Departments of 1 Oral Physiology and 2 Biochemistry, School of Dentistry, Meikai University, Sakado, Saitama 350-0283, Japan
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ABSTRACT |
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Na+-K+- ATPase -subunits
in basolateral membrane vesicles (BLMVs) purified from rat parotid
glands were 32P-labeled within 5 s by incubation with
[
-32P]ATP at 37°C in the presence of cAMP, but no
labeling occurred without cAMP. Phosphorylation of
Na+-K+-ATPase was associated with a decrease in
its activity. This
-subunit phosphorylation disappeared when BLMVs
were briefly incubated with cAMP and subsequent washing before the
incubation with [
-32P]ATP, indicating that catalytic
subunit of protein kinase A (PKA) associated to BLMVs via binding with
its RII regulatory subunit anchored on the membrane. In the
absence of cAMP, a PKA catalytic subunit readily reassociated with the
membrane-bound RII subunit. HT-31 peptide inhibited the
Na+-K+-ATPase phosphorylation by membrane-bound
endogenous PKA, indicating an involvement of A-kinase anchoring protein
(AKAP). AKAP-150 protein in BLMVs was shown by immunoblotting and an
RII overlay assay and was coimmunoprecipitated by anti-RII antibody.
These results show that Na+-K+-ATPase of rat
parotid gland acinar cells is regulated in vivo by membrane-anchored
PKA via AKAP rather than by free cytosolic PKA.
adenosine 3',5'-cyclic monophosphate; A-kinase anchoring protein; electrochemical gradient; parotid gland
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INTRODUCTION |
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A TYPICAL MEMBRANE-BOUND
ENZYME, Na+-K+-ATPase, consists of at
least two subunits, the catalytic -subunit and the glycosylated
-subunit (22, 23, 36, 41). By converting the
energy of ATP, Na+-K+-ATPase produces an
electrochemical gradient of Na+ and K+ across
the plasma membrane (42). Thus
Na+-K+- ATPase maintains the membrane
potential of excitable nerve (32, 43, 49) and muscle
tissues (32, 49) and is also involved in the reabsorption
of Na+ in the kidneys (22, 23, 36) and in the
salivary glands (24, 26, 27). It was reported that
Na+-K+-ATPase activity in intact cells was
inhibited by a protein phosphatase inhibitor, phospho-DARPR-32
(phosphorylated form of a dopamine- and cAMP-regulated 32-kDa protein)
(1). Purified Na+-K+-ATPase was
shown to be phosphorylated in vitro by the catalytic subunit of protein
kinase A (PKA) exogenously added (3). The phosphorylation
site of Na+-K+-ATPase by PKA was identified to
be Ser-943 of its
-subunits (for rat
Na+-K+-ATPase) (3, 15). The
activity of purified Na+-K+-ATPase was
decreased by in vitro phosphorylation of the ATPase by exogenous PKA
(3). Na+-K+-ATPase in the cells
was also shown to be phosphorylated by the stimulation of the cells
with forskolin (2, 17) and cAMP analogs (9,
18). Thus Na+-K+-ATPase is thought to be
downregulated by cAMP via PKA-dependent phosphorylation
(3).
The action of many hormones is mediated by the generation of the intracellular cAMP. The predominant effect of cAMP is to activate a cAMP-dependent protein kinase (PKA) (34, 45-47). Four molecules of cAMP bind each dormant PKA holoenzyme, activating the kinase by releasing the catalytic subunits from the regulatory subunit-cAMP complex. Two classes of regulatory subunits exist: RI and RII, which form the type I and type II PKA holoenzymes, respectively. Since the PKA catalytic subunit has rather broad substrate specificity, various proteins can be phosphorylated by the kinase in vitro regardless of the physiological significance. Phosphorylation of various cellular proteins was also observed in vivo when cells were treated with membrane-permeable cAMP analogs or forskolin and incubated for a rather long period of time, for example, 30 min or more (2, 3, 9, 17, 18). Therefore, for transducing physiological signals, PKA has to somehow perform preferential phosphorylation of its specific target substrate in vivo. However, it is not yet definitely clear how each target protein is specifically phosphorylated in vivo in response to the increase in cAMP via the activation of PKA, which has broad substrate specificity. Scott et al. (37-39), Coghlan et al. (11), and Mochly-Rosen (33) demonstrated the role of A-kinase anchoring protein (AKAP), a specific protein that anchors the PKA regulatory subunit RII but not RI (and thereby the catalytic subunit bound to RII) on the membrane near its specific target proteins. Thus the cellular location of PKA is dictated by the regulatory subunit: the RI isoform is thought to be primarily cytoplasmic, whereas a significant proportion of the RII isoform associates with the plasma membrane, cytoskeletal components, endoplasmic reticulum, secretory granules, and nuclei (12, 13, 29). The anchoring of PKA near its substrate may thus permit rapid phosphorylation of a specific substrate protein on the membrane in response to an increase in intracellular cAMP.
