From The Department of Internal Medicine, ¶ Department of
Veterans Affairs Medical Center and University of
Texas Southwestern Medical Center, Dallas, Texas 75235
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ABSTRACT |
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Regulation of the renal Na/H exchanger NHE-3 by
protein kinase A (PKA) is a key intermediate step in the hormonal
regulation of acid-base and salt balance. We studied the role of NHE-3
phosphorylation in this process in NHE-deficient AP-1 cells transfected
with NHE-3 and in OKP cells expressing native NHE-3. A
dominant-negative PKA-regulatory subunit completely abolished the
effect of cAMP on NHE-3 activity demonstrating a role of PKA in the
functional regulation of NHE-3 by cAMP. NHE-3 isolated from
cAMP-treated cells showed lower phosphorylation by purified PKA
in vitro suggesting that NHE-3 is a PKA substrate in
vivo. Although changes in NHE-3 whole protein phosphorylation is
difficult to detect in response to cAMP addition, the tryptic
phosphopeptide map of in vivo phosphorylated NHE-3 showed a
complex pattern of constitutive and cAMP-induced phosphopeptides. To
test the causal relationship between phosphorylation and activity, we
mutated eight serines in the cytoplasmic domain to glycine or alanine.
Single or multiple mutants harboring S552A or S605G showed no PKA
activation or reduced regulation by PKA activation. Ser-552 and Ser-605
were phosphorylated in vivo. However, multiple mutations of
serines other than Ser-552 or Ser-605 also reduced the functional PKA
regulation. We conclude that regulation of NHE-3 by PKA in
vivo involves complex mechanisms, which include phosphorylation
of Ser-552 and Ser-605.
Mammalian plasma membrane Na/H exchangers
(NHEs)1 use downhill inward
Na+ gradients to extrude H+ from cells. Six
genetic NHE isoforms have been identified with specific pharmacologic
characteristics and tissue distributions (reviewed in Ref. 1). NHE-3 is
limited to transporting epithelia such as the kidney and
gastrointestinal tract (2, 3). In the kidney, NHE-3 is expressed
exclusively on the apical membrane of the proximal tubule and the thick
ascending limb where it mediates absorption of a significant fraction
of the filtered NaCl and NaHCO3 (4-6). NHE-3 plays a
critical role in renal regulation of extracellular fluid volume and
acid-base balance (7). Acute hormonal regulation of NHE-3 involves
multiple intracellular cascades including activation of adenylyl
cyclase (1). The NHE-3 isoform when expressed in fibroblasts is
inhibited by cAMP analogues or forskolin (8-10). Although the NHE-1
isoform is cAMP insensitive, chimeric insertion of the NHE-3
cytoplasmic domain confers cAMP sensitivity to the NHE-1 transporting
domain (11). Partial deletions of the cytoplasmic domain of NHE-3
reduce although more drastic truncations abolish the effect of cAMP on
NHE-3 activity (10, 11). The NHE-3 cytoplasmic domain is a substrate
for protein kinase A (PKA) in vitro (10) and NHE-3
phosphorylation is increased by cAMP or forskolin addition in
vivo (10, 12). Kurashima and co-workers (12) showed that Ser-605
and Ser-634 are both important for forskolin to inhibit NHE-3 activity,
although only Ser-605 is phosphorylated in vivo. This study
shows that PKA directly phosphorylates NHE-3 and inhibits its activity
via complex mechanisms. Phosphorylation of Ser-552 and Ser-605 were
increased by cAMP addition and appeared to be critical for functional
inhibition of NHE-3, although other regions of the transporter are
likely to be involved in PKA regulation.
Cell Lines--
Rat NHE-3 was tagged (C-terminal) with
hexahistidines (NHE-3/6H) by polymerase chain reaction, sequenced, and
cloned into the mammalian expression plasmid pcDNA3 (Invitrogen,
Carlsbad, CA). Plasmids were transfected into Na/H exchanger-deficient
AP-1 cells (derived from Chinese hamster ovarian fibroblasts) (gift from Dr. Sergio Grinstein, Toronto, Ontario, Canada) using Lipofectin (Life Technologies, Inc.), and double-selected for G418 resistance (400 µg/ml) and H+ survival. Cells were maintained in minimum
essential Eagle's medium (Sigma) with 10% fetal bovine serum (Life
Technologies, Inc.) and 200 µg/ml G418. G418 was replaced by 100 units/ml penicillin and 100 µl/ml streptomycin two passages before
experimentation. Individual clones were selected from pooled
transfectants by limiting dilution. The function of NHE-3/6H was not
different from nontagged NHE-3 (pCMV5/NHE3) expressed in AP-1 cells
(10) in terms of basal activity, antigenic expression, Na, resting
pHi and ethylisopropyl amiloride kinetics, and response to cAMP
addition (data not shown). OKP cells (gift from K. Hruska, St. Louis,
MO), were maintained in Dulbecco's modified Eagle's medium
supplemented with 4.5 mg/ml glucose, 100 units/ml penicillin, and 100 µl/ml streptomycin. Confluent monolayers were rendered quiescent by serum removal (16-24 h, AP-1P cells; 36-48 h, OKP cells), and PKA was
activated by the addition of cell permeant 8-Br-cAMP.
