From the Kidney Disease Program, Department of
Medicine, University of Louisville, Louisville, Kentucky
40202 and ¶ Louisville Veterans Affairs Hospital,
Louisville, Kentucky 40206
Received for publication, November 19, 2002, and in revised form, December 17, 2002
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ABSTRACT |
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Parathyroid hormone inhibits sodium-phosphate
cotransport in proximal renal tubule cells through activation of
several kinases. We tested the hypothesis that the activity of these
kinases was coordinated by an A kinase anchoring protein (AKAP) by
demonstrating that the type II sodium-phosphate cotransporter (NaPi-4)
physically associated with an AKAP and that this association was
necessary for regulation of phosphate transport by parathyroid hormone. Immunoprecipitation with anti-NaPi-4 antiserum and glutathione S-transferase pull-down with GST-NaPi-4 showed that NaPi-4
associated with AKAP79, protein kinase A catalytic and regulatory
subunits, and the parathyroid hormone receptor in opossum kidney
cells. When the regulatory subunit of protein kinase A was uncoupled from the AKAP by a competing peptide, parathyroid hormone lost the
ability to inhibit phosphate transport. This result was confirmed by
co-transfecting HEK293 cells with the sodium-phosphate cotransporter and wild type AKAP, a mutant AKAP79, or the empty vector. 8-Bromo-cAMP was able to inhibit phosphate transport in cells expressing the wild
type AKAP79 but not empty vector or mutant AKAP79. We conclude that
parathyroid hormone inhibits proximal renal tubule
sodium-phosphate cotransport through a signaling complex dependent upon
an AKAP.
Phosphate balance is maintained primarily by regulation of
sodium-dependent phosphate reabsorption by type II
sodium-phosphate cotransporters in the proximal renal tubule (1-4).
Parathyroid hormone (PTH)1
inhibits the function and expression of type II sodium-phosphate cotransporters through activation of several signal transduction pathways (5-7). Stimulation of PTH receptors leads to activation of
protein kinase A (PKA) through coupling to the stimulatory guanine
nucleotide regulatory protein Gs. Simultaneously, PTH receptor stimulation activates protein kinase C (PKC) through coupling
to Gq. Direct activation of either PKA or PKC causes inhibition of sodium-dependent phosphate transport in renal
proximal tubule, similar to the effect of PTH itself. The significance of this dual signaling is not clear; nor are the mechanisms for coordinating the two pathways.
Activation of similar signaling pathways by other stimuli such as
dopamine does not result in identical regulation of sodium-phosphate cotransport or cotransporter expression (8). The mechanism for this
agonist-specific effect on phosphate despite activation of ostensibly
similar second messengers is not understood. Several mechanisms to
explain agonist-specific functional effects have been proposed,
including activation of different enzyme isoforms, coupling to
different G proteins, or activation of different unrecognized signals.
Recently, interest has focused on the role of scaffolding and anchoring
proteins as a means to compartmentalize and individualize agonist
effect on intracellular processes (9-11). A kinase anchoring proteins
(AKAPs) are a family of proteins that express a well conserved sequence
that binds to the regulatory subunit of PKA (PKA RII) (12-15). Each
AKAP also expresses a sequence that targets it to a specific
subcellular position and so directs signal transduction to a unique
locale. Some AKAPs, such as AKAP79 and gravin, bind multiple kinases
and phosphatases (16-20). AKAPs play a role in the regulation of
multiple transport proteins, including sodium channels (21), potassium
channels (22), and calcium channels (23-25). Recently, Klussmann
et al. (26), using a competing peptide to block AKAP binding
to PKA regulatory subunits, demonstrated that forskolin activated PKA
but could not stimulate translocation of water channels to the apical
membrane. Thus, tethering of PKA to a specific cellular site by an AKAP
was critical to the ability of hormone-activated PKA to regulate water
transport. Lamprecht et al. (27) reported that ezrin, an
AKAP (28), regulates cAMP inhibition of NHE3, the sodium-hydrogen
exchanger, in opossum kidney (OK) cells.
These findings suggested to us the possibility that PTH regulated the
expression and function of the sodium-phosphate cotransporter through a
signaling complex assembled by an AKAP. We tested this hypothesis by
examining the physical association of the sodium-phosphate cotransporter with an AKAP and other signal transduction proteins. We
also examined the effect of disruption of the AKAP-PKA binding on PTH
regulation of sodium-phosphate cotransport. These studies were
performed in OK cells, a continuous cell line derived from the Virginia
opossum that exhibits several characteristics of mammalian renal
proximal tubule including a polarized morphology, apical expression of
sodium-phosphate cotransporters (NaPi-4), and regulation of phosphate
uptake by PTH, PKA, and PKC.
Wild type OK cells were a generous gift of Dr. Steve Scheinman
(Syracuse Health Science Center, Syracuse, NY). AKAP79/150 affinity-purified polyclonal antibody and recombinant PKA RII protein
were a generous gift from Dr. Christine Loh (ICOS Corp., Bothell, WA)
(29). Dr. Heini Murer and Dr. Jurg Biber (University of Zurich, Zurich,
Switzerland) generously provided the cDNA for NaPi-4. Dr. John D. Scott (Oregon Health Science University, Portland, OR) kindly provided
the wild type and dominant negative mutant AKAP79 cDNA constructs
(25).
Cell Culture--
OK cells were grown to confluence in
monolayers in 175-cm2 flasks in culture medium consisting
of Eagle's medium with Earle's salts (Invitrogen) supplemented
with 10% heat inactivated fetal calf serum, 4 mM
glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin, pH 7.4, as previously described (30). The cells from passages 82-88 were used
for experiments at 100% confluence.
