Statistics. Values are given as means ± SE, statistical comparisons between two groups were performed by Student's t-test, and comparisons among several groups were done by analysis of variance. P < 0.05 was considered significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mutation of Ser-943 to Ala-943 attenuates the regulation of Na+-K+-ATPase by PDBu. To assess the possible role in intact cells of the PKA phosphorylation site in the regulation of Na+-K+-ATPase activity by PKC, COS cells were transfected either with wild-type alpha 1-subunit of rat Na+-K+-ATPase or with an alpha 1-subunit in which Ser-943 was mutated to Ala. Na+-K+-ATPase activity in membranes isolated from these transfected cells was assayed at various concentrations of Na+ (Table 1). The PKC activator PDBu (10-6 M) caused a significant inhibition of Na+-K+-ATPase in cells expressing wild-type enzyme. The inhibitory effect of PDBu was completely abolished by the highly specific PKC inhibitor bisindolylmaleimide (2 × 10-6 M; data not shown) (35). The inhibition of enzyme activity caused by PDBu was less pronounced in cells expressing the Ala-943 mutant compared with that observed for cells expressing wild-type Na+-K+-ATPase. The inhibition at 5, 10, and 70 mM Na+ was 56.6 ± 7.2, 45.0 ± 7.0, and 24.2 ± 5.7% for wild-type enzyme and 23.0 ± 6.0, 17.0 ± 2.7, and 13.9 ± 3.0% for the Ala-943 mutant. At 5 and 10 mM Na+ the differences in the PDBu inhibition between wild-type enzyme and the Ala-943 mutant were significant (P < 0.01). Although a difference was also observed at saturating (70 mM) Na+ concentration, the difference was statistically insignificant (P > 0.05). At all Na+ concentrations assayed, ouabain-insensitive ATPase activity measured in membranes prepared from cells expressing either wild-type or mutant Na+-K+-ATPase was not significantly affected by PDBu (data not shown). Because PDBu-induced changes in enzyme activity were more pronounced at nonsaturating than saturating Na+ concentrations, the assay of Na+-K+-ATPase activity was performed at 10 mM Na+ in all subsequent experiments.

                              
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  		Table 1.   Effect of PDBu on Na+-K +-ATPase activity in membranes isolated from COS cells stably expressing wild-type or S943A mutant Na+-K +-ATPase

PDBu does not stimulate the cAMP/PKA pathway in COS cells. It seemed possible that the effect of PDBu was achieved by activation of the cAMP/PKA system, as has been described for other cell types (23). To investigate this, we measured cytosolic cAMP concentrations and the activity of PKA in COS cells following PDBu treatment. PDBu at a concentration of 10-6 M did not increase cAMP levels; in fact, it caused a slight but insignificant inhibition of cytosolic cAMP production (Table 2). Furthermore, PDBu did not increase the PKA activity. The activity of PKA in PDBu-treated cells was not significantly different from the activity of vehicle-treated cells (Table 3; P > 0.05). As a control, we observed that Sp-5,6-DCl-cBIMPS (10-4 M), a cell-permeant cAMP analog (31), significantly increased PKA activity (Table 3; P < 0.01).

                              
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  		Table 2.   Effects of PDBu and forskolin on accumulation of intracellular cAMP

To exclude the possibility that PDBu might affect the state of phosphorylation of Ser-943 of Na+-K+-ATPase, we studied the effect of this drug using an antibody that detects the Ser-943-phosphorylated but not dephosphorylated form of the enzyme (12, 19). Cells expressing wild-type enzyme were pretreated with Sp-5,6-DCl-cBIMPS (10-4 M), PDBu (10-6 M), or OAG (10-6 M), another specific PKC activator, and proteins in lysates were analyzed by SDS-PAGE and immunoblotting (Fig. 1). An increase in phosphorylation of the alpha -subunit of Na+-K+-ATPase (molecular mass ~110 kDa) was observed after treatment with Sp-5,6-DCl-cBIMPS but not with PDBu or OAG.