In the present study, using Na+-K+-ATPase-rich basolateral membrane vesicles (BLMVs) purified from rat parotid gland acinar cells, we investigated how Na+-K+-ATPase is phosphorylated in response to the increase in cAMP. We found that the Na+-K+-ATPase-rich basolateral membrane contained the holoenzyme form of PKA anchored on the membranes via the RII regulatory subunit and that BLMVs indeed contained a functional AKAP subtype, AKAP-150, which could be coimmunoprecipitated by the anti-RII antibody. The PKA anchored to the BLMVs by AKAP/RII quickly phosphorylated Na+-K+-ATPase on the membrane with the addition of cAMP in the presence of ATP, resulting in a decrease in Na+-K+-ATPase activity. The PKA catalytic subunit reversibly associated with the basolateral membranes in the absence of or on removal of cAMP. We thus conclude that the membrane-anchored PKA, rather than the free cytosolic one, may play an important role in the regulation of Na+-K+-ATPase in the basolateral membrane of parotid gland acinar cells by various signaling molecules that employ cAMP as an intracellular messenger.
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MATERIALS AND METHODS |
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Preparation of BLMVs. BLMVs were prepared from the parotid glands of 10-wk-old male Wistar strain rats (31) (Tokyo Laboratory Animal Science, Tokyo, Japan). The glands were minced and suspended in homogenization buffer [10 mM HEPES/Tris (pH 7.4), 10% sucrose, 1 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)] at 4°C, and floating tissue fragments were removed by aspiration. The minced parotid tissue was then homogenized in a Polytron (Kinematica model PT-2000 and aggregate PTA type 10S; Luzern, Switzerland) with one 10-s burst at power level five. The homogenate was centrifuged at 2,500 g for 5 min, after which the supernatant was filtered through a fine 155-µm nylon mesh and centrifuged at 22,000 g for 20 min. The pellet was suspended in a few milliliters of homogenization buffer and passed through a 30-gauge needle, and then the suspension was diluted with the buffer to 10 ml/g starting parotid tissue. Percoll was added in this suspension for a final concentration of 16%. This material was centrifuged in 15-ml aliquots at 41,500 g for 40 min. The small band (BLMV fraction) on top of the dense white band was collected and diluted 10-fold with 10 mM HEPES buffer that contained 100 mM mannitol, 100 mM KCl, and 1 mM EDTA, adjusted to pH 7.4 with Tris. The BLMV suspension was then centrifuged at 48,000 g for 20 min, and the pellet was resuspended in the same buffer without Percoll to remove the Percoll. The resulting suspension was centrifuged again, and the final pellet was stocked after having been suspended in the same buffer.
Phosphorylation of
Na+-K+-ATPase
in BLMVs.
Eight micrograms of BLMVs were incubated for 30 s at 37°C with
40 µM [-32P]ATP (10 Ci/mmol) or 1 mM
[
-32P]ATP (1 Ci/mmol) in 40 µl of incubation mixture
that contained 20 mM sodium acetate, 60 mM potassium acetate, 60 mM
KCl, 10 mM magnesium acetate, 1 mM EGTA, 100 mM mannitol, 0.0025%
Triton X-100, 10 mM HEPES (adjusted to pH 7.4 with Tris), and 10 µM
cAMP (or 16 units of PKA catalytic subunit). After the reaction had been stopped by the addition of 200 µl of 18% trichloroacetic acid
(TCA), the resulting precipitate was washed with distilled water and
boiled with SDS-PAGE buffer that contained 0.0625 M Tris · HCl
(pH 6.8), 1% SDS, and 0.25% 2-mercaptoethanol. The sample was applied
to an SDS-PAGE (4-20% gradient gel) according to Laemmli
(28). The phosphorylation pattern of BLMVs was examined by autoradiography.
Measurement of endogenous cAMP-dependent protein kinase activity
in BLMVs.
Eight micrograms of BLMV was incubated for 15 s with 1 mM
[-32P]ATP (0.01 Ci/mmol) and 10 µM cAMP in the same
incubation mixture as described in Phosphorylation of
Na+-K+-ATPase
in BLMVs. After the reaction had been stopped by TCA, radioactivity of the resulting total precipitate was measured. For
preparation of a standard curve for the 32P incorporation
into total BLMV proteins, BLMVs were incubated with various amounts of
the exogenous PKA catalytic subunit (0-16 units) in the absence of
cAMP under similar conditions. Endogenous cAMP-dependent protein kinase
activity was calculated based on the linear part of the standard curve.
Preparation of anti-1 antiserum.
An anti-
1 antiserum was prepared by the method described
in our previous report (24-27). In brief,
membrane-bound Na+-K+-ATPase was prepared from
whole kidneys of male rats by the method of Jorgensen
(22). The
-subunit was then separated from the membrane-bound Na+-K+-ATPase by SDS-PAGE (5%
acrylamide) using Laemmli's buffer system (28).
Anti-
1 antiserum was obtained by immunization of a
rabbit with the
1 protein (24-27).
Western blot analysis of
Na+-K+-ATPase.
