 |
INTRODUCTION |
NHE11 is the ubiquitous
form of the Na+/H+ exchanger, responsible for
regulation of intracellular pH and cell volume. NHE1 has increased
activity in hypertensive patients and in animal models of hypertension,
and the exchanger can be stimulated by various exogenous factors. In
several examples of increased NHE1 activity, the primary change is not
in NHE1 expression but in post-translational regulation of activity. In
immortalized cells from hypertensive patients, enhanced NHE1 activity
appears to reflect increased Vmax rather than
increased NHE1 mRNA (1). Similarly, vascular smooth muscle cells
(VSMC) from spontaneously hypertensive rats (SHR) show an increased
Vmax compared with their normotensive Wistar-Kyoto (WKY) counterparts but do not overexpress NHE1 mRNA or
protein (2-4). Furthermore, there is no evidence for genetic linkage
between the Na+/H+ exchanger gene and human
hypertension (5), and no mutations have been detected in the entire
coding region of NHE1 in SHR compared with WKY (6). Hence, increased
NHE1 activity in hypertension is best explained by altered
intracellular regulation of the exchanger. In support of this concept,
phosphorylation of NHE1 by growth factors was increased in the SHR
compared with the WKY (7).
NHE1 is activated in cells stimulated with numerous agonists. Epidermal
growth factor, thrombin, phorbol esters, and serum were first found to
stimulate Na+/H+ exchanger phosphorylation
concurrently with a rise in intracellular pH (8). p160 ROCK mediates
lysophosphatidic acid-induced activation of NHE1 via carboxyl-terminal
phosphorylation (9). Angiotensin II activates extracellular
signal-regulated kinase (ERK), which in turn stimulates NHE1
phosphorylation and activity (10, 11). In fact, ERK1/2 activation
appears to be a common denominator in the process of NHE1 activation by
several growth factors, since MEK1/ERK1/2 pathway inhibition with
PD98059 or use of a dominant negative ERK1 construct blocks activation
of the exchanger by
1-adrenergic receptor agonists,
serum, endothelin, platelet-derived growth factor, thrombin, and
phorbol esters among others (12-16). Interestingly, in lymphoblasts
from hypertensive patients, increased NHE1 activity and phosphorylation
correlated with enhanced ERK1/2 activity, although there were no
apparent differences in the level of tyrosine phosphorylation of
ERK between cells from hypertensive and normotensive patients (17).
Indeed, the relative activity of the ERK pathway in hypertension
remains controversial (18, 19).
We showed previously that activation of NHE1 by serum was dependent on
phosphorylation of serine 703 (Ser(P)703) by p90 ribosomal
S6 kinase (RSK), which in turn was directly regulated by ERK1/2 (20).
Interestingly, the NHE1 sequence at Ser(P)703 (RIGSDP) is
similar to a consensus sequence (RSXSXP) specific for binding of 14-3-3. Indeed, 14-3-3 ligands share a common binding determinant that mediates their interaction with 14-3-3. First described for Raf-1, the consensus recognition motif RSXpSXP (where X designates any amino acid and pS represents phosphoserine)
applies to most known binding partners of 14-3-3 (21). In the
overwhelming majority of interactions documented so far, 14-3-3 necessitates a phosphorylated serine for binding. For example,
mutants of Raf-1 where Ser259 or Ser621
are mutated to alanine have reduced binding to 14-3-3 (22). An arginine
in the
3 or
4-position is also crucial, as is the proline at +2,
although in some instances changes in the proline may be tolerated (23,
24). The phosphoserine contacts 14-3-3 by salt bridges to side chains
contained within the aliphatic groove of 14-3-3. In the case of
14-3-3
, mutations of residues within this groove (K49E, R56E, or
R127E) diminish interaction with binding partners Raf1 and Bcr
(25).