Site-directed Mutagenesis--
Specific serines in the
cytoplasmic domain of NHE-3 were mutated singularly or in combination
using a modification of the double-stranded method of Deng and
Nickoloff (15) (QuickChangeTM kit, Stratagene, La Jolla,
CA). The 8 mutations were: S513G, S552A, S575A, S605G, S634A, S661A,
S690G, and S804G. After annealing the pcDNA3/NHE-3/6H parent vector
with mutagenic oligonucleotides, strands were extended with
Pfu DNA polymerase. Host-derived wild type methylated
templates were digested with DpnI and plasmid DNA's were
isolated from transformed XL1-B Escherichia coli. Multiple mutations were created sequentially. Each mutation was confirmed by
direct sequencing. New cell lines were generated as described above.
Na/H Exchanger Activity Assays--
NHE-3 activity was measured
fluorimetrically in confluent cells on glass coverslips with the
pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(-6)-carboxyfluorescein as sodium-dependent cell pH recovery (dpHi/dt)
after acid loading with the K/H ionophore nigericin (13). For sodium kinetics, sodium was replaced iso-osmotically with choline. Buffer capacity was calculated by measuring In Vivo Phosphorylation and Isolation of NHE-3--
NHE-3
phosphorylated in transfected AP-1 cells was isolated by either
immunoprecipitation or nickel-affinity chromatography. After incubation
in phosphate-free Dulbecco's modified Eagle's medium (30 min), cells
were labeled with [32P]orthophosphate (200-330 µCi/ml;
120 min) and 8-Br-cAMP or vehicle was added. After washing with
ice-cold Tris-buffered saline, cells were lysed with ice-cold RIPA
buffer (in mM: 150 NaCl, 80 sodium fluoride, 50 Tris-HCl,
pH 8.0, 5 EDTA, 1 EGTA, 25 sodium pyrophosphate, 1 sodium
orthovanadate; Nonidet P-40 1% (v/v), deoxycholate 0.5% (w/v), SDS
0.1% (w/v); in µg/ml: 100 phenylmethylsulfonyl fluoride, 4 leupeptin, 4 aprotinin, 10 pepstatin). The slurry was cleared by
centrifugation (109,000 × gmax @ 50,000 rpm; 25 min; 4 °C; Beckman TLX: TLA 100.3 rotor), and NHE-3 was
immunoprecipitated with antiserum 1314 (14) (1:500 dilution) and
protein A-Sepharose. After washing with RIPA buffer, the
antibody-antigen complex was eluted in SDS buffer (5 mM
Tris-HCl, pH 6.8, 10% (v/v) glycerol, 1% (w/v)
For studying surface NHE-3, only OKP cells were used. Repeated attempts
to isolate surface NHE-3 from AP-1 cells yielded variable results
ranging from low to no recovered biotinylated NHE-3 despite abundant
total cellular NHE-3. Surface NHE-3 from four 100-mm dishes of
confluent OKP monolayers were pooled to generate an adequate signal for
one phosphopeptide map. Cells were labeled with 32P and
incubated with 100 µM 8-Br-cAMP, surface proteins were
labeled with biotin (in mM: 10 triethanolamine, pH 7.4, 2 CaCl2, 150 NaCl; 1.5 mg/ml NHS-SS-biotin, (Pierce)) (16)
for 30 min at 4 °C. After quenching (in mM: 140 Na2HPO4, pH 7.4, 0.1 CaCl2, 1 MgCl2, 100 glycine), cells were lysed in RIPA buffer (in
mM: 150 NaCl, 50 Tris-HCl, pH 7.4, 0.5 EDTA, 80 sodium
fluoride, 25 sodium pyrophosphate, 1 sodium orthovanadate; in %: 1 (v/v) Triton X-100, 0.5 (w/v) deoxycholate, 0.1 (w/v) SDS; in µg/ml:
250 phenylmethylsulfonyl fluoride, 10 leupeptin, 10 aprotinin, 20 pepstatin) and equilibrated with streptavidin-agarose at 4 °C on a
rotary mixer overnight. After washing with RIPA, biotinylated surface
proteins were released from the biotin-streptavidin-agarose complex by
100 mM dithiothreitol at room temperature, and the
liberated proteins retrieved as a supernatant after centrifugation. The
supernatant was diluted 1:100 with RIPA buffer, spun-out with a
Centricon-10 filter (Amicon, Beverly, MA), and reconstituted with RIPA
without dithiothreitol. The plasma membrane 32P-labeled
NHE-3 was immunoprecipitated as described above.
Specific Inhibition of PKA in Vivo--
AP-1 NHE-deficient cells
were transfected with pcDNA-3/NHE-3 with or without the plasmid
REVA/B (gift from Dr. Stanley McKnight, Seattle, WA) (17).
REVA,B is a dominant-negative mutant regulatory subunit of
PKA (PKA-RSU), which binds stoichiometrically to the catalytic subunit
(PKA-CSU) but is devoid of both cAMP binding sites and, hence, does not
release its pseudo substrate inhibition even in high ambient cAMP
concentrations. 40% confluent AP-1 cells were transfected using
LipofectAMINETM (Life Technologies, Inc.) with the
following combination of plasmids: 1) pcDNA3 vector (20 µg); 2)
pcDNA3/NHE-3/6H (10 µg), pcDNA3 (10 µg); 3)
REVA/B (10 µg), pCDNA3 (10 µg); and 4)
pcDNA3/NHE-3/6H (10 µg) and REVA/B (10 µg). A 5-min
pulse of 5 µM 8-Br-cAMP was given to the cells 40 h
post-transfection to allow the dominant-negative PKA-RSU to engage the
native PKA-CSU. At 48-h post-transfection, cells were treated with
either 100 µM 8-Br-cAMP or vehicle for 30 min and NHE-3
activity was measured by 22Na uptake.