Immunoprecipitation--
The OK cell plates were washed with
Hanks' balanced saline solution twice and once with 50 mM
mannitol, 5 mM Tris-HCl buffer, pH 7.4. The cells were
lysed with 50 mM Tris-HCl buffer, pH 7.4, and the lysate
(100 µg of protein) was incubated with preimmune serum for 1 h
at room temperature on a rotator. 10 µl of Protein A-Sepharose beads
were added and incubated for another 1 h at room temperature. The
sample was centrifuged for 5 min at 5000 rpm in a microcentrifuge. The
pellet was discarded, and the supernatant was incubated with 10 µl of
antiserum against NaPi-4 (30) or antibody against AKAP79/150 overnight
at 4 °C. 10 µl of Protein A-Sepharose beads were added and
incubated on a rotator for 1 h at 4 °C. The samples were
centrifuged for 5 min at 5000 rpm. The pellet was washed with Hanks'
balanced saline solution three times and subjected to SDS-PAGE.
Immunoprecipitation Using Seize-X Beads--
The anti-NaPi-4
antiserum (antibody) was immobilized on Protein A-Sepharose beads using
the Seize X Protein A immunoprecipitation kit (Pierce) according to the
manufacturer's protocol. Briefly, 0.4 ml of the ImmunoPure plus
Immobilized Protein A-Sepharose beads were washed twice with
binding/wash buffer (0.14 M NaCl, 0.008 M
Na2PO4, 0.002 M potassium
phosphate, 0.01 M KCl, pH 7.4). 0.4 ml of 1:1 diluted (in
binding buffer) antiserum was added to the beads and incubated on a
rotator for 1 h at room temperature. The beads were washed three
times with 0.5 ml of binding buffer, and 0.4 ml of the binding buffer
was added. 2 mg of the disuccinimidyl suberate was dissolved in 80 µl
of Me2SO, and 25 µl of disuccinimidyl suberate reagent
was added to the beads and incubated on a rotator for 1 h at room
temperature. The beads were centrifuged and washed three times with 0.5 ml of quenching buffer (25 mM Tris-HCl, 0.15 M
NaCl, pH 7.2). The beads were then washed with elution buffer (Pierce)
until no protein was detected at 280 nm in the eluent. The beads were
then washed with the quenching buffer twice. OK cells were lysed in
immunoprecipitation lysis buffer containing 20 mM Tris-HCl,
pH 7.4, 137 mM NaCl, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM 4-(2-aminoethyl)-benzenesulfonyl
fluoride, 20 mM NaF, 1 mM
Na2VO3, 1% Nonidet P-40, and 1% Triton X-100.
The lysate was centrifuged at 10,000 × g for 10 min.
The supernatant (200 µg of protein) diluted to 0.4 ml with binding
buffer was added to the beads and incubated overnight at 4 °C. The
beads were centrifuged, and the flow-through was collected and stored at Immunoblot Assay--
The immunoprecipitated OK cell lysates
were solubilized in Laemmli sample buffer, subjected to 10% SDS-PAGE,
and transferred electrophoretically to either nitrocellulose
(Trans-Blot; Bio-Rad) or polyvinylidene difluoride (PolyScreen;
PerkinElmer Life Sciences) as described previously (30). After
blocking, the membranes were incubated overnight at 4 °C with
primary antibodies (NaPi-4, 1:5000; AKAP79/150, 1:5000; PTH receptor,
1:1000 dilution) in 5% milk in TTBS. Location of specific antibodies
was detected by incubation with peroxidase-labeled goat anti-rabbit IgG
at 1:10,000 dilution for NaPi-4 and AKAP79/150 and anti-mouse IgG at a
1:2000 dilution for PTH receptor in 5% milk in TTBS, followed by
development with enhanced chemiluminescence (Renaissance; PerkinElmer). The bands imaged by chemiluminesence were analyzed by
densitometry. The films were scanned using a Personal Densitometer SI
(Amersham Biosciences).
RII Overlay Assay--
Proteins were separated on 10% SDS-PAGE
and transferred to nitrocellulose. AKAPs were detected using
recombinant RII Phosphorylation Assay--
The immunoprecipitates were washed
three times with Hanks' balanced saline solution and incubated for
2 h, 1 h, and 30 min in 30 µl of kinase buffer containing
25 mM HEPES, 25 mM Preparation of GST and GST-NaPi-4-Glutathione-Sepharose
Beads--
NaPi-4 full-length or deletion mutant (DM-4) was shuttled
from NaPi-4 pCR2.1 plasmid into the EcoRI site of
GST-pGEX-3. The ligated GST-pGEX3-NaPi-4 cDNA was transformed into
E. coli DH5 GST Pull-down Assay--
OK cells grown on six-well plates
(Corning) were washed with Hanks' balanced saline solution and lysed
with 200 µl of immunoprecipitation lysis buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% (v/v)
Triton X-100, 0.5% (v/v) Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 20 mM sodium orthovanadate, 20 mM NaF, 5 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 21 µg/ml aprotinin, and 5 µg/ml leupeptin. The lysates were precleared by incubation with GST-glutathione-Sepharose beads for
2 h at 4 °C. Following this preclearing step, GST-NaPi-4, GST
vector, or glutathione-Sepharose beads were added to the lysates and
incubated at 4 °C overnight on a rotator. The beads were washed three times with Krebs Plus buffer, and 40 µl of 2× Laemmli buffer was added to each tube. The samples were boiled for 5 min and then
subjected to 10% SDS-PAGE. Proteins were transferred onto nitrocellulose for immunoblot analysis.