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  		Fig. 1.   Effect of Sp-5,6-DCl-cBIMPS (DCl), phorbol 12,13-dibutyrate (PDBu), and 1-oleoyl-2-acetoyl-sn-glycerol (OAG) on state of phosphorylation of Ser-943 of Na+-K+-ATPase alpha -subunit. Cells stably expressing rat Na+-K+-ATPase alpha 1 were treated at 37°C for 15 min with vehicle (Ctr), 10-4 M Sp-5,6-DCl-cBIMPS, 10-6 M PDBu, or 10-6 M OAG. After drug treatment, cells were lysed. Samples with equal amounts of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and probed with an antibody specific for Ser-943-phosphorylated Na+-K+-ATPase. A: representative immunoblot showing changes in Ser-943 phosphorylation of Na+-K+-ATPase alpha -subunit. B: quantitative analysis of changes in Ser-943 phosphorylation of Na+-K+-ATPase alpha -subunit in 4 separate experiments. * P < 0.01 vs. control.

                              
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  		Table 3.   Effects of PDBu and Sp-5,6-DCl-cBIMPS on activity of PKA

PKC-dependent inhibition of Na+-K+-ATPase activity is potentiated by stimulation of PKA activity. The less-pronounced inhibition of Na+-K+-ATPase by PKC observed for cells expressing the Ala-943 mutant may be attributable either to lack of phosphorylation of Ser-943 or to a change of structure due to substitution of Ser-943 by Ala-943. To address this issue, regulation of Na+-K+-ATPase activity by PDBu was measured after preincubation with Sp-5,6-DCl-cBIMPS, using the conditions that resulted in increased Ser-943 phosphorylation (see Fig. 1). Cells expressing wild-type Na+-K+-ATPase were pretreated with vehicle or Sp-5,6-DCl-cBIMPS (10-4 M) for 20 min before a subthreshold dose (10-8 M) of PDBu was added. In vehicle-pretreated cells, PDBu (10-8 M) decreased the activity of Na+-K+-ATPase by <10% (Fig. 2). However, when the cells were pretreated with Sp-5,6-DCl-cBIMPS (10-4 M), the inhibitory effect of PDBu on Na+-K+-ATPase activity was increased more than twofold (Fig. 2). Thus the inhibition of Na+-K+-ATPase in response to PDBu was significantly higher in Sp-5,6-DCl-cBIMPS-pretreated cells than in vehicle-pretreated cells (P < 0.05). These results suggest that the state of Ser-943 phosphorylation modulates the inhibition of Na+-K+-ATPase in response to PDBu. In other studies (data not shown), pretreatment of cells expressing wild-type enzyme with H-89, a specific PKA inhibitor (13), significantly attenutated the inhibitory effect of PDBu (Cheng, unpublished observations).


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  		Fig. 2.   Sp-5,6-DCl-cBIMPS pretreatment enhances inhibition of activity of Na+-K+-ATPase by PDBu. Confluent cultures of cells expressing wild-type Na+-K+-ATPase were preincubated with vehicle or 10-4 M Sp-5,6-DCl-cBIMPS for 20 min at 37°C. After a brief wash, a near-threshold dose (10-8 M) of PDBu was added, and cells were incubated for 10 min. Cell membranes were then isolated, and Na+-K+-ATPase activity was measured. Data were obtained from 4 independent experiments. Sp-5,6-DCl-cBIMPS pretreatment alone caused a 10.6% inhibition of activity of Na+-K+-ATPase. This effect of Sp-5,6-DCl-cBIMPS had been subtracted from combined effect of Sp-5,6-DCl-cBIMPS + PDBu. Thus data presented in this figure represent net effect of PDBu. * P < 0.05, significantly different from vehicle-pretreated.