In Vitro Phosphorylation of NHE-3 by PKA--
For sequential
in vivo and/or in vitro or back phosphorylation
experiments, AP-1 cells transfected with NHE-3/6H were treated with 100 µM 8-Br-cAMP or control medium as described above except no 32PO4 was added. NHE-3 was purified by
nickel-affinity chromatography. The NHE-3 immobilized on Ni-NTA columns
was partially renatured using an 8 to 0 M linear urea
gradient (same as wash buffer above but with urea gradient) and
retained as an insoluble complex on solid support. The beads were
dialyzed in phosphate-buffered saline at 4 °C overnight and
subjected to direct phosphorylation by purified PKA-CSU. The dialyzed
NHE-3/Ni-NTA-agarose complex was washed with 1× kinase buffer (in
mM: 50 Hepes-Tris, pH 7.0, 0.3 dithiothreitol, 5 MgCl2, 1 EGTA) and aliquoted in equal amounts into
Eppendorf tubes. In vitro phosphorylation was initiated in
each tube by addition of 1× kinase buffer supplemented with 50 µCi
[ Phosphoamino Acid Analysis and Tryptic Phosphopeptide
Mapping--
Purified phosphoproteins were eluted from the acrylamide
gel with 50 mM NH4HCO3,
trichloroacetic acid-precipitated, hydrolyzed by boiling in 6 M HCl, and the 32P-labeled amino acids were
electrophoretically resolved in a Hunter TLC electrophoresis unit (HTLE
7000, CBS Scientific Co, Del Mar, CA) (first dimension: 2.2% formic
acid, 7.8% acetic acid, pH 1.9; second dimension: 5% acetic acid,
0.5% pyridine, pH 3.5) along with cold standards (P-Ser, P-Thr,
P-Tyr). Phosphoamino acids were identified by autoradiography and
alignment with ninhydrin-stained phosphoamino acid standards (18).
Tryptic phosphopeptide mapping was performed as described by Boyle
(18). The purified in vivo phosphorylated NHE-3 was
fractionated by SDS-PAGE and transferred to nitrocellulose membrane.
Membrane pieces containing NHE-3 protein were localized by
autoradiography, excised, and incubated in 100 mM acetic
acid containing 0.5% (w/v) polyvinylpyrrolidone at 37 °C for 45 min. After washing with deionized water and 0.05 M
NH4HCO3 solution, membranes were incubated in
15 µg TPCK (N-tyrosyl-L-phenylalanine chloromethyl ketone)-treated trypsin (Worthington) in 300 µl of 0.05 M NH4HCO3 in a 37 °C shaking
waterbath. 15 µg of TPCK-treated trypsin was added after 2 and
12 h. After repeated deionized water washes and lyophilization,
the dried sample was resuspended in 20 µl of electrophoresis buffer
(per liter: 25 ml of 88% (w/v) formic acid, 78 ml of glacial acetic
acid, 897 ml of deionized water, pH 1.95), spotted on a cellulose TLC
plate (Merck, Darmstadt, Germany), and electrophoresis was performed on
a Hunter thin-layer electrophoresis apparatus with the above buffer (30 min; 1.0 kV). Separation of the peptides in the second dimension was
achieved by ascending chromatography (375:250:75:300 (v/v)
n-butanol, pyridine, glacial acetic acid, deionized water).
The phosphopeptides were visualized by either phosphorimaging or
autoradiography, and the signal of individual phosphopeptides were
quantified by densitometry. To eliminate variations in cpm's loaded
and exposure times, the intensity of each phosphopeptide on a given TLC
plate was normalized to the intensity of an invariant constitutive
phosphopeptide B on the same plate. Quantitative differences between
control and cAMP was assessed statistically by ANOVA.
Effect of PKA Activation on NHE-3 Activity and
Phosphorylation--
Fig. 1 shows that
cAMP inhibited NHE-3 activity in a time- (Fig. 1A) and
dose-dependent (Fig. 1B) fashion in both AP-1
and OKP cells. In both AP-1 (Fig.
2A) and OKP (Fig.
2B) cells, the Vmax was reduced by
approximately 40% (con versus cAMP: AP-1P; 0.86 versus 0.55 pH units/min, p < 0.05, OKP;
1.05 versus 0.61 pH units/min, p < 0.05, t test) with no change in KNa (con
versus cAMP: AP-1P;14 mM versus 12 mM, p = 0.52, OKP; 20 mM
versus 16 mM, p = 0.67, t test). We have previously shown that 200 µM
cAMP increases NHE-3 phosphorylation but lower doses were not examined (10). When we performed a dose response of the effect of cAMP on NHE-3
phosphorylation, we found no consistently detectable effect on NHE-3
whole protein phosphorylation in both AP-1 and OKP cells until 100 µM of cAMP was used (Fig.