Samples were subjected to SDS-PAGE, and the separated proteins were
electrophoretically transferred onto polyvinylidene difluoride (PVDF)
membrane filters in 10 mM 3-[cyclohexylamino]-1-propanesulfonic acid
and 10% methanol (pH 11) (4). The filter was then stained immunochemically with the anti-1 antiserum
(24-27). In brief, filters were blocked with 5% skim
milk in T-TBS (10 mM Tris · HCl, pH 7.4, containing 150 mM NaCl
and 0.05% Tween 20) at room temperature for 45 min and then incubated
for 2 h with the anti-
1 antiserum in T-TBS that
contained 5% skim milk. After being washed with T-TBS, these filters
were incubated for 1 h with anti-rabbit IgG goat serum conjugated
with horseradish peroxidase (HRP). The filters were then washed, and
the signal was detected with enhanced chemiluminescence (ECL) Western
blotting detection reagents (Amersham RPN 2106; Amersham, UK).
Phosphopeptide mapping of
[32P]phospho-Na+-K+-ATPase
-subunit.
[32P]phospho-BLMV protein phosphorylated in the presence
of cAMP was subjected to SDS-PAGE, and the gels were dried without fixation. The 92-kDa signal, the putative
[32P]phospho-Na+-K+-ATPase
-subunit, was cut from the gel and swollen with 16 µl (original
volume of cut gels) of 125 U/ml V8 protease in 100 mM ammonium
carbonate (pH 7.8) (50). The gel pieces were incubated at
37°C for 3 h, and the reaction was then stopped by boiling for 5 min. The peptides extracted from the gel pieces were applied to
tricine-SDS-PAGE (16% acrylamide) after being boiled in
tricine-SDS-PAGE buffer (35).
Measurement of Na+-K+-ATPase activity and protein contents. The enzyme activity was measured by the modified method of Igarashi et al. (21) with the following components (at final concentrations) in the reaction mixture: 30 mM NaCl, 10 mM KCl, 5 mM MgCl2, 1 mM EGTA, 5 mM ATP, 100 mM mannitol, and 10 mM HEPES, adjusted to pH 7.4 with Tris. Two hundred microliters of reaction mixture containing the enzyme preparation (16 µg protein) were incubated at 37°C for 10 min. The reaction was stopped by the addition of 200 µl of 35% TCA. The inorganic phosphate (Pi) liberated (total ATPase activity) was measured by the procedure of Fiske and Subbarow (16). Ouabain-nonsensitive ATPase activity was measured in the same way as for measurement of total ATPase activity except that 2 mM ouabain was present. The ouabain-sensitive ATPase activity calculated from the two assays was considered as Na+-K+-ATPase. The amount of enzyme liberating 1 µmol of Pi per minute was defined as 1 unit. Protein amounts were determined by the use of a protein assay kit (Bio-Rad) with bovine serum albumin serving as a standard (5).
RNA preparation. Total RNAs were prepared by the method of Chimczynski and Sacchi (10) using acid guanidinium thiocyanate-phenol-chloroform. Approximately 100-mg amounts of rat brain, kidney, and parotid gland were separately homogenized in 2 ml of guanidinium thiocyanate denaturing solution with the Polytron aggregate. The homogenate was mixed with 0.2 ml of 2 M sodium acetate (pH 4.0), 2 ml of water-saturated phenol, and 0.4 ml of 49:1 chloroform/isoamyl alcohol, stood for 15 min on ice, and then centrifuged. RNAs in the aqueous phase were precipitated with isopropanol. These RNAs were dissolved in denaturing solution again and reprecipitated with isopropanol. RNA precipitates were washed with 75% EtOH, air dried, and dissolved in water.
RT-PCR and DNA sequencing.
RT-PCR was carried out with an RNA PCR kit (AMV) version 2.1, code no.
R019A (Takara Shuzo, Biomedical Group, Shiga, Japan). Primers used to
detect isoforms of rat AKAP are listed in Table 1. PCR was performed with the GeneAmp
9600 PCR system (Perkin Elmer, Foster City, CA) according to the
following schedule: denaturation, annealing, and elongation at 94°C
for 30 s, 60°C for 30 s, and 72°C for 90 s,
respectively, for 30 cycles.
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RII overlay assay for AKAP. The presence of AKAPs in BLMVs of the rat parotid gland was detected by an RII overlay assay (20, 30, 40). BLMV protein was separated by SDS-PAGE and transferred to a PVDF membrane filter, which was then blocked with 5% skim milk in T-TBS and washed three times for 10 min with T-TBS. The filter was next incubated for 1 h with 5 nM RII regulatory subunit at room temperature in T-TBS and washed three times for 10 min with T-TBS. For detection of RII regulatory subunit binding, the filter was incubated for 2 h with anti-RII goat polyclonal IgG in T-TBS that contained 5% skim milk and was washed with T-TBS, followed by a 1-h incubation with anti-goat IgG conjugated to HRP. Signals were detected with ECL plus Western blotting detection reagents (Amersham RPN 2132).