Transfection--
HEK293 cells were maintained in DMEM (Cellgro,
Herndon, VA) supplemented with 10% fetal calf serum and 1% penicillin
and streptomycin. The cells were split a day prior to transfection at a
concentration such that the cells were 60-80% confluent. HEK293 cells
were transfected with the indicated plasmids using LipofectAMINE
(Invitrogen) according to the manufacturer's protocol. The cells were
washed with OPTI medium 24 h before treatment with 8-Br-cAMP.
Phosphate Uptake--
Phosphate transport was measured by
determination of radiolabeled phosphate uptake as previously described
(30). Each assay was performed in triplicate and averaged, and the mean
was considered as a single data point.
Statistics--
Data are shown as mean ± S.E. The
n values shown represent the number of separate experiments.
Each experiment was done in triplicate. p value is
calculated using SigmaStat software utilizing Student's t
test. A p value less than 0.05 was a priori
considered statistically significant.
Association of NaPi-4 and an AKAP--
To determine whether any
AKAPs associated with NaPi-4, we immunoprecipitated OK cell membranes
with antiserum to NaPi-4 and performed immunoblot analysis for
AKAP79/150 and AKAP149 (Fig. 1). The
AKAP79/150 antibody identified bands at 79 and 60 kDa. AKAP149
monoclonal antibody did not identify a band. Ezrin antibody inconsistently and only faintly identified a band at the 79-kDa position (data not shown). Immunoblots of OK cell lysates and crude
membrane preparations for AKAP79/150 are shown in Fig.
2. Only very faint bands are seen in the
whole cell lysates; however, the crude membranes exhibited strong bands
at 60 kDa and a 79/80-kDa doublet. We therefore focused our attention
on AKAP79. To determine which of the bands identified by the AKAP79/150
polyclonal antibody in NaPi-4 immunoprecipitates were AKAPs, we
performed an RII overlay assay on the immunoprecipitated proteins in
the presence and absence of a competing peptide,
DLIEEAASRSVDAVIEQVKAAGAY, or a nonfunctional analogue,
DLIEEAASRPVDAVIEQVKAAGAY (Fig. 3). In this assay, the immunoblot
membrane is incubated in buffer containing the regulatory subunit of
PKA (RII), which should bind any AKAP, followed by immunoblot for RII.
The RII overlay assay identified the 79-kDa protein (left
lane). The addition of the competing peptide virtually
abolished RII binding (middle lane), whereas the
addition of the inactive analogue had no effect on RII binding
(right lane). The RII overlay assay also
demonstrated significant nonspecific binding as noted by the heavy
lower molecular weight bands. The intensity of these bands was not
diminished by either the competing peptide or the nonfunctional
analogue. There was an additional band identified at about 150 kDa by
the RII overlay assay; however, the intensity of this band was
diminished by both the competing peptide and the nonfunctional
analogue. These findings suggest that in the NaPi-4 immunoprecipitates, only the 79-kDa band is specifically an RII-binding protein
(i.e. an AKAP).
To exclude the possibility of a nonspecific interaction between the
polyclonal antiserum and the OK cell membrane proteins, we
immunoprecipitated OK cell membranes with preimmune anti-NaPi-4 serum,
immune serum, and immune serum preincubated overnight with cognate
peptide. Subsequent immunoblot with the ICOS AKAP79/150 antibody
demonstrated that the preimmune serum did not immunoprecipitate AKAP79
and that preincubation of the immune serum with peptide markedly
decreased the ability to immunoprecipitate AKAP79 (Fig. 4). Note in the accompanying panel that
preincubation of the immune serum with immunizing peptide markedly
diminished the ability of the antiserum to immunoprecipitate NaPi-4. To
confirm the AKAP79/NaPi-4 association, we immunoprecipitated OK cell
membranes with the ICOS 79/150 antibody and blotted for NaPi-4. We
detected NaPi-4 in the ICOS antibody immunoprecipitates (Fig.
5). Immunoprecipitation with both NaPi-4
and AKAP79/150 antibody resulted in several nonspecific bands,
especially at molecular sizes of 50 kDa or less. We therefore repeated
the NaPi-4 immunoprecipitation using antibody covalently linked to
beads. Using this technique, as shown in Fig.
6, we confirmed the fact that NaPi-4
antibody immunoprecipitates an AKAP recognized by AKAP79 antibody.
Identification of Proteins That Co-immunoprecipitate with
NaPi-4--
We next determined whether NaPi-4 and the AKAP associated
with other cellular signaling components. We reasoned that if an AKAP
were present, the catalytic as well as the regulatory subunit of PKA
would be present in the NaPi-4 immunoprecipitates. We
immunoprecipitated OK cells with NaPi-4 antiserum after preclearing the
lysates with preimmune serum and then incubated the immunoprecipitate
with radiolabeled ATP in the presence or absence of exogenous cAMP. Autoradiography of the separated proteins demonstrated three
phosphorylated bands when cAMP was present in the assay but not when
cAMP was omitted from the assay (Fig. 7).
The control experiments demonstrated that under these reaction
conditions, activated PKA was capable of phosphorylating myelin basic
protein and that this phosphorylation was blocked by the PKA inhibitor,
IP20. If the catalytic subunit were present, we reasoned that
the regulatory subunit of PKA would also be present in NaPi-4
immunoprecipitates. Fig. 8 shows that immunoprecipitation with NaPi-4 or AKAP79 antibody blotted positively for the regulatory subunit, PKA RII.