3). Even at 100 µM, changes
in total NHE-3 phosphorylation was variable. This was in sharp
contradistinction to the effect of cAMP on NHE-3 activity, which was
evident at doses as low as 1 and 10 µM for OKP and AP-1
cells, respectively.
Role of PKA in Regulating NHE-3 Function--
One hypothesis is
that the effect of cAMP may be independent of A kinase activity. Direct
gating by cyclic nucleotides without involvement of protein kinase has
been described for cation channels (19). To test this hypothesis, we
inhibited endogenous PKA-CSU with a dominant-negative mutant regulatory
subunit (PKA-RSU) in a transient transfection system rather than with
pharmacologic inhibitors, which may have nonspecific effects. Because
cells that did not take up REVA/B (plasmid for mutant
PKA-RSU) also did not receive NHE-3, all 22Na uptake can be
assumed to originate from cells that received both plasmids. Fig.
4 summarizes the data. Only cells
transfected with NHE-3 expressed significant 22Na uptake
indicating that the measured flux reflected NHE-3 activity and in the
absence of REVA/B, NHE-3 activity was suppressed by cAMP
addition as expected. In the presence of the dominant-negative PKA-RSU,
cAMP had no effect on NHE-3 activity indicating that the cAMP effect on
NHE-3 activity is mediated by PKA-CSU.
Phosphorylation of NHE-3 by PKA--
If PKA is necessary for the
regulation of NHE-3 by cAMP, another hypothesis that can explain the
dissociation between activity and whole protein phosphorylation is that
PKA phosphorylates a protein other than NHE-3, which in turn modulates
NHE-3 activity. NHE-RF for example is a phosphoprotein that regulates
NHE-3 function (20-22). To address whether NHE-3 is phosphorylated by
PKA in vivo, we used the indirect approach of back
phosphorylation. If the PKA sites on NHE-3 are occupied by
nonradioactive PO4 because of in vivo
phosphorylation before cell lysis, these sites will be unavailable to
accept 32PO4 in the subsequent in
vitro reaction with purified PKA-CSU. When we harvested NHE-3 from
either cAMP-activated or control cells and subjected purified NHE-3 to
in vitro phosphorylation by PKA, we found a significantly
smaller degree of 32PO4 incorporation into
NHE-3 from cAMP-activated compared with control cells (Fig.
5). These findings are compatible with
the hypothesis that PKA indeed directly phosphorylates NHE-3 in
vivo. It is important to note that the current data do not rule
out the fact that PKA may phosphorylate proteins other than NHE-3 and
kinases other than PKA may also phosphorylate NHE-3.
Phosphoamino Acid and Tryptic Phosphopeptide Mapping of in Vivo
Phosphorylated NHE-3--
The most likely explanation for the
dissociation between activity and whole protein phosphorylation is
presence of constitutive phosphorylated residues on NHE-3 that are not
regulated by cAMP addition. To address this, we performed tryptic
phosphopeptide maps using varying doses of cAMP. We observed increased
intensity of phosphopeptides D, E, F, and J after cAMP addition (Fig.
6A). Although there are
variations from map to map, with over 30 maps performed on AP-1 cells,
it is evident that phosphopeptide B is constitutively phosphorylated.
Because the absolute signal of a specific phosphopeptide on a given map
is affected by a variety of factors such as the efficiency of retrieval
from the total protein as well as the exposure time; one way to
summarize the results from all the maps quantitatively is to express
the intensity of each phosphopeptide as a ratio to the constitutive
phosphopeptide B on the same plate. The results from all the
phosphopeptide maps performed on wild type cells are summarized in Fig.
6B. Phosphopeptides J and E were significantly increased by
[cAMP] Regulation of Na/H Exchange Activity in Mutant NHE-3
(NHE-3mut)--
If PKA phosphorylates NHE-3, the critical
question is whether phosphorylation of NHE-3 is functionally important
for its regulation. Because only serines are phosphorylated, we mutated
8 serines individually and in combination in the cytoplasmic domain
that conform to the classical PKA consensus target motif (23) and screened for the ability of cAMP to regulate NHE-3 activity. Single amino acid mutations per se do not significantly affect
whole cell NHE-3 protein expression (Fig.
8A). However, four or more point mutations did decrease NHE-3 expression with the lowest expression in the six-point mutant (Fig. 8B). NHE-3
transcript levels were not affected by single or multiple mutations
(not shown). Fig. 9A shows
tracings of several representative mutants, and Fig. 9, B
and C, summarize the functional data from all the mutants.
Among the single mutants, NHE-3mut/S552A failed to show a
response to cAMP addition. NHE-3mut/S605G showed much
reduced inhibition by cAMP (control versus cAMP
p = 0.28). NHE-3mut/S634A showed a slightly
attenuated but nonetheless intact response to cAMP. This finding
suggests phosphorylation of Ser-552 and Ser-605 are key steps in
modulating NHE-3 activity. One concern with this data is whether the
lack of regulation is truly due to the mutant NHE-3 or because of
cellular context. This is particularly critical for S552A because
Kurashima and co-workers (12) found intact cAMP regulation with the
same mutation. To determine that the lack of cAMP sensitivity actually
stems from S552A rather than a missing cellular factor, we studied
NHE-3mut/S552A against a heterogenous cellular background
with two approaches. We isolated individual clones from the pooled
NHE-3mut/S552A transfectants, and we transiently
transfected NHE-3mut/S552A into AP-1 cells at different
passages (Table I). As a positive
control, we compared NHE-3mut/S552A to one of the
cAMP-responsive single serine mutants NHE-3mut/S690G. As
shown in Table I, all NHE-3mut/S552A-transfected cells
remained cAMP insensitive, whereas all NHE-3mut/S690G-transfected cells maintained cAMP
sensitivity. Despite the potentially diverse cellular context, cAMP
responsiveness segregated with the mutation.