Coimmunoprecipitation of AKAP-150 with PKA regulatory subunit. BLMVs were solubilized in lysis buffer (1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 40 mM sodium pyrophosphate, 50 mM KH2PO4, 10 mM sodium molybdate, 2 mM sodium orthovanadate, 6 µM dithiothreitol, 300 µM PMSF, 100 µM L-1-tosylamide 2-phenylethyl chloromethyl ketone, 1.5 µM pepstatin, 1.5 µM leupeptin, and 20 mM Tris · HCl, pH 7.4). Anti-RII antibody was preconjugated to protein G beads as follows: protein G beads were first washed with lysis buffer that contained 1% ovalbumin. Anti-RII antibody or preimmune goat serum was then incubated for 1 h in the lysis buffer that contained ovalbumin and was washed with the same buffer. The protein G beads preconjugated with anti-RII IgG were mixed with the lysate of BLMVs and incubated for 4 h at 4°C. The beads were collected by centrifugation and washed three times with lysis buffer, with the tube exchanged for a new one for the last wash. The final pellet of beads was extracted with SDS-PAGE sample buffer and then applied to SDS-PAGE. The separated proteins were transferred to a PVDF filter electrically, and AKAP-150 was detected by Western blot analysis with anti-AKAP-150 antibody.
Reagents. ATP-Tris salt and PKA catalytic subunit (P-8289) were purchased from Sigma Chemical (St. Louis, MO). Precast Tris-glycine and tricine polyacrylamide gels were obtained from Novex. SDS and 2-mercaptoethanol were products of Wako Pure Chemical Industries (Osaka, Japan). PVDF transfer membrane filters were from Millipore SA (Molsheim, France). Peroxidase conjugated anti-rabbit IgG goat serum was procured from Seikagaku (Tokyo, Japan). Anti-AKAP-95, -150, and -220 antibodies and anti-goat IgG HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against PKA RII subunit was obtained from Upstate Biotechnology. HT-31 peptide (6), Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val-Lys-Ala-Ala-Gly-Ala-Tyr, was synthesized by Sawady Technology (Tokyo, Japan).
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RESULTS |
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Phosphorylation of BLMV proteins by endogenous PKA anchored on the
membrane.
BLMVs purified from rat parotid glands did not contain any significant
amounts of cytosolic proteins, including the free PKA catalytic subunit
(31). This was supported by the result that membrane
proteins were only faintly phosphorylated by a short-term (30-s)
incubation without cAMP in the presence of [-32P]ATP
(Fig. 1, A and
B, lane 1). Phosphorylation of some of these faintly labeled proteins, such as the 110-kDa band shown later in Fig.
10, seemed to occur independently of cAMP and PKA. However, with the
addition of cAMP (10 µM), protein phosphorylation was clearly
detected by the 30-s incubation at 37°C: mainly four protein bands
with molecular masses of 300 kDa, 180 kDa, 92 kDa, and 50 kDa were
phosphorylated (Fig. 1A, lane 2). As described
later (Fig. 10), phosphorylation of the 92-kDa protein
(Na+-K+-ATPase
-subunit) became detectable
5 s after the addition of cAMP. The phosphorylation of these
proteins was inhibited by PKI-(5-24) peptide, a
specific inhibitor peptide of PKA, but the phosphorylation of the
110-kDa protein was not inhibited (Fig. 1A, lane
3). These results indicate the presence of membrane-anchored PKA
in the holoenzyme form in the purified BLMVs.
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Identification of the 92-kDa phosphoprotein as
Na+-K+-ATPase
-subunit.
Since Na+-K+-ATPase is one of the major
proteins in purified BLMV preparations and its
-subunit has a
molecular weight of 92 kDa, the 32P-labeled 92-kDa protein
on the SDS-PAGE was assumed to be the Na+-K+-ATPase
-subunit. To confirm this
assumption, we examined the 92-kDa 32P phosphoprotein by
using a polyclonal antiserum raised against the
1-isoform of the Na+-K+-ATPase
-subunit (24-27).
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Dissociation of the endogenous PKA catalytic subunit.
As we demonstrated (Fig. 1), PKA in the holoenzyme form is associated
with the BLMV. We then examined whether the catalytic subunit of PKA or
the regulatory subunit was anchored on the membrane. BLMVs were
incubated with cAMP (10 µM) for 5 min to separate the PKA holoenzyme
into the catalytic subunit and regulatory subunits, and the membrane
was washed. Subsequently, the BLMVs were incubated with
[-32P]ATP to assess the
protein-phosphorylating activity. As shown in Fig.
4, the BLMVs thus pretreated with cAMP
and washed lost the activity for phosphorylating membrane proteins
including Na+-K+-ATPase, even in the presence
of cAMP.
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Reconstitution of the PKA holoenzyme by anchoring of its catalytic
subunit to the BLMV.