NaPi-4 is regulated by hormone-stimulated PKC as well as PKA.
Additionally, AKAP79/150 is capable of binding protein kinase C and
phosphatases along with PKA. Therefore, we examined the NaPi-4
immunoprecipitates for the presence of several protein kinase C
isoforms and phosphatases. Immunoblots of immunoprecipitates separated
by SDS-PAGE failed to reveal any PKC isoforms, including PKC
In preliminary experiments (data not shown), we demonstrated the
presence of the PTH receptor in NaPi-4 immunoprecipitates by
two-dimensional gel separation and mass spectroscopy of tryptic digests, showing 30% peptide coverage. Since PTH is one of the major
physiologic regulators of sodium-phosphate cotransport, we examined the
NaPi-4 immunoprecipitates for evidence of the PTH receptor by
immunoblot analysis of one-dimensional gels. As seen in Fig.
9, immunoblot for the PTH receptor
revealed the presence of a single band in NaPi-4 immunoprecipitates.
This band was present but of far lesser density in immunoprecipitates
from preimmune serum.
The ability of NaPi-4 antibody to immunoprecipitate the PTH receptor
and AKAP79/150 suggests that all three proteins can be found in renal
brush border membranes (BBM). Therefore, we prepared BBM from OK cells
grown on permeable supports and blotted for NaPi-4, the PTH receptor,
and AKAP79/150. As shown in Fig. 10, there was about 6-8-fold enrichment of NaPi-4 expression and the PTH
receptor in the BBM when compared with expression in crude cell
preparations; however, there was no difference in AKAP79/150 expression
between lysates and BBM.
We corroborated the association of NaPi-4 with AKAP79/150 and the PTH
receptor by performing GST pull-down assay on OK cell lysates using a
full-length NaPi-4-GST fusion protein linked to glutathione-Sepharose
beads. Immunoblot analysis of the separated proteins from the GST
pull-down assay confirmed the presence of AKAP79/150 and the PTH
receptor (Fig. 11). We confirmed that
the band identified by the AKAP79/150 antibody was an AKAP by an RII overlay assay, demonstrating RII binding of the same band. Similar analysis of pull-downs with GST vector alone failed to reveal any of
the proteins. We performed a pull-down with a NaPi-4-GST fusion
protein, where the C-terminal of the NaPi-4 was deleted (DM-4).
Pull-downs using DM-4 showed the expected absence of staining for
NaPi-4 using the C-terminal NaPi-4 antiserum. Immunoblots using
an N-terminal antibody confirmed the actual presence of NaPi-4 in the
pull-downs (data not shown). The C-terminal deletion mutant GST
pull-downs showed a marked decrease in the amount of AKAP79 and a
corresponding decrease in the intensity of the RII overlay assay. These
data corroborate our immunoprecipitation data, demonstrating a physical
association between NaPi-4, PKA RII, the PTH receptor, and an AKAP
similar to the human AKAP79/150. They also suggest that the C terminus
of NaPi-4 is required for full binding of the AKAP.
Functional Significance of the AKAP-NaPi-4 Association--
To
determine whether the AKAP-NaPi-4 association was of functional
significance, we uncoupled PKA from AKAPs by incubating OK cells with
the blocking peptide stearated at its N terminus or with an inactive
stearated analogue for 30 min at 37 °C. The cells were then treated
with either vehicle or with 10
To confirm that AKAP79 (or a similar AKAP) is involved in the
regulation of NaPi-4, we co-transfected HEK293 cells with NaPi-4 cDNA and with the wild type or mutant AKAP79 or vector alone (Fig. 13). 48 h post-transfection, the
cells were treated with vehicle or 10 Renal proximal tubule phosphate transport is regulated by many
physiologic stimuli through multiple signaling pathways. Dietary phosphate deprivation, insulin, and growth factors increase phosphate reabsorption, whereas dietary phosphate excess, PTH, and dopamine decrease phosphate reabsorption. Under typical physiologic conditions, dietary ingestion of phosphate exceeds demands, necessitating net renal
excretion of phosphate. PTH, a major regulator of renal phosphate
homeostasis, inhibits phosphate transport through activation of PKA and
PKC, resulting in acute and chronic decrease in apical membrane
expression of type II sodium-phosphate cotransporters. Other activators
of PKC such as epinephrine and angiotensin II can actually increase,
not decrease, phosphate reabsorption in this tubule segment. The
complex nature of this regulatory process suggested that PTH-specific
phosphaturia may be accomplished by compartmentalization of
PTH-stimulated signaling pathways with target substrates. An AKAP
seemed a likely candidate for this function, as several AKAPs have been
reported to bind multiple kinases and phosphatases. Additionally,
although both PKA and PKC can mimic the phosphaturic action of PTH,
several studies have suggested that the role of PKA is more important
than that of PKC. We reasoned that an AKAP could bind PKA and possibly
PKC and phosphatases in close proximity to the OK cell type II
sodium-phosphate cotransporter, thus ensuring specificity of action.
Our studies support our hypothesis that PTH regulation of
sodium-phosphate cotransport is dependent on an AKAP. First, we demonstrated that NaPi-4 is physically associated with an AKAP. Immunoprecipitation of OK cell lysates with polyclonal NaPi-4 antiserum
yielded the presence of an AKAP recognized by polyclonal antibody
directed against AKAP79/150. Immunoprecipitation with either preimmune
serum or antiserum preincubated with NaPi-4 peptide failed to
demonstrate the AKAP79/150 band. This same band also stained positively
in an RII overlay assay, confirming that the protein recognized by
AKAP79/150 antibody also could bind the regulatory subunit of PKA,
a cardinal feature of AKAPs.