The results were less clear in the multiple mutants. All the multiple
mutants harboring Ser-552 were not regulated by cAMP addition
confirming the importance of Ser-552. However,
NHE-3mut/S575A/S661A/S690G/S804G which contains neither
Ser-552 nor Ser-605, also showed no inhibition by cAMP addition.
Tryptic Phosphopeptide Map of NHE-3mut/S552A and
NHE-3mut/S605G--
If Ser-552 and Ser-605 are indeed
critical for inhibition of NHE-3 activity as phosphoserines, they
should be phosphorylated in vivo by cAMP addition. We
compared phosphopeptide maps of these two NHE-3mut's to
wild type NHE-3 in response to cAMP. Fig.
10 shows the phosphopeptide maps of
wild type NHE-3, NHE-3mut/S552A, and
NHE-3mut/S605G. Phosphopeptide J, which was increased by
8-Br-cAMP in wild type NHE-3 is absent in NHE-3mut/S552A,
and phosphopeptide E, which was also increased by 8-Br-cAMP in wild
type NHE-3, was absent from NHE-3mut/S605A indicating that
both Ser-552 and Ser-605 are phosphorylated in vivo.
We confirmed the previous finding of inhibition of
Vmax by cAMP in native NHE-3 in renal epithelial
cells (24-26) and NHE-3 heterologously expressed in fibroblasts
(8-12). The lack of detectable NHE-3 whole protein phosphorylation is
comparable with the report by Kurashima and co-workers (12) although we
observed discernible but variable changes in NHE-3 whole protein
phosphorylation with 100 µM 8-Br-cAMP. As some cation
channels are directly gated by cyclic nucleotides (19), we queried if
such can be a model for NHE-3 regulation by cAMP. When we inactivated
the catalytic subunit of PKA, 8-Br-cAMP no longer regulated NHE-3
activity indicating that the cAMP effect is
kinase-dependent. Also congruent with A kinase-mediated
action is the fact that unlike the cyclic nucleotide-gated channels
(27), the primary sequence of NHE-3 does not conform to the
The cytoplasmic domain of NHE-3 contains numerous PKA consensus target
sites and NHE-3 is rapidly phosphorylated by PKA in vitro
(10). Although NHE-3 is a phosphoprotein in vivo (10, 12),
there is no a priori reason to assume that PKA directly phosphorylates NHE-3 in vivo. The basis of the back
phosphorylation assay rests on the presumption that certain common
sites are preserved for in vivo and in vitro
kinase reactions and multiple kinases do not converge on the exact same
serine residues. Our results suggest that PKA likely phosphorylates
NHE-3 in the cell. It is possible that the regulation of NHE-3 by cAMP
involves phosphorylation of proteins other than NHE-3 by PKA and that
PKA may activate other kinases that phosphorylate NHE-3. Kurashima and
co-workers (12) showed that the lack of change in total NHE-3 protein
phosphorylation in vivo is because of multiple
constitutively phosphorylated sites masking the change in the regulated
sites. We also found that phosphopeptides B, C, and D are largely
unregulated in vivo. In contrast, phosphopeptide E (Ser-605)
and J (Ser-552) were increased at 1 and 10 µM 8-Br-cAMP,
respectively. The dose-response profiles of phosphopeptides E and J
actually correlate well with that of changes in NHE-3 activity in
response to cAMP.
If phosphorylation of Ser-552 and Ser-605 are important, then
elimination of phosphorylation should abolish functional regulation. All the single and double point mutants showed comparable antigen expression and baseline transport activity but the quadruple mutants showed decreased and the six-point mutant showed markedly attenuated expression of both NHE-3 protein and activity. Because NHE-3 transcript levels are not affected by the mutations, one has to postulate that the
multiple serine mutations significantly affected translation and/or
protein half-life. This study does not address the mechanism of these
changes. Ser-552 and Ser-605 were both phosphorylated in
vivo and when mutated singly to nonphosphorylatable residues, functional regulation by PKA was abrogated in both mutants. Kurashima and co-workers (12) have reported the importance of Ser-605 and Ser-634
in the functional regulation of NHE-3 by PKA although Ser-634 appeared
not to be a phosphoserine. Our phosphopeptide map and functional
response of the NHE-3mut/S605A is very similar to the
previous report. A major disparate finding is that we found Ser-552 to
be critical for PKA regulation, whereas Kurashima and co-workers (12)
found normal functional regulation of NHE-3mut/S552A by
cAMP. In addition, Cabado and co-workers (11) from the same group found
that truncation of the cytoplasmic domain at amino acid 579 rendered
NHE-3 nonresponsive to cAMP. The difference may reside in the fact that
Ser-552, which is phosphorylated in our cells, was not phosphorylated
in the previous report as is evident by the absence of phosphopeptide J
(12). It is conceivable that multiple PKA target phosphoserines may
participate in mediating the functional inhibition of NHE-3 and
different host cells may use different sets of serines. The basis for
the differential NHE-3 phosphorylation is unclear, because both
laboratories used AP-1 cells from the same source. Because AP-1 cells
are mutants derived and selected from the parental Chinese hamster
ovary cell, repeated passages and clonal expansion might have created
different phenotypes.