Since the involvement of AKAP in the phosphorylation of
Na+-K+- ATPase by endogenous PKA was
suggested, we further examined the functional role of AKAP in the
regulation of the Na+-K+-ATPase phosphorylation
(Fig.5). First, the endogenous PKA
holoenzyme bound to BLMVs was separated into its catalytic and
regulatory subunits by incubating it with cAMP, and the catalytic
subunits were washed away as described in the previous section,
resulting in the loss of Na+-K+-ATPase
phosphorylation. However, the BLMVs thus treated were able to
restore the phosphorylation of Na+-K+-ATPase by
the membrane-bound PKA holoenzyme when the vesicles were incubated with
the exogenous PKA catalytic subunit followed by extensive washing for
removal of the free catalytic subunits. It should be emphasized that
the phosphorylation of Na+-K+-ATPase observed
in the PKA-reconstituted BLMV was dependent on the presence of cAMP,
indicating that the PKA responsible for the
Na+-K+-ATPase phosphorylation was derived from
its holoenzyme form on the membrane. Furthermore, the reconstitution
process of BLMV with the PKA catalytic subunit was inhibited by the
PKI-(5-24) peptide, indicating that the binding of
the PKA catalytic subunit to the BLMV was mediated by the RII
regulatory subunit, which was anchored to the membrane by AKAP. The
PKI-(5-24) peptide is known to bind to the
same site of the PKA catalytic subunit as does the regulatory subunit
(8) and thereby inhibits the catalytic activity and the
binding of the regulatory subunit to the catalytic subunit. Thus our
data indicate that the PKA catalytic subunit reversibly associated with
the BLMV, mediated by its regulatory subunit/AKAP, in the absence of
cAMP.
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mRNAs of AKAP in rat parotid gland.
To determine whether AKAP genes are actually expressed in rat parotid
glands, we examined mRNAs for AKAP in the tissue by RT-PCR. On the
basis of the cDNA sequences registered in the Query GenBank database,
National Center for Biotechnology, two sets of each primer pair were
designed and prepared for three AKAP subtypes: AKAP-95, AKAP-150, and
AKAP-220 (Table 1). With all of the primer pairs, which were expected
to produce large PCR products (553 bp for AKAP-95, 558 bp for AKAP-150,
and 251 bp for AKAP-220) and small PCR products (199 bp, 199 bp, and
200 bp for AKAP-95, AKAP-150, and AKAP-220, respectively), DNA products of the expected sizes were obtained when RT-PCR was carried out with
RNAs prepared from rat brain, kidney, and parotid gland, though the
mRNA levels for AKAP-95 and AKAP-220 in the parotid gland seemed to be
less abundant than the level of AKAP-150 mRNA (Fig.
6, A and B). The
larger PCR products were tested by the nested PCR method using the
respective primer pairs for the smaller PCR products (Fig.
6C). In an additional independent experiment, the DNA
sequence of each smaller PCR product in Fig. 6B was analyzed and confirmed to be identical to the corresponding portion of the cDNA
sequence for AKAP-95, AKAP-150, or AKAP-220 (data not shown).
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Detection of AKAP-150 protein in BLMVs of rat parotid gland.
Since the expression of AKAP mRNAs in the parotid gland suggested the
presence of AKAP proteins in the BLMVs, we examined BLMV protein by
immunoblotting with antibodies to AKAP-95, AKAP-150, and AKAP-220.
Anti-AKAP-150 antibody revealed a specific signal at a molecular weight
of 150 kDa on a blotting membrane of BLMV, and the antibody also
recognized a signal at the same molecular size on the membrane of rat
brain homogenate (Fig. 7). On the other
hand, signals for AKAP-95 and AKAP-220 proteins were not detectable on
the blotting membrane of BLMV (not shown).
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Effect of HT-31 peptide on
Na+-K+-ATPase
phosphorylation by membrane-anchored PKA.
We examined the effect of the HT-31 peptide on
Na+-K+- ATPase phosphorylation on BLMVs. The
HT-31 peptide is known to competitively inhibit RII binding to AKAP
(6, 20, 48, 52, 53). After the endogenous PKA catalytic
subunit in BLMVs was removed by cAMP and washed away, the BLMVs were
incubated in the presence or absence of the HT-31 peptide. BLMVs were
then incubated with the exogenous PKA catalytic subunit to reconstitute
the membrane-anchored PKA holoenzyme. BLMVs thus treated were tested
for cAMP-stimulated phosphorylation of
Na+-K+-ATPase on the membrane. As shown in Fig.
9, the HT-31 peptide decreased the level
of Na+-K+-ATPase phosphorylation by
membrane-anchored PKA, indicating the involvement of AKAP in the
regulation of Na+-K+-ATPase phosphorylation by
cAMP/PKA.
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Time course of
Na+-K+-ATPase
phosphorylation by endogenous and exogenous PKA.