We next identified other components of this signaling complex. We
demonstrated the presence of the catalytic subunit of PKA along with
the regulatory subunit in NaPi-4 immunoprecipitates by the ability of
the immunoprecipitates to support phosphorylation after the addition of
cAMP and radiolabeled ATP. By immunoblot analysis, we showed the
presence of the PTH receptor and protein phosphatase 2b, but not any
PKC isoforms, in NaPi-4 immunoprecipitates.
Because immunoprecipitation reactions can result in nonspecific protein
interactions, even when appropriate controls are performed, we
confirmed the association of NaPi-4, AKAP79/150, and the PTH receptor
by performing a GST pull-down assay. These assays demonstrated the
presence of AKAP79/150 and the PTH receptor in GST pull-downs using
GST-NaPi-4-linked glutathione beads but not in pull-downs using GST
vector beads. Based on these identical results using two different
methodologies, we conclude that NaPi-4 and the PTH receptor are linked
in close proximity to each other in a signaling complex containing an
AKAP. This conclusion is supported by a recent abstract presented by
Gisler et al. (32). Using a yeast two-hybrid assay, they
demonstrated that NaPi-Cap1 acts as a scaffold associating the rat
sodium-phosphate cotransporter, NaPi-2, with an AKAP,
D-AKAP2. These two proteins share 40% sequence homology. Thus, whether the NaPi-4-associated signaling complex involves two
different AKAPs or whether the similarity between the two proteins was
sufficient that the AKAP79 antibody recognized D-AKAP2 remains to be determined.
Finally, we demonstrated the functional importance of the AKAP/PKA RII
association in PTH regulation of phosphate transport by disrupting the
association with a competing peptide. The loss of the ability of PTH to
inhibit phosphate uptake in the presence of the competing peptide, but
not an inactive analog of that peptide, suggests very strongly that the
integrity of the PTH action is dependent on the AKAP/PKA RII
association. It is tempting to conclude that the association of
AKAP79/150 with PKA is the critical interaction; however, the competing
peptide blocks all AKAP/PKA interactions and is therefore not specific
for AKAP79/150. The transfection experiments, however, confirm that
AKAP79 or a similar AKAP could serve in this capacity.
These data raise several questions. First, association of the PTH
receptor with NaPi-4 suggests that PTH receptors are localized on the
apical membrane of proximal tubule. Classical teaching holds that PTH
receptors have a basolateral localization; however, studies suggest the
presence of PTH receptors on apical membranes. Traebert and colleagues
(33) have shown in microperfused proximal renal tubules that apical
perfusion with PTH inhibits phosphate transport similar to basolateral
incubation with PTH. It is likely then that PTH receptors are present
on both membranes. Our data suggest that the PKA-stimulated and
PKC-stimulated actions of PTH may be to some extent spatially
separated. PTH regulates the activity of the Na-K-ATPase, a protein
that is confined to the basolateral membrane and indirectly influences
sodium-phosphate cotransport through regulation of intracellular sodium
concentration. PTH, acting on apical receptors through activation of
PKA, might function primarily to regulate NaPi-4 trafficking in and out
of the apical membrane, whereas PTH activation of basolateral receptors might function to inhibit Na-K-ATPase through activation of both PKA
and PKC.
Our data suggest that the AKAP/PKA association is essential for PTH
inhibition of phosphate transport but do not address the mechanism by
which this is accomplished. Type II sodium-phosphate cotransporters are
constantly shuttled through a one-way path from synthesis, insertion
into the apical membrane, removal into a lysosome, and degradation.
Regulation of the number of active transporters is accomplished by
altering either the rate or the bulk flow of proteins through this
pathway. Acute changes in transporter number occur by increasing the
rate of insertion or removal from the apical membrane. Chronic changes
either increase or decrease the overall quantity of proteins by
changing the rate of synthesis. This theorized hypothesis for
sodium-phosphate cotransporter trafficking is supported by several
pieces of data (34-40). Sodium-phosphate cotransporters have been
identified on endosomal vesicles (38). Pretreatment of OK cells with
colchicine, which blocks microtubule-dependent processes
(39), or C3 exotoxin, which inactivates Rho, a small molecular weight
GTPase implicated in membrane trafficking (40), partially blocks the
ability of PTH to inhibit phosphate transport. Inhibition of the
lysosomal degradative pathways in OK cells by leupeptin increases total
cellular expression of cotransporters (36, 37). In the presence of
leupeptin, PTH inhibits phosphate transport but does not decrease
transporter expression. Confocal imaging reveals that the transporters
have been removed from the apical membrane into a subapical
compartment. These transporters cannot be reinserted into the apical
membrane for reuse, indicating that PTH initiates an irreversible chain
of events leading to protein destruction.