One possible interpretation for the lack of regulation of
NHE-3mut/S552A is that some cellular cofactor(s) are
missing in that particular AP-1 recipient that harbors the mutant NHE-3
and that the cAMP-insensitive phenotype stems from the cell rather than
NHE-3mut/S552A. Such an explanation is unlikely for several
reasons. All pooled transfectants of single or multiple mutant NHE-3s
were generated about the same time from the same parental AP-1 cells. In addition, when we tried to examined NHE-3mut/S552A in as
heterogenous an AP-1 cellular background as we can create, we found
that the cAMP-nonresponsive phenotype prevailed.
The lack of regulation of NHE-3mut/S575/S661/S690/S804 is
harder to explain because none of the serines were functionally
important when examined as single mutants. One can speculate on several explanations. One is that there are multiple regulatory phosphoserines with P-Ser-552 and P-Ser-605 having the most dominant effects with
lesser contribution from the other phosphoserines. Although singular
mutations of serines other than Ser-552 and Ser-605 do not affect the
overall response, mutation of four minor phosphorylation sites may be
sufficient. A second possibility is that these serines participate in
specific protein-protein interactions with regulatory factors such as
NHE-RF (20, 21, 29-31), E3KARP (22), or calmodulin (32) that modulate
NHE-3 function, and this interaction is disrupted by the mutations. A
third possibility is that the mutations distort the protein structure
to such an extent that it nonspecifically alters NHE-3 function and its
regulation. Crystallographic evidence indicates that one can
significantly alter the protein structure of a domain of a protein by a
single amino acid mutation from tyrosine to phenylalanine, which
essentially only removes a single hydroxyl group (33). The present
paper does not distinguish the three stated possibilities. We did
however observe intact regulation of all mutants including the four- to
six-point mutants by acute hyperosmolality (25-40% inhibition) (data
not shown). This is compatible with the ability of hyperosmolality to
inhibit NHE-3 activity despite radical truncations of the cytoplasmic domain (34).
In summary, we hypothesize that the functional regulation of NHE-3 by
PKA does not converge on single covalent modifications such as
phosphorylation of a specific serine. We submit a model where PKA
phosphorylates NHE-3 on Ser-552 and Ser-605 in addition to other
serines. Phosphorylation of Ser-552 and Ser-605 each play a major
although not exclusive role in the functional regulation of NHE-3,
which involves specific interaction of various regions of the
cytoplasmic domain of NHE-3 with other regulatory cofactors.
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
pHi in response to a 20 mM NH4Cl pulse at the trough pH. The addition
of 8-Br-cAMP did not alter
2',7'-bis-(2-carboxyethyl)-5-(-6)-carboxyfluorescein calibration or
buffer capacity in AP-1 or OKP cells (data not shown). For
22Na uptake studies, monolayers on 24-well dishes were
acidified by NH3/NH4+ prepulse/withdrawal
(pulse in mM: 10 Hepes·HCl, pH 7.4, 30 NH4Cl, 100 choline Cl, 5 KCl; withdrawal in mM:10 Hepes·HCl pH
7.4, 130 choline chloride, 5 KCl), and 22Na flux was
initiated at 20 °C (in mM: 130 choline chloride, 2 CaCl2, 1 MgCl2, 10 Hepes·HCl, pH 7.4, 1 ouabain, 1 22NaCl (1 µCi/ml), 50 nM
ethyl-isopropyl-amiloride), and terminated by washing with
phosphate-buffered saline (4 °C). Cells were lysed in 0.1 M NaOH for protein concentration determination (Bradford) and scintillation counting.
-mercaptoethanol,
0.1% (w/v) SDS, 0.01% (w/v) bromphenol blue), resolved on SDS-PAGE
and transferred to nitrocellulose membrane. For nickel-affinity
purification of NHE-3, cells were lysed in buffer B1 (in
mM: 8,000 urea, 100 Na2PO4, 10 imidazole, pH 8.0; in % (v/v): glycerol 30, Triton-X 100 0.5), sheared
with a 25-gauge needle, combined with buffer B2 (1:1, v/v) (composition same as B1 except no glycerol), clarified by centrifugation
(109,000 × gmax @ 50,000 rpm, 25 min,
Beckman TLX: TLA 100.3 rotor), and equilibrated with Ni-NTA resin
(Qiagen, Chatsworth, CA) by rocking for 1 h at room
temperature. The resin was washed with 5× bed volumes of wash buffer
(in mM: 8,000 urea, 100 Na2PO4, pH
8.0, 50 imidazole; in % (v/v): 0.5 Triton-X 100), and NHE-3/6His was eluted (in mM: 8,000 urea, 100 Na2PO4, 500 imidazole, pH 8.0) for SDS-PAGE.
Immunoblots were performed with anti-peptide antiserum 1568 (1:2,000)
(14) using enhanced chemiluminescence (Amersham Pharmacia Biotech), and
the 32P content of NHE-3 was visualized by autoradiography
on the same filters after decay of enhanced chemiluminescence. Both
antigenic and 32P signals were quantified by densitometry,
and changes in phosphorylation were normalized to the antigenic signal.