We examined the time course of phosphorylation of the
Na+-K+-ATPase -subunit (92-kDa protein) by
membrane-anchored endogenous PKA and by exogenously added PKA. The
phosphorylation reaction was terminated by the addition of TCA at
various time points after the addition of [
-32P]ATP
and cAMP (Fig. 10,
A-C). Furthermore, we measured the intensity of 92-kDa
signals on autoradiogram films by computer-assisted image analysis
(Fig. 10D). Phosphorylation of
Na+-K+-ATPase by endogenous PKA was cAMP
dependent, became clearly detectable in 5 s, and reached a plateau
level within 15 s (Fig. 10, B and D).
Phosphorylation by exogenous PKA was also detectable in 5 s, but
reached a plateau level much more slowly, i.e., by 30 s (Fig. 10,
C and D).
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Downregulation of
Na+-K+-ATPase
activity via phosphorylation of the enzyme by PKA anchored to BLMVs.
We further examined the effect of phosphorylation of the ATPase by
membrane-anchored PKA on the activity of the enzyme (Fig. 11). When
Na+-K+-ATPase was phosphorylated by incubating
the BLMVs with ATP and cAMP, the ATPase activity was decreased to
~80% of that of the control. The addition of
PKI-(5-24) peptide along with cAMP blocked this
cAMP-dependent inhibition of Na+-K+-ATPase,
whereas PKI-(5-24) peptide itself did not have any
stimulatory or inhibitory effect in this experimental system. Thus the
activity of Na+-K+-ATPase was quickly
downregulated by the action of membrane-anchored PKA in response to the
addition of cAMP, in other words, in response to an increase in the
cAMP level.
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DISCUSSION |
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Salivary glands consist of acini, striated ducts, and convoluted
tubules. Since the parotid gland contains many acinar cells, it is a
convenient model for the study of the mechanism and regulation of
epithelial fluid and electrolyte secretion. Generally, fluid secretion
is regulated by the combined action of four membrane transport systems,
i.e., Na+-K+-ATPase,
Na+-K+-2Cl cotransporter,
Ca2+-activated K+ channel in basolateral
membranes, and an apical conductive pathway for Cl
,
presumably involving Ca2+-activated Cl
channels (51). Thus Na+-K+-ATPase
plays a vital role in maintaining homeostasis of epithelial fluid and
electrolyte secretion as well as in generating membrane potential in
excitable tissues.
In the present study, we investigated the regulatory effect of cAMP on
Na+-K+-ATPase of rat parotid gland acini via
the endogenous PKA anchored on the cell membranes.
Na+-K+-ATPase-rich BLMVs used in this study
were purified from rat parotid glands and did not contain any
significant amounts of free catalytic subunit of PKA. Therefore,
without the addition of cAMP, only faint bands of 32P
phosphoprotein were detected on the autoradiogram (Fig. 1). However,
with the addition of cAMP, protein phosphorylation occurred mainly on
four proteins (300 kDa, 180 kDa, 92 kDa, and 50 kDa), including the
Na+-K+-ATPase -subunit (92 kDa) in the
BLMVs, and was inhibited by PKI-(5-24) peptide,
indicating that the BLMV preparation contained membrane-anchored
endogenous PKA in its holoenzyme form.
The phosphorylation profile of BLMV proteins obtained with the membrane-anchored PKA in the presence of cAMP (Fig. 1A) was similar to that of the exogenous PKA catalytic subunit, except for the 23-kDa protein phosphorylated by the latter system (Fig. 1B). This difference might be due to the difference in availability and specificity of the catalytic subunit of PKA for this protein substrate in the endogenous system and in the exogenous system; i.e., the 23-kDa protein might be distant from the anchored endogenous PKA compared with the other proteins, 300 kDa, 180 kDa, 50 kDa, and Na+-K+-ATPase. Therefore, the endogenous catalytic subunit, which was released from the regulatory subunit anchored on the membrane with the addition of cAMP did not adequately phosphorylate the 23-kDa protein during the relatively short period of incubation (30 s). However, the 23-kDa phosphorylation by endogenous PKA indeed became detectable when the incubation time was prolonged (not shown).
Among all proteins in the BLMV, the 32P-labeled 92-kDa
protein, having a molecular weight identical to that of the -subunit of Na+-K+-ATPase, was the only protein reactive
with an antiserum raised against the
1-subunit of
Na+-K+-ATPase of rat (Fig. 2). This polyclonal
antibody was specific for the
1-isoform of the
-subunit (24-27) and inhibited the
Na+-K+- ATPase activity (24).
Therefore, Na+-K+-ATPase is thought to be
phosphorylated by endogenous PKA anchored on the membrane. This was
confirmed by the analysis of V8 proteolytic products from the 92-kDa
protein. The 32P signal was exclusively detected on the
proteolytic peptide fragment of 20 kDa in peptides detected with the
anti-
1 antibody (Fig. 3), indicating that only the
1-subunit of Na+-K+-ATPase was
phosphorylated even if other proteins with the same molecular weight
were contained in the 92-kDa band.