Uncoupling AKAP from PKA could impair normal base-line turnover of
NaPi-4 by interfering with targeting of the sodium-phosphate cotransporter into the apical membrane. This explanation seems unlikely, since the basal rate of phosphate transport was neither increased nor decreased in OK cells incubated with the competing peptide, suggesting that the number of transporters did not change. On
the other hand, basal phosphate uptake was higher in the HEK293 cells
transfected with NaPi-4 and wild-type AKAP79 when compared with cells
transfected with NaPi-4 alone or with NaPi-4 and mutant AKAP79. This
observation does suggest a potential role for the AKAP in membrane
targeting. The AKAP could bring the PTH receptor in close proximity to
PKA, facilitating the ability of PTH to activate PKA. The transfection
experiments in HEK293 cells, where Pi transport was
inhibited by cAMP and not by PTH, however, suggests that the AKAP/PKA
RII interaction is critical for a more downstream step. Co-transfection
of the dominant negative AKAP79, which is unable to bind PKA RII,
blocked inhibition of phosphate transport by 8-Br-cAMP, a direct
activator of PKA not requiring PTH receptor. Another possibility is
that AKAP/PKA RII dissociation could prevent PKA from phosphorylating
an as yet unidentified substrate necessary for regulation of NaPi-4.
Data presented here show that PKA is capable of phosphorylating three
substrates in NaPi-4 immunoprecipitates. In previous experiments, we
have demonstrated that PKA does not directly phosphorylate NaPi-4.
Jankowski et al. (41) have demonstrated that the type II
sodium-phosphate cotransporter exists as a phosphoprotein and that
treatment of OK cells with PTH resulted in a decrease in NaPi-4
phosphorylation. Thus, it is very unlikely that any of the three
phosphorylated substrates are NaPi-4, and the targets for AKAP-directed
PKA phosphorylation remain unknown.
We do not know what regulates the assembly or disassembly of
this signaling complex. Under some clinical conditions, such as dietary
phosphate deprivation, the phosphaturic action of PTH is inhibited.
This effect of diet could potentially be mediated by AKAP/PKA
dissociation. PTH itself could regulate AKAP expression or localization.
In summary, we have produced evidence that PTH regulation of type II
sodium-phosphate cotransporters in proximal renal tubule cells is
dependent on the integrity of a signaling complex composed of the PTH
receptor, PKA regulatory and catalytic subunits, a phosphatase, an
AKAP, and the type II sodium-phosphate cotransporter. The role of this
complex in the regulation of phosphate homeostasis remains to be elucidated.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C for further analysis. The beads were washed 8-10 times with the quenching buffer. The proteins bound to the anti-NaPi-4 beads
were eluted in 190 µl of the elution buffer and collected in 10 µl
of 1 M Tris buffer, pH 9.5. Elution was repeated at least three times. 20 µl of the eluted samples were mixed with 5 µl of
sample buffer (5×) without reducing agents. The sample was boiled for
5 min, and proteins were separated by 10% SDS-PAGE, transferred to
nitrocellulose membrane, and either blotted for NaPi-4 or processed for
RII overlay assay. For PTH receptor immunoblot, the samples were
incubated at 37 °C for 10 min instead of boiling for 5 min.
(ICOS, Bothell, WA). The nitrocellulose membrane was
incubated with 5% milk, 0.2% bovine serum albumin in 20 mM Tris, 145 mM NaCl, pH 7.4 (TBS) at room
temperature for 1 h to inhibit nonspecific binding and
then incubated overnight at 4 °C with 10 nM recombinant
RII protein. The membrane was then washed four times with TTBS and incubated for 2 h at room temperature with affinity-purified
anti-RII antibodies at 1:5000 dilution in 5% milk, 0.2% bovine serum
albumin in TBS. The nitrocellulose membrane was washed again with TTBS four times, and the location of AKAPs was detected by incubation with
peroxidase-labeled goat anti-rabbit IgG at a 1:5,000 dilution in 5%
milk, 0.2% bovine serum albumin in TBS, followed by development with
enhanced chemiluminescence (Renaissance; PerkinElmer Life Sciences).
-glycerophosphate, 25 mM MgCl2, 2 mM dithiothreitol, and
0.1 mM sodium vanadate, pH 7.4, and 3 µl of
[
-32P]ATP (1 mCi/ml) in the presence or absence of 0.5 µM cAMP. A positive control with 0.5 µM
myelin basic protein and 2 µl of active catalytic unit of PKA in 30 µl of kinase buffer and a negative control (positive control plus 2 µl of the PKA inhibitory peptide TTYADFIASGRTGRRNAIHR) were
run simultaneously with the test samples. The reaction was stopped by
adding 1× Laemmli buffer and heating the samples at 95 °C for 5 min. The samples were run on 10% SDS-PAGE, and phosphorylation
was detected by autoradiography.
. Positive colonies were selected by
restriction endonuclease mapping using restriction enzymes
EcoRI, SacI, and MluI for
orientation. The constructs were also confirmed by sequencing.
The GST-pGEX-3-NaPi-4 or GST-pGEX-3-DM-4 plasmid was transformed into
E. coli BL21PlysS, and the expression and purification of
GST and GST-NaPi-4 or GST-DM-4 fusion protein was performed as
previously described by Zu et al. (31). Briefly, a fresh
overnight culture of E. coli BL21 transformed with either
GST-pGEX-3, recombinant GST-pGEX-3-NaPi-4, or GST-pGEX-3-DM-4 was
diluted 1:10 in 2× YT medium containing ampicillin (100 µg/ml) and
grown at 37 °C. After this reached an absorption of 0.6 at 600 nm,
ispropyl-
-D-thiogalactopyranoside was added to a final
concentration of 0.1 mM, and the bacterial growth was
continued for 4 h at 37 °C. Cells were harvested, washed once
with phosphate-buffered saline, and lysed in phosphate-buffered saline
containing 0.5% Triton X-100 and 5 mM Pefabloc by mild sonication on ice. The samples were centrifuged at 10,000 × g for 15 min at 4 °C. Samples (10 ml) of bacterial
supernatant were rocked for 30 min at 4 °C with 800 µl of
glutathione-Sepharose beads previously washed three times by and
resuspended in phosphate-buffered saline with 0.5% Triton X-100. The
beads were washed 8-10 times with 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, and 50 mM KCl and resuspended in
phosphate-buffered saline with 0.5% Triton X-100.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (52K):
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Fig. 1.