Based on sequence similarity between our proposed 14-3-3 binding site
in NHE1 and those identified in known 14-3-3 ligands, we hypothesized
that phosphorylation of NHE1 at serine 703 would create a binding site
for 14-3-3. In addition, we postulated that 14-3-3 binding would
modulate NHE1 activity. In the present study, we show that 14-3-3
associates with NHE1 in cells stimulated by serum, and using mutants of
NHE1 we determined that this interaction occurs at serine 703. Functionally, binding of 14-3-3 to the exchanger was necessary for
activation of NHE1 by serum, an effect that correlated with diminished
dephosphorylation of Ser(P)703 observed when NHE1 is bound
to 14-3-3.
 |
MATERIALS AND METHODS |
Far Western Analysis--
Preparation of overlapping GST fusion
proteins spanning amino acids 516-815, comprising the entire
cytoplasmic domain of NHE1, was described previously (11). Five
constructs, NHE1-(516-630), NHE1-(625-747), NHE1-(747-815),
NHE1-(625-670), and NHE1-(625-714), were used (Fig. 1). GST-14-3-3
was a gift from Dr. A. J. Muslin (Washington University School of
Medicine, St. Louis, MO). After transformation of GST constructs into
the BL21 strain of Escherichia coli, cultures
were grown to the sublog phase and induced for 3 h at 37 °C
with 0.5 mM
isopropyl-
-D-thiogalactopyranoside (Sigma). Cells were
collected, sonicated, and centrifuged. The supernatants were incubated
with glutathione-Sepharose beads (Amersham Pharmacia Biotech) overnight
at 4 °C, and bound fusion proteins were washed extensively with
fusion protein lysis buffer (50 mM Tris, pH 8, 1 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.25%
Nonidet P-40). Purified GST-NHE1 fusion proteins were stimulated with
RSK (20 mM Tris, pH 7.4, 12 mM
MgCl2, 100 mM NaCl, 5 units of p90 RSK2 (Upstate Biotechnology Biotechnology, Inc., Lake Placid, NY), 10 mM ATP) and then resolved on SDS-PAGE and transferred to
nitrocellulose membranes. GST-14-3-3 fusion proteins were labeled with
[
-32P]ATP using protein kinase A buffer (20 mM Tris, pH 7.4, 12 mM MgCl2, 100 mM NaCl, 10 µl of protein kinase A, 10 mCi of
[
-32P]ATP), and used to probe the membranes.
Alternatively, membranes containing GST-14-3-3 were probed with
[
-32P]ATP-labeled GST-NHE1 constructs.
Cell Culture--
PS127A cells (Chinese hamster lung fibroblasts
that overexpress human NHE1) and PS120 cells (Chinese hamster lung
fibroblasts deficient in NHE1) were gifts of Dr. J. Pouysségur
(University of Nice, France). All cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum (FCS). Cells were serum-starved (0% FCS) overnight prior to experiments.
Preparation of Cell Lysates--
Control PS127A and PS120 cells
and cells stimulated with 20% FCS for 5-40 min, with or without
PD98059 (Calbiochem), were harvested using NHE1 lysis buffer (30 mM Tris, pH 8, 10 mM NaCl, 5 mM
EDTA, 10 g/liter polyoxyethylene-8-lauryl ether, 1 mM
o-phenanthroline, 1 mM indoacetamide, 10 mM NaF, 5 mM Na3VO4, 10 mM sodium pyrophosphate). Cells were immediately frozen on
ethanol/dry ice, and the cell lysates were then thawed on ice, scraped,
sonicated, and centrifuged at 14,000 × g at 4 °C
for 15 min. Supernatants were used immediately.
14-3-3 Pull-down--
Cell lysates containing 500 µg of
protein were incubated overnight at 4 °C with 10 µg of GST-14-3-3
bound to glutathione-Sepharose. The beads were washed two times with 1 ml of NHE1 lysis buffer, once with 1 ml of LiCl wash buffer (500 mM LiCl, 100 mM Tris-Cl, pH 7.6, 0.1% Triton
X-100, and 1 mM dithiothreitol), and two times more with 1 ml of lysis buffer before the addition of boiling Laemmli buffer.
Proteins were resolved on SDS-PAGE and transferred to nitrocellulose
membranes for Western analysis. Anti-NHE1 antibody was obtained from Chemicon.