In general, both methods of purification yielded similar results in
terms of changes in phosphorylation.
-32P]ATP, 0.1 mM ATP, 25 units of
PKA-CSU, and rocked in room temperature for the designated time period.
The reaction was stopped by addition of 5× reaction volumes of stop
buffer (1% (w/v) SDS, 10 mM EDTA, 10 mM
imidazole, pH 8.0), transferred to chromatography columns and washed
with another 5× bed volume of stop buffer. The phosphorylated NHE-3
was eluted with 500 mM imidazole with 0.1% SDS and
subjected to SDS-PAGE, transferred to nylon membranes, and
autoradiography and immunoblot was performed as described above.
RESULTS
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Fig. 1.
Time (A) and dose dependence
(B) of cAMP addition on NHE-3 activity in AP-1
(solid bar) and OKP (open bar)
cells. 8-Br-cAMP were added to cells, and NHE-3 activity was
measured fluorimetrically as sodium-dependent cell pH
recovery. Asterisk indicates significant difference
(p < 0.05, ANOVA) from control. Bars and errors bars
represent mean ± S.E.; n = 4 for each time point
and dose. n = number of experiments for time
dependence: 0 min, OKP n = 5, AP-1 n = 6; 10 min, OKP n = 4, AP-1 n = 3; 20 min, OKP n = 3, AP-1 n = 4; 30 min, OKP
n = 7, AP-1 n = 9; 40 min, OKP
n = 4, AP-1 n = 7. n = number of experiments for dose dependence: 0 µM, OKP
n = 5, AP-1 n = 6; 10 6
M, OKP n = 4, AP-1 n = 5;
10
5 M, OKP n = 4, AP-1
n = 4; 10
4 M, OKP
n = 7, AP-1 n = 9; 2-10
4
M, OKP n = 5, AP-1 n = 7.
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Fig. 2.
Effect of cAMP on NHE-3 sodium kinetics.
, control;
, 100 µM 8-Br-cAMP for 30 min.
A, AP-1P cells (con versus 8-Br-cAMP:
Vmax; 0.86 versus 0.55 pH units/min,
p < 0.05, KNa; 14 mM versus 12 mM, p = 0.520). B, OKP cells (con versus 8-Br-cAMP:
Vmax; 1.05 versus 0.61 pH units/min,
p < 0.05, KNa; 20 mM versus 16 mM, p = 0.67). Symbols and errors bars represent mean ± S.E.
n = number of experiments: n = 3 for
each cell line treated with vehicle or 8-Br-cAMP for each [Na] except
for [Na] = 5, n = 4 for each.
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Fig. 3.
Dose-response of cAMP on NHE-3 activity and
phosphorylation. AP-1P cells (closed symbols) and OKP
cells (open symbols). The y-axis shows NHE-3
phosphorylation (square symbols) and activity
(circles) expressed as percent of control. Changes in
phosphorylation were normalized to NHE-3 antigen. NHE-3 activity was
measured fluorimetrically as sodium-dependent cell pH
recovery and is the same data as in Fig. 1B. Symbols and
errors bars represent mean ± S.E. n = number of
experiments for phosphorylation dose response: AP-1: control,
n = 9; 1 µM, n = 3; 10 µM, n = 3, 100 µM.
n = 9. OKP cells: control, n = 4; 1 µM, n = 3; 10 µM,
n = 4, 100 µM. n = 6.
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Fig. 4.
Acute regulation of NHE-3 by cAMP: effect of
dominant-negative regulatory subunit of PKA (REVA/B).
NHE-deficient AP-1 cells were transfected with the following plasmids:
pcDNA, expression vector; NHE-3; and REVA/B,
dominant-negative regulatory subunit of PKA. Native PKA was activated
by 100 µM 8-Br-cAMP for 30 min, and NHE-3 activity was
measured by 22Na uptake and normalized to total cellular
protein per well. Data from one experiment are shown. A total of three
experiments were performed with similar results.
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Fig. 5.
Sequential in vivo and
in vitro phosphorylation of NHE-3. AP-1P cells
were treated with 8-Br-cAMP or vehicle. 6His-tagged NHE-3 was purified,
immobilized on Ni-NTA beads, and subjected to phosphorylation by
purified PKA in the presence of [ -32P]ATP. The
phosphorylated protein was eluted off the beads, fractionated by
SDS-PAGE, transferred to filters, and analyzed with autoradiography and
immunoblot. One experiment is shown. Three independent experiments
showed similar results.
1 and 10 µM, respectively, and phosphopeptide
F was increased at 100 µM cAMP. Because NHE-3 is more
abundant in intracellular compartments than in plasma membrane in AP-1
cells (12), we examined whether the measured inhibition of NHE-3
activity, which reflects plasma membrane NHE-3, is associated with
changes in cell surface NHE-3 phosphorylation. Extremely low and
variable levels of retrievable biotinylatable NHE-3 in AP-1 cells
precluded a meaningful conclusion in these cells. However, when we
isolated surface biotinylated NHE-3 from OKP cells, multiple
cAMP-induced NHE-3 phosphopeptides were detected (Fig. 6C).
The pattern of phosphorylation of OK surface NHE-3 was identical to
that of total cellular NHE-3 (not shown). Next, we determined the
phosphoamino acid composition of the in vivo phosphorylated
protein in AP-1 cells to be almost exclusively serines (Fig.