OK cell lysates were immunoprecipitated with
NaPi-4 antiserum and blotted for several AKAP79/150s (lane
1) or AKAP149 (lane
2). AKAP79/150 antibody stained two bands.
AKAP149 failed to detect any bands in the NaPi-4 immunoprecipitates.
Blots are representative of three separate experiments.
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[in a new window]
Fig. 2.
OK cell lysates and crude membrane
preparations were blotted for AKAP79/150. Lysates revealed only
faint bands, whereas crude membrane preparation showed bands at 60 kDa
and a doublet at 79-80 kDa.
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[in a new window]
Fig. 3.
OK cell lysates were immunoprecipitated with
NaPi-4 antiserum as described under "Materials and Methods."
The proteins were separated by SDS-PAGE and transferred to
nitrocellulose membranes, and RII overlay was performed (see
"Materials and Methods") in the presence of vehicle
(left lane), competing peptide that blocks the
binding of AKAP-PKA RII (middle lane), or the
inactive analogue (right lane). RII overlay
identified a protein at the same molecular size as did the AKAP79/150
antibody. RII binding was inhibited by the competing peptide but not
the inactive analogue. Blots are representative of three separate
experiments.
View larger version (42K):
[in a new window]
Fig. 4.
The OK cell lysates were immunoprecipitated
with preimmune serum, NaPi-4 antiserum, and NaPi-4 antiserum
preincubated with cognate peptide as described under "Materials and
Methods." The proteins were separated by SDS-PAGE, transferred
to nitrocellulose membrane, and blotted for AKAP79/150. The
arrow indicates the AKAP79/150 band. Immunoprecipitation
with preimmune serum did not show an AKAP79/150 band.
Immunoprecipitation with NaPi-4 antiserum blotted positively for
AKAP79/150. The intensity of the AKAP79/150 band was markedly decreased
in immunoprecipitates of NaPi-4 antiserum preincubated with peptide.
Blots are representative of three separate experiments. The
top panel demonstrates that preincubation of
NaPi-4 antiserum with immunizing peptide significantly decreases the
ability of NaPi-4 antiserum to immunoprecipitate NaPi-4 (far
right lane). Preimmune serum likewise does not
immunoprecipitate NaPi-4 (far left
lane). NaPi-4 in immunoprecipitates runs as a broad band
between 90 and 110 kDa (middle lane).
View larger version (21K):
[in a new window]
Fig. 5.
OK cell lysates were immunoprecipitated with
AKAP79/150 antibody or antibody preincubated with recombinant AKAP79 as
described in "Materials and Methods." The proteins were
separated by SDS-PAGE, transferred to nitrocellulose membranes, and
blotted for NaPi-4. The arrow indicates the NaPi-4 band,
clearly present as a broad band in cells immunoprecipitated with
AKAP79/150 antiserum (right lane) but markedly
decreased in cells immunoprecipitated with AKAP79/150 antiserum
preincubated with AKAP79 (left lane). Blots are
representative of three separate experiments.
View larger version (33K):
[in a new window]
Fig. 6.
The OK cell lysate was immunoprecipitated
with NaPi-4 antiserum using the Seize-X IgG kit from Pierce according
to the manufacturer's protocol. The proteins from column
flow-through, column first wash, and the first eluted sample were
separated by 10% SDS-PAGE and transferred to nitrocellulose membrane.
The nitrocellulose membrane was blotted against antibodies for NaPi-4
and AKAP79, and an RII overlay assay was performed. The
arrows indicate NaPi-4, AKAP79, and RII overlay positive
bands. Blots are representative of three different experiments.
View larger version (32K):
[in a new window]
Fig. 7.
OK cells were immunoprecipitated with NaPi-4
antibody. The beads were washed and an in vitro
phosphorylation was carried out as described under "Materials and
Methods." Following phosphorylation, the proteins were separated by
SDS-PAGE and autoradiographed. The presence of three phosphorylated
bands in the presence of cAMP indicates the presence of catalytically
active PKA catalytic subunit. Results are representative of three
separate autoradiographs.
View larger version (40K):
[in a new window]
Fig. 8.
OK cells were immunoprecipitated with either
NaPi-4 or AKAP79/150 antibody and immunoblotted for PKA RII.
Immunoprecipitation with both NaPi-4 and AKAP79/150 antibody showed a
positive signal for the presence of PKA RII. Blots are representative
of three different experiments.
1,
-
2, -
, -
, and -
. The immunoprecipitates, however, did blot
positively for protein phosphatase 2b (data not shown).
View larger version (31K):
[in a new window]
Fig. 9.
OK cells were immunoprecipitated with either
preimmune serum or NaPi-4 antiserum, the beads were washed, and 40 µl of 2× Laemmli buffer was added. The samples
were incubated at 37 °C for 10 min. The proteins were separated with
10% SDS-PAGE, transferred to nitrocellulose membrane and blotted
against PTH receptor antibodies. The arrow indicates the
position of the PTH receptor positive band. Blots are representative of
three different experiments.
View larger version (41K):
[in a new window]
Fig. 10.