It is worth noting that phosphorylation of BLMV proteins was carried out in the presence of 0.0025% Triton X-100, since Na+-K+-ATPase is a typical membrane-bound enzyme with eight transmembrane domains (41). This detergent concentration was slightly higher than the concentration (0.00125%) that gave the maximum ATPase activity, and it did not denature the Na+-K+-ATPase or change the membrane structure. Although a much higher concentration of Triton X-100 (e.g., 0.05% [15]) is very successful for the purpose of protein phosphorylation, such a high concentration caused a vast increase in the nonspecific protein phosphorylation and entire loss of the Na+-K+-ATPase activity (unpublished observations). As mentioned formerly, prolonged incubation also caused an increase in phosphorylation of the nonspecific substrates such as the 23-kDa protein. Thus the detergent concentration as well as the incubation time period is a quite important factor in examining the regulation of membrane-bound enzymes by protein phosphorylation.
The catalytic subunit of PKA is assumed to be associated with the BLMV by binding to its regulatory subunit, since Scott et al. (37-39) and Coghlan et al. (11-13) reported the existence of an AKAP that anchored the regulatory subunit to the membrane. We tested this assumption by preincubating the BLMVs with cAMP (Fig. 4). The PKA catalytic subunit was removed from the BLMVs by dissociating it from its regulatory subunit, indicating that the regulatory subunit, but not the catalytic subunit, was directly anchored to the membrane, and supporting the idea that AKAP is involved in the in vivo phosphorylation of Na+-K+-ATPase by PKA in the rat parotid gland.
Therefore, by performing RT-PCR with primers for AKAP subtypes that are
expressed in other rat tissues (11-13, 29, 39), we
examined whether or not AKAP mRNA was present in the acinar cells of
the rat parotid gland. By this procedure, we detected three subtypes of
AKAP mRNAs: those for AKAP-95, AKAP-150, and AKAP-220 (Fig. 6). The
mRNAs for AKAP-95 and AKAP-220 were much less abundant than for
AKAP-150. We also examined AKAP proteins in BLMVs and detected AKAP-150
protein by immunoblot analysis (Fig. 7). AKAP-95 and AKAP-220 proteins
were present in a less than detectable amount, if present at all. The
presence of functional AKAP-150 protein in the BLMVs was further
evidenced by the result of an RII overlay assay for AKAP: the exogenous
RII regulatory subunit bound to the AKAP-150 blotted onto the filter
(Fig. 8). Furthermore, AKAP-150 was coimmunoprecipitated with the RII
subunit by the anti-RII antibody (data not shown), i.e., the AKAP-150 in BLMV was associated with the endogenous RII subunits of PKA. These
results indicate that the PKA RII regulatory subunit was associated
with the basolateral membrane via AKAP-150 and that AKAP-150 was
functional for anchoring PKA in rat parotid gland acinar cells. The
presence of functional AKAP protein, AKAP-150, in BLMVs, together with
the fact that incubation of BLMVs with cAMP released free catalytic
subunits of PKA, further supports the idea that AKAP is involved in the
in vivo phosphorylation of the Na+-K+-ATPase
-subunit by PKA in the rat parotid gland.
It has already been reported that Na+-K+-ATPase
was phosphorylated in vitro by the PKA catalytic subunit exogenously
given (3, 17). Na+-K+-ATPase was
also shown to be phosphorylated in vivo by endogenous PKA when cells
were treated with forskolin (2, 17) or membrane-permeable cAMP analogs (9, 18). Nevertheless, the actual process by which Na+-K+-ATPase is phosphorylated and
regulated in cells by hormones or neurotransmitters that elevate the
intracellular cAMP level is not yet definitely clear. Since the time
period of cAMP elevation in cells by signaling molecules is generally
thought to be rather short, if a certain enzyme was regulated by
PKA-dependent phosphorylation in response to the transient and
short-term increase in cAMP level, it should be quickly and
specifically phosphorylated by the kinase. As we demonstrated here,
rapid phosphorylation of the -subunit of
Na+-K+-ATPase in BLMVs by membrane-anchored
endogenous PKA was indeed the case: the phosphorylation was detectable
within 5 s on cAMP stimulation in the presence of
[
-32P]ATP and reached the plateau level in 15 s
(Fig. 10). It should be emphasized that the
-subunit phosphorylation
rate by the endogenous PKA in the early phase, for example, during the
first 5 s of incubation, was much faster than that of the
exogenously added PKA. In these experiments, the activity per one assay
of the endogenous PKA was estimated to be 0.2-0.3 U/8 µg of
BLMVs (Table 2), and that of the exogenously added PKA was estimated to
be 16 U/8 µg of BLMVs.
Furthermore, in the absence (or on the removal) of cAMP, the PKA
catalytic subunit was able to reversibly bind to the BLMV via its
regulatory subunit/AKAP complex, as was demonstrated in Fig. 5. This
PKA anchoring process was clearly inhibited by the PKI peptide. The PKI
peptide is a part of the regulatory subunit of PKA and binds to the
catalytic subunit of PKA at the same site where the regulatory
subunit/AKAP complex binds to the catalytic subunit, but PKI does not
contain the amino acid sequence needed for binding with AKAP. Thus in
the experimental system employed in Fig. 5, PKI blocked the
phosphorylation of the -subunit of Na+-K+-ATPase by inhibiting the anchoring of
PKA to the BLMV and not by directly preventing the phosphorylation
reaction, since PKI was washed out before the phosphorylation reaction,
indicating the functional implication of AKAP in the regulation of
Na+-K+-ATPase activity.