7). Prolonged exposure revealed a small
amount of phosphothreonine, which when quantified by scintillation
represented 0.5-1% of the counts. Phosphoamino acid analysis of OK
cells showed predominantly phosphoserines with 2% phosphothreonine
(data not shown).
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Fig. 6.
Phosphopeptide map of NHE-3 in AP-1 and OKP
cells: Response to 8-Br-cAMP. A, tryptic phosphopeptide
maps of immunoprecipitated 32P-labeled NHE-3 from AP-1
cells treated with different [cAMP]. 32P-NHE-3 was
digested with trypsin, resolved by electrophoresis and thin-layer
chromatography, and visualized by autoradiography. Phosphopeptides are
labeled by alphabet letters. B, quantitative summary of dose
response of phosphopeptide maps of NHE-3 in AP-1 cells. Intensity of
phosphopeptides C, D, E, F, and J were normalized to the intensity of
the corresponding phosphopeptide B on the same thin-layer
chromatography plate. Symbols and errors bars represent mean ± S.E. n = number of experiments: control,
n = 9; 1 µM, n = 3; 10 µM, n = 3; 100 µM,
n = 9. Asterisk indicates p = 0.05 when compared with the value of control by ANOVA. C,
cAMP-induced phosphorylation of surface NHE-3 in OK cells. OK cells
were pulsed with 32P, 100 µM 8-Br-cAMP was
added, and surface NHE-3 was immunoprecipitated from the biotinylatable
fraction of the cell lysate, digested with trypsin, and resolved in two
dimensions. Two independent maps showed similar results.
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Fig. 7.
Phosphoamino acid analysis of NHE-3
phosphorylated in vivo. Cells were pulsed with
[32P]orthophosphate, 100 µM 8-Br-cAMP was
added, and the immunoprecipitated NHE-3 was subjected to acid
hydrolysis, resolved on two-dimensional electrophoresis, and subjected
to autoradiography for phosphoamino acid analysis. Three independent
experiments showed predominantly phosphoserines.
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Fig. 8.
Immunoblot of NHE-3 from AP-1 cells
expressing NHE-3mut. 20 µg of membrane protein
from each of the cell lines were resolved by SDS-PAGE and probed with
anti-NHE-3 antiserum 1568. Single and multiple serine mutations are
indicated on the figure. Single mutants (A) and multiple
mutants (B). Two independent experiments showed similar
results.
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Fig. 9.
Effect of cAMP on NHE function
NHE-3muts in mutants with serine mutations. Point
mutants of NHE-3 was expressed in AP-1 NHE-deficient fibroblasts. NHE-3
activity in control and 8-Br-cAMP (100 µM, 30 min)-treated cells were measured fluorimetrically as
sodium-dependent cell pH recovery. Asterisk
indicates p < 0.05 by unpaired t test
(control versus cAMP in same cell line). A,
representative tracings from six selected clones; B, summary
of all single mutants. n = number of experiments: WT,
n = 21; S513G, n = 4; S552A,
n = 11; S575A, n = 9; S605G,
n = 9; S634A, n = 4; S661A,
n = 6; S690G, n = 4; S804G,
n = 6. C, summary of all multiple mutants.
n = number of experiments: WT, n = 21, S513G/S552A, n = 4; S513A/S552A/S661A,
n = 3; S661A/S690G/S804G, n = 4;
S513G/S552A/S575A/S605G, n = 5;
S552A/S575A/S661A/S690G/S804G, n = 5;
S513G/S552A/S575A/S661A/S690/S690G/S804G, n = 5.
NHE-3 activity in AP-1 cells transfected rat NHE-3
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Fig. 10.
Phosphopeptide map of in vivo
phosphorylated wild type and mutant NHE-3. Cells were pulsed
with 32P and treated with 8-Br-cAMP. NHE-3 was
immunoprecipitated, and the tryptic peptides were resolved by
electrophoresis and thin-layer chromatography and visualized by
autoradiography. A, WT versus
NHE-3mut/S552A. n = 5; B, WT
versus NHE-3mut/S605G, n = 3.
DISCUSSION
-roll/
-helix motif derived from the catabolite gene activator protein model (28). In addition, a recombinant fusion protein of
maltose-binding protein/NHE-3 cytoplasmic domain failed to bind
3H-cAMP (data not shown).
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Stan McKnight for providing reagents, to Dr. Melanie Cobb and Dr. Robert Alpern for helpful discussions, and to Dr. Michel Baum and Dr. Robert Alpern for careful reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the Research Service of the Department of Veterans Affairs and the National Institutes of Health (DK48482).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 a National Institutes of Health Training Grant (T32 DK07257-17).
To whom correspondence should be addressed: Dept. of Internal
Medicine, 5323 Harry Hines Blvd., Dallas, TX 75235-8856. Tel.: 214-648-3152; Fax: 214-648-2071; E-mail: omoe{at}mednet.swmed.edu.
The abbreviations used are: NHE, Na/H exchanger; PKA, protein kinase A; PAGE, polyacrylamide gel electrophoresis; CSU, catalytic subunit; RSU, regulatory subunit; TPCK, N-tyrosyl-L-phenylalanine chloromethyl ketone; ANOVA, analysis of variance; WT, wild type.
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REFERENCES |
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