Plasmid Construction and Cell Transfections--
Human NHE1
cDNA was a gift from Dr. L. Fliegel (University of Alberta,
Canada). Serine 703 was mutated to alanine using polymerase chain
reaction as described earlier (20). All other mutations were done using
a QuikChange mutagenesis kit (Stratagene). Briefly, sense and antisense
primers were designed to contain the desired mutation, indicated below
by the lowercase letters, and to anneal to the same sequence on
opposite strands of the plasmid. The following sense primers were used
(antisense primers were the identical sequence in reverse):
CGCATCGGCTCAGACgCACTGGCCTATGAGCCG (NHE1 P705A),
ACCAGGCAGCGGCTGCGGgCCTACAACAGACACACGCTG (NHE1 S648A), GTCATCACCATCGACCCGGCgTCCCCGCAGTCACCCGAGTC (NHE1 S723A),
ACCCGGCTTCCCCGCAGgCACCCGAGTCTGTGGAC (NHE1 S726A),
TCCCCGCAGTCACCCGAGgCTGTGGACCTGGTGAATG (NHE1 S729A), and
TCTCTGTTGCCTACgAGAATGTGGTAGGTGC (14-3-3 K49Q). The primers were
extended with Pfu polymerase, resulting at length in the generation of mutated plasmids containing the desired nicks, and DpnI was added to the final product to digest the parental
DNA. After the correct sequence was confirmed, the cDNA was
subcloned into pcDNA/3.1(+) (Invitrogen Inc.) to transfect
mammalian cells or into pGEX-2TK (Amersham Pharmacia Biotech) to
express it as GST fusion protein.
PS120 cells were seeded at 40% confluence onto 60-mm dishes 24 h
prior to transfection and transfected with 4 µg of plasmid DNA using
the LipofectAMINE Plus method (Qiagen). After 3 h of incubation
with the DNA-lipid complexes, the cells were refed with
serum-containing medium. Two days post-transfection, the transfected
cells were trypsinized and seeded onto three 100-mm dishes in medium
containing 1 mg/ml neomycin. After 1 week of selection, cell colonies
were picked and expanded into individual cultures. Transfected PS120
cells were maintained in Dulbecco's modified Eagle's medium, 10% FCS
with 1 mg/ml neomycin, and every 2 weeks cells were subjected to acid
selection to maintain high level expression of NHE1 as described
previously (20). In brief, cells were exposed to 50 mM
NH4Cl in TBSS (135 mM NaCl, 5 mM
KCl, 1.5 mM CaCl2, 1.0 mM
MgCl2, 200 mg/dl glucose, 20 mM HEPES, 20 mM Tris, pH 7.4) for 1 h followed by low NaCl/choline
Cl solution buffer (130 mM choline Cl, 5 mM
NaCl, 5 mM KCl, 1.5 mM CaCl2, 1.0 mM MgCl2, 200 mg/dl glucose, 20 mM
HEPES, 20 mM Tris, pH 6.5) for 1 h.
PP1
Protection Assay--
GST-NHE1-(625-747) fusion protein
was phosphorylated by p90 RSK2 in HMK buffer (20 mM Tris,
pH 7.4, 12 mM MgCl2, 100 mM NaCl) at 37 °C overnight. One µg of GST-NHE1 was incubated with 40 µg of GST or 10 µg of GST-14-3-3 at 20 °C for 1 h, and then
protein phosphatase 1
was added for 5-60 min. The phosphatase
reaction was stopped by the addition of boiling Laemmli buffer.
Proteins were resolved on SDS-PAGE, and phosphorylation state was
evaluated by autoradiography.