We also examined the effect of HT-31 peptide on the cAMP/PKA-dependent Na+-K+-ATPase phosphorylation. The HT-31 peptide has an amino acid sequence identical to the part of AKAP that binds with the RII subunit, and therefore the peptide can competitively inhibit the binding of RII to AKAP (6, 20, 48, 52, 53). Incubation of BLMVs with the HT-31 peptide resulted in a decrease in cAMP-stimulated Na+-K+-ATPase phosphorylation by membrane-bound PKA (Fig. 9), also supporting the involvement of AKAP in the regulation of Na+-K+-ATPase via its cAMP/PKA-dependent phosphorylation.
Although the rate of phosphorylation of the
Na+-K+-ATPase -subunit by the endogenous PKA
was much faster than that of the exogenously added PKA, the maximal
levels of the
-subunit phosphorylation by AKAP-associated endogenous
PKA (0.2-0.3 units) and exogenous PKA (16 units) were calculated
to be 23.1 and 32.1 fmol Pi incorporated per milligram of
protein of BLMV, respectively (Table 2). Taking into consideration that
a certain part of AKAP-associated PKA might be for phosphorylating
membrane proteins other than Na+-K+-ATPase, the
above values indicate that PKA associated with
Na+-K+-ATPase-specific AKAP efficiently and
quickly phosphorylated the
-subunit in the presence of an adequate
amount of cAMP. It is likely that the level of PKA-dependent
protein phosphorylation is regulated by the level of cAMP rather than
by the arbitrary phosphorylation efficiency of PKA once activated.
Therefore, the results might suggest that ~70% (23.1/32.1) of the
Na+-K+-ATPase molecules on the BLMV are
associated with the PKA holoenzyme via AKAP. The
Na+-K+-ATPase activity was downregulated by
phosphorylation of the
-subunit by endogenous PKA, as its activity
was decreased to 80% by the action of endogenous PKA (Fig. 11).
Bertorello et al. (3) reported that maximally 40% of the
Na+-K+-ATPase activity was inhibited when it
was phosphorylated in vitro in the presence of an excess amount of the
PKA catalytic subunit exogenously added. Our result of 20% inhibition
of the Na+-K+-ATPase activity by endogenous
PKA, where ~70% of the
-subunit was estimated to be
phosphorylated, might be also compatible with the result of 40%
inhibition by Bertorello et al. (3).
The results obtained in the present study indicated that, on the
addition of cAMP, the -subunit of
Na+-K+-ATPase in BLMV was quickly and
selectively phosphorylated by the membrane-anchored endogenous PKA with
a high efficiency, resulting in the inhibition of its activity. The
presence of a functional AKAP subtype, AKAP-150, in BLMVs was also
demonstrated. By the removal of cAMP, the free catalytic subunit of PKA
reversibly became anchored to the BLMV via binding with the regulatory
subunit/AKAP complex. PKI blocked the
Na+-K+-ATPase phosphorylation by inhibiting
this process of PKA anchoring to the membrane, even though it was not
contained in the phosphorylation reaction mixture. Inhibition of RII
association with AKAP by the HT-31 peptide also resulted in a decrease
in membrane-bound PKA-dependent phosphorylation of
Na+-K+-ATPase. Thus
Na+-K+-ATPase might be downregulated in vivo by
signaling molecules that employ cAMP as an intracellular messenger via
the action of PKA anchored on the membrane by AKAP rather than by the
free cytosolic ones with a broad substrate specificity.
The functional relevance of the interaction between Na+-K+-ATPase and the AKAP-150 subtype is of great interest and should be further elucidated because Na+-K+-ATPase is an essential component in maintaining homeostasis of epithelial fluid and electrolyte secretion, as well as in generating membrane potential in excitable tissues such as neurons, and because AKAP-150 is reported to be abundant in Purkinje cells and in neurons of olfactory bulb, basal ganglia, cerebral cortex, and other forebrain regions (19).
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ACKNOWLEDGEMENTS |
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We thank Dr. R. James Turner for helpful suggestions and encouragement during the course of this work.
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FOOTNOTES |
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This work was supported in part by Ministry of Education, Science, and Culture, Japan Grants-in-Aid 07672027, 10671748, and 11671851 and by a grant from the Miyata Foundation, Meikai University.
Address for reprint requests and other correspondence: K. Kurihara, Dept. of Oral Physiology, School of Dentistry, Meikai Univ., 1-1 Keyaki-Dai, Sakado-Shi, Saitama 350-0283, Japan (E-mail: kkinji{at}dent.meikai.ac.jp).
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.
Received 15 July 1999; accepted in final form 12 June 2000.
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