Brush border membranes
(BBM) were prepared from the OK cells grown on
inserts. The BBM proteins and crude lysate proteins were separated
by SDS-PAGE, transferred to nitrocellulose membranes, and blotted for
NaPi-4, AKAP79/150, and the PTH receptor. There was significantly
higher NaPi-4 and PTH receptor expression in the BBM, whereas there was
no difference in AKAP expression between the lysate and BBM. Blots are
representative of three different experiments. A, NaPi-4
immunoblot; B, AKAP 79 immunoblot; C, PTH
receptor immunoblot; Lane 1, lysate; Lane 2,
BBM.
View larger version (65K):
[in a new window]
Fig. 11.
GST pull-down assay was performed as
described under "Materials and Methods." The presence of
NaPi-4, AKAP79/150, and PTH receptor in the GST-NaPi-4 beads and the
absence of these proteins in glutathione beads alone, the GST-vector
beads, and GST-DM4 beads confirm the association of NaPi-4 with the
AKAP and PTH receptor. RII overlay confirmed that the protein stained
positive for the AKAP79/150 antibody is an AKAP. Ezrin stained positive
in GST-vector, GST-NaPi-4, and GST-DM4 beads, indicating that its
association is nonspecific to NaPi-4. Blots are representative of three
different experiments.
7 M PTH-(1-34)
for 2 h, followed by measurement of radiolabeled phosphate uptake.
As shown in Fig. 12, PTH decreased
32Pi uptake in OK cells. Pretreatment with
competing peptide inhibited the ability of PTH to decrease the
32Pi uptake, whereas pretreatment with the
inactive analogue did not.
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[in a new window]
Fig. 12.
OK cells were grown to confluence and
pretreated with 100 µg/ml stearated competing
peptide or the inactive analogue for 30 min. The cells were
treated with 10 7 M PTH for 2 h, and
phosphate uptake was measured as described under "Materials and
Methods." Under basal conditions, PTH decreased phosphate uptake by
35%. The effect of PTH was inhibited by preincubation with the
competing peptide. Preincubation with the inactive analogue had no
effect on PTH-induced decrease in phosphate uptake. Data represent
mean ± S.E. for four experiments. *, p < 0.005 by Student's t test.
4 M
8-Br-cAMP, and phosphate uptake was measured (Fig.
14). Control HEK293 cells exhibited a
phosphate uptake of 5.3 ± 1.0 pmol/min/mg protein. Cells
transfected with NaPi-4 showed a 20% increase in basal phosphate
uptake (6.4 ± 1.2 pmol/min/mg protein). Cells transfected with
NaPi-4 plus wild type AKAP showed a 35% increase in basal phosphate
uptake (7.3 ± 1.7 pmol/min/mg protein), whereas cells transfected
with NaPi-4 plus the mutant AKAP showed no increase in basal phosphate
uptake. In cells transfected with NaPi-4 alone, 8-Br-cAMP decreased
Pi uptake by 38%, compared with cells transfected with
vector (13%). In cells transfected with wild-type AKAP79 alone,
8-Br-cAMP increased Pi uptake by 14%, whereas in cells transfected with mutant AKAP79, 8-Br-cAMP decreased Pi
uptake by 23%. However, when the cells were co-transfected with NaPi-4 and wild type AKAP79, 8-Br-cAMP inhibited Pi uptake by
63%. 8-Br-cAMP inhibited Pi uptake by only 8% in cells
co-transfected with NaPi-4 and the dominant negative AKAP mutant.
View larger version (44K):
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Fig. 13.
HEK293 cells were grown to 60-80%
confluence; transfected with NaPi-4, human wild-type AKAP79, or
dominant negative mutant AKAP79 alone; or co-transfected with NaPi-4
and either wild type or mutant AKAP79. 48 h
post-transfection, the cells were lysed, membranes were isolated, and
the proteins were separated by 10% SDS-PAGE. The separated proteins
were transferred to nitrocellulose and blotted for NaPi-4 or AKAP79.
The blots confirm successful co-expression of NaPi-4 and both AKAP79
proteins (representative blots from three experiments).
View larger version (21K):
[in a new window]
Fig. 14.
HEK293 cells were grown to 60-80%
confluence and transfected with NaPi-4, human wild-type AKAP79, or
mutant AKAP79 alone or co-transfected with NaPi-4 and either wild-type
or mutant AKAP79. Phosphate uptake was measured 48 h
post-transfection after incubation with either vehicle or 8-Br-cAMP.
Inhibition of phosphate uptake by 8-Br-cAMP was maximal in
NaPi-4/wild-type AKAP79 double transfectants. Co-expression of NaPi-4
and the dominant negative mutant AKAP79 markedly decreased inhibition
of phosphate uptake by 8-Br-cAMP (average of two experiments).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENT |
---|
We acknowledge the excellent technical assistance of Nina Lesousky.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Veterans Affairs Merit Review Board (to E. D. L.).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.
§ Recipient of an American Heart Association Ohio Valley Affiliate Fellowship.
To whom correspondence should be addressed: University of
Louisville, Kidney Disease Program, Baxter Research Bldg., 570 S. Preston St., Pod 102, Louisville, KY 40202. Tel.: 502-852-0014; Fax:
502-852-4384; E-mail: Eleanor.Lederer@kdp.louisville.edu.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M211775200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PTH, parathyroid hormone; PKA, protein kinase A; AKAP, A kinase anchoring protein; PKC, protein kinase C; OK, opossum kidney; 8-Br-cAMP, 8-bromo-cyclic AMP; BBM, brush border membrane(s).
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REFERENCES |
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