Intracellular pH (pHi)
Measurement--
Na+/H+ exchange was
determined by ethyl isopropyl amiloride-sensitive pHi recovery
following acid loading as described previously using the fluorescent
pH-sensitive dye, acetoxymethyl ester of
2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) (Molecular Probes, Inc., Eugene, OR). Fluorescent measurements (excitation at 490 and 450 nm with emission at 530 nm) were made with a
PTI Delta scan spectrofluorometer. Briefly, cells were grown on glass
coverslips and growth-arrested at 70-80% confluence by incubation in
0% FCS/Dulbecco's modified Eagle's medium for 24 h prior to
use. Cells were loaded with 3 µM acetoxymethyl ester of
BCECF-AM in serum-free Dulbecco's modified Eagle's medium at 37 °C for 30 min, washed twice, and incubated in a HEPES-Tris balanced salt solution (130 mM NaCl, 5 mM KCl,
1.5 mM CaCl2, 1.0 mM
MgCl2, and 20 mM HEPES buffered to pH 7.4 with
Tris base) for 30 min. All experiments were then performed at 25 °C.
Cells were acid-loaded by incubation with Tris-buffered salt solution
(130 mM NaCl, 5 mM KCl, 1.5 mM
CaCl2, 1.0 mM MgCl2, 20 mM HEPES buffered with Tris base to pH 7.4) containing 20 mM NH4Cl, followed by Tris-buffered salt
solution with 0 mM NH4Cl. Cell pH recovery was
recorded over the next 10 min. Afterward, the nigericin/high K+ technique with 20 µM nigericin in KCl
solution (130 mM KCl, 10 mM HEPES, pH 7.4-6.5)
was used to calibrate the relationship between excitation ratio
(F490/F450) and
pHi. In serum stimulation experiments, 20% FCS was added to
Tris-buffered salt solution and nigericin solutions. The rate of
pHi recovery was converted to mmol of H+/min/liter
of cells (JH) by multiplying by the buffering power.
Buffering power was calculated from the change in cell pHi
observed upon NH4Cl addition. The data were then plotted as
mmol of H+/min/liter of cells (JH)
versus pHi.
Statistics--
Values presented are means ± S.E.
Student's t test or analysis of variance was used when
appropriate. p values of <0.05 were considered statistically significant.
 |
RESULTS |
GST-NHE1 Constructs That Encompass Ser703 Bind
14-3-3--
We have previously demonstrated that serum stimulates the
phosphorylation of NHE1 at serine 703 (20). This serine is contained within the sequence RIGSDP, which is similar to established recognition motifs of 14-3-3 proteins. To determine whether 14-3-3 binds to NHE1,
we constructed three GST fusion proteins corresponding to amino acids
516-630, 625-747, or 748-815, together comprising the entire
cytoplasmic tail of the exchanger (Fig.
1). Using these GST fusion proteins in a
far Western assay, we determined that 14-3-3 binds the region
encompassing amino acids 625-747, whereas its association with the
other two portions of the cytoplasmic tail or to GST alone was
negligible (Fig. 1). Two additional GST fusion proteins spanning amino
acids 625-670 or 625-714 were used to define the region that binds
14-3-3. Only GST-NHE1-(625-714) interacted with 14-3-3, indicating
that 14-3-3 binds NHE1 between amino acids 670 and 714. Appropriately,
this region contains serine 703 and our proposed 14-3-3 binding
motif.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1.
GST-14-3-3 binds NHE1-(625-714) and
NHE1-(625-747). Five different GST-NHE1 constructs,
GST-NHE1-(516-630), GST-NHE1-(625-747), GST-NHE1-(748-815),
GST-NHE1-(625-670), and GST-NHE1-(625-714), spanning the entire
cytoplasmic tail of the exchanger, were prepared. These constructs were
resolved on SDS-PAGE and probed with labeled GST-14-3-3. Equal protein
loading was confirmed by Ponceau staining (not shown). Autoradiography
revealed that GST-14-3-3 only bound GST-NHE1-(625-714) and
GST-NHE1-(625-747). Identical results were obtained by probing
GST-14-3-3-containing membranes with labeled GST-NHE1 constructs (not
shown). Hence, 14-3-3 interacts with NHE1 at residues located between
amino acids 670 and 714, containing Ser703 and the proposed
14-3-3 binding motif RIGSDP.
|
|
Serum-stimulated Association of 14-3-3 with NHE1 in Cultured Cells
Is Blocked by PD98059--
It is well documented that serum and growth
factors stimulate NHE1 activity and phosphorylation. To confirm that
14-3-3 associates with NHE1 in cells, we stimulated PS127 fibroblasts
that overexpress NHE1 with 20% FCS and immunoprecipitated NHE1 from
the cell lysates. Coprecipitation of 14-3-3 with NHE1 was enhanced in
serum-stimulated cells, whereas NHE1 immunoprecipitation was equivalent
in all conditions (Fig. 2A).