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  		Fig. 3.   PDBu-dependent inhibition of Na+-K+-ATPase in cells expressing an Asp-943 mutant. Confluent cultures of cells stably expressing wild-type (WT) Na+-K+-ATPase or the Asp-943 mutant in which Ser-943 of Na+-K+-ATPase alpha 1 was mutated to Asp (S943D) were treated with PDBu (10-6 M) at 37°C for 15 min, and Na+-K+-ATPase activity was measured in isolated membranes at 10 mM Na+. For comparison, results were obtained in the same experiment for cells expressing the Ala-943 mutant (S943A). Data were obtained from 5 independent experiments. Na+-K+-ATPase activity measured in membranes isolated from cells expressing the Asp-943 mutant was 2,360 nmol Pi · mg protein-1 · h-1. * P < 0.01 compared with wild-type enzyme.

Mutation of Ser-943 to Asp-943 does not reduce the inhibitory effect of PDBu. To further analyze the role of phosphorylation of Ser-943 of Na+-K+-ATPase, Ser-943 was converted to a negatively charged amino acid, Asp, in an attempt to mimic the phosphorylated form of Ser-943. The Asp-943 mutant was then expressed in COS cells, and the effect of PDBu on enzyme activity was examined. In contrast to the results obtained for the Ala-943 mutant, replacement of Ser-943 by Asp-943 did not attenuate the inhibitory effect of PDBu (Fig. 3). The inhibitory effect of PDBu was enhanced slightly but not to a statistically significant extent. Ouabain-insensitive ATPase activity was not significantly affected by PDBu in cells expressing the Asp-943 mutant (data not shown).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have shown previously that the PKC phosphorylation site Ser-23 is essential for PKC-induced inhibition of the rat Na+-K+-ATPase alpha 1-isoform. Mutation of Ser-23 to Ala abolished the inhibition of the enzyme by PKC (36). In the present study, we show that inhibition of Na+-K+-ATPase by PDBu, an activator of PKC, is also influenced by the phosphorylation state of the PKA site, Ser-943. Evidence for the importance of phosphorylation of Ser-943 by PKA in the regulation of Na+-K+-ATPase by PKC was obtained from two types of studies. First, mutation of Ser-943 to the nonphosphorylatable amino acid Ala-943 (to mimic dephosphoserine) significantly attenuated the PDBu-dependent inhibition of Na+-K+-ATPase activity. Second, when PKA was activated and Ser-943 phosphorylation increased, PDBu-dependent inhibition was significantly potentiated. The latter finding supports the concept that the Ser-to-Ala mutation did not have a nonspecific effect on conformation of the enzyme. The intracellular concentration of cAMP, the activity of PKA, and the state of phosphorylation of Ser-943 were not significantly elevated by PDBu, indicating that the effect of PKC activation on Na+-K+-ATPase activity was not mediated by activation of the cAMP/PKA signaling pathway as reported in some other cells (23).

To further evaluate the role of phosphorylation of Ser-943 in the regulation of Na+-K+-ATPase activity by PKC, Ser-943 was converted to a negatively charged amino acid, Asp, in an attempt to mimic the phosphorylated forms of Ser-943. The PKC-dependent inhibition of Na+-K+-ATPase activity was only slightly enhanced, but not to a statistically significant extent compared with the wild-type enzyme (see Fig. 3), which suggests that introduction of the structurally similar Asp residue at position 943 acts only as a partial mimic of the phosphorylated Ser. The reason for this partial imitation might be ascribed to the fact that the Asp residue carries only one negative charge in its amino acid, whereas when the Ser residue is phosphorylated it normally carries two negative charges.

An interaction between PKA and PKC in the regulation of Na+-K+-ATPase activity has previously been observed in rat tissues (5, 6, 8). In rat proximal convoluted tubules, modulation of Na+-K+-ATPase by DA2 dopaminergic receptors, which are primarily linked to activation of PKC, occurs when the cAMP/PKA pathway is simultaneously activated (5, 6). Such a modulatory role for the cAMP/PKA pathway was also observed in studies of neurons (8). On the basis of the results of these and other studies, we have suggested a role for PKA-dependent phosphorylation of the phosphatase inhibitor proteins, DARPP-32 and inhibitor-1, and subsequent inhibition of protein phosphatase-1, in the regulation of the phosphorylation of Na+-K+-ATPase (1-3, 22). The present study provides an additional biochemical mechanism to account for the interaction between PKA and PKC in the regulation of Na+-K+-ATPase.

