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 |
We have previously shown that the rat
Na+-K+-ATPase
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
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
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
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 |
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
-subunit and a smaller glycosylated
-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
-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
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
1-isoform Ser-23 is
phosphorylated (18, 29); in the Bufo
marinus
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
1-subunit, might influence the
effect of PKC on the activity of this ion pump.
 |
MATERIALS AND METHODS |
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
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
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
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
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
1-subunit had been transfected,
expressed in a stable way, and assembled with the monkey
-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(
-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid (EGTA), 30 Tris · HCl, pH 7.4, 3 Tris-ATP, and
tracer amounts of
[
-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
-subunit was evaluated with an antibody that
selectively detects the Ser-943-phosphorylated, but not
dephosphorylated, form of Na+-K+-ATPase
-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
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
-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
[
-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
[
-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-
-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),
[
-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 |
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
1-subunit of rat
Na+-K+-ATPase
or with an
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).
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
-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
-subunit. Cells stably expressing rat
Na+-K+-ATPase
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
-subunit. B: quantitative analysis
of changes in Ser-943 phosphorylation of
Na+-K+-ATPase
-subunit in 4 separate experiments.
* P < 0.01 vs. control.
|
|
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
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 |
We have shown previously that the PKC phosphorylation site Ser-23 is
essential for PKC-induced inhibition of the rat
Na+-K+-ATPase
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.
 |
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