To compensate for variable quality of NHE1 antibody lots, we also
verified 14-3-3 association with NHE1 using glutathione-Sepharose-bound
GST-14-3-3 in a pull-down assay. Binding of NHE1 to 14-3-3 was
increased 5.2 ± 0.4-fold in cells stimulated for 5 min and
remained elevated (3.9 ± 0.3-fold) after 40 min (Fig.
2B). In comparison, GST-Sepharose alone did not pull down the exchanger from serum-stimulated cells.

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 2.
GST-14-3-3 binding to NHE1 from
serum-stimulated cells is inhibited by PD98059. PS127 cells were
stimulated with 20% FCS for 0 (control) or 5-40 min, and NHE1 was
immunoprecipitated (IP) from the cell lysates
(A). Precipitated proteins were separated by SDS-PAGE,
transferred to polyvinylidene difluoride, and immunoblotted
(IB) with anti-14-3-3 or anti-NHE1 antibody. Coprecipitation
of 14-3-3 with the exchanger was greater in serum-stimulated cells than
in controls, whereas NHE1 precipitation was equivalent in all
conditions. B, cell lysates from serum-stimulated cells were
also used in a pull-down assay with GST or GST-14-3-3 bound to
glutathione-Sepharose. PPT, precipitated. NHE1 from
serum-stimulated cells bound GST-14-3-3 but not GST. Furthermore, 1-h
pretreatment with the MEK1 inhibitor PD98059 (50 µM)
inhibited serum-stimulated association of NHE1 with GST-14-3-3. Results
are mean ± S.E., n = 4-5. *, p < 0.05; **, p < 0.01 versus control (0 min).
|
|
We have found that serum stimulation of cells leads to phosphorylation
of serine 703 and that this phosphorylation is greatly diminished in
the presence of the MEK1 inhibitor PD98059. We investigated whether
MEK1 inhibition also interferes with binding of 14-3-3 to NHE1. PS127
cells were incubated with PD98059 for 1 h prior to serum
stimulation. Fig. 2B confirms that 50 µM
PD98059, shown to prevent agonist-stimulated ERK1/2 and RSK activation
and Na+/H+ exchange (12, 14, 15, 26),
completely abolished serum-stimulated association of 14-3-3 with NHE1.
1 µM PD98059 had no effect (data not shown). Based on the
findings that 14-3-3 binds a region spanning amino acids 670-714,
containing serine 703, and that both association of 14-3-3 with NHE1
and phosphorylation of serine 703 in serum-stimulated cells is
sensitive to PD98059, we sought to confirm that Ser(P)703
lies within the 14-3-3 binding motif of NHE1.
Serum Fails to Stimulate Association of 14-3-3 with NHE1 Mutated at
Serine 703 or Proline 705--
To establish that 14-3-3 binds NHE1 at
serine 703, PS120 cells (which are deficient in NHE1) were stably
transfected with the NHE1 S703A mutant described previously (20). Serum
stimulation failed to promote interaction of 14-3-3 with NHE1 S703A, as
demonstrated by a pull-down assay (Fig.
3). Hence, Ser703 plays a
major role in the interaction between NHE1 and 14-3-3, and
phosphorylation of this serine is likely to be a requirement for
association of these two proteins.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 3.
NHE1 S703A and NHE1 P705A mutants fail to
interact with GST-14-3-3. PS 120 cells were stably transfected
with wild type NHE1 (NHE wt), NHE1 S703A, or NHE1 P