The mechanism by which PKA phosphorylation affects the regulation of Na+-K+-ATPase by PKC remains to be elucidated. Phosphorylation of rat renal Na+-K+-ATPase by PKA or PKC has been shown to alter the affinity of the enzyme for Na+ (19, 25) and K+ (29) and to change the equilibrium between the Na+-binding E1 form of the enzyme and the K+-binding E2 form of the enzyme (29). These studies suggest that a change in conformation occurs following PKA or PKC phosphorylation. It would be interesting to determine whether the change in conformation induced by PKA phosphorylation renders the Na+-K+-ATPase a better substrate for phosphorylation by PKC or a poorer substrate for dephosphorylation of the PKC site by a relevant protein phosphatase(s). It is possible that the carboxy-terminal H8-H10 segment (in which the PKA site is located) of Na+-K+-ATPase is structurally in contact with the amino-terminal H1-H2 segment (in which the PKC site is located). There is some evidence to support such a possibility (33). Finally, it cannot be excluded that Ser-943 phosphorylation acts synergistically with Ser-23 phosphorylation to produce inhibition of the enzyme.

The present observation that the response of Na+-K+-ATPase to PKC is conditioned by the phosphorylation state of the PKA site leads to speculation that the response of Na+-K+-ATPase to PKA might be affected by the state of phosphorylation of the PKC site. Thus the level of Na+-K+-ATPase activity may be a complicated function of the various signal transduction pathways that affect the state of phosphorylation of different phosphoacceptor sites in this critical enzyme.

    ACKNOWLEDGEMENTS

This work was supported by Grant 03644 from the Swedish Medical Research Council (to A. Aperia) and by National Institutes of Health Grant MH-40899 (to A. C. Nairn and P. Greengard).

    FOOTNOTES

Address for reprint requests: A. Aperia, Dept. of Woman and Child Health, St. Göran's Children's Hospital, Karolinska Institute, S-11281 Stockholm, Sweden.

Received 10 April 1997; accepted in final form 28 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Aperia, A., J. Fryckstedt, L.-B. Svensson, H. C. Hemmings, Jr., A. C. Nairn, and P. Greengard. Phosphorylated Mr 32,000 dopamine- and cAMP-regulated phosphoprotein inhibits Na+,K+-ATPase activity in renal tubule cells. Proc. Natl. Acad. Sci. USA 88: 2798-2801, 1991[Abstract/Free Full Text].

2.   Aperia, A., U. Holtbäck, M.-L. Syrèn, L.-B. Svensson, J. Fryckstedt, and P. Greengard. Activation/deactivation of renal Na+,K+-ATPase: a final common pathway for regulation of natriuresis. FASEB J. 8: 436-439, 1994[Abstract].

3.   Aperia, A., F. Ibarra, L.-B. Svensson, C. Klee, and P. Greengard. Calcineurin mediates alpha -adrenergic stimulation of Na+,K+-ATPase activity in renal tubule cells. Proc. Natl. Acad. Sci. USA 89: 7394-7397, 1992[Abstract/Free Full Text].

4.   Beguin, P., A. T. Beggah, A. V. Chibalin, P. Burgener-Kairuz, F. Jassier, P. M. Mathews, B. C. Rossier, and K. Geering. Phosphorylation of the Na,K-ATPase alpha -subunit by protein kinase A and C (PKC) in vitro and in intact cells: identification of a novel motif for PKC-mediated phosphorylation. J. Biol. Chem. 269: 24437-24445, 1994[Abstract/Free Full Text].

5.   Bertorello, A., and A. Aperia. Inhibition of proximal tubule Na+,K+-ATPase activity requires simultaneous activation of DA1 and DA2 receptors. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F924-F928, 1990[Abstract/Free Full Text].

6.   Bertorello, A. M., and A. Aperia. Both DA1 and DA2 receptor agonists are necessary to inhibit NaKATPase activity in proximal tubules from rat kidney. Acta Physiol. Scand. 132: 441-443, 1988[Medline].

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AJP Cell Physiol 273(6):C1981-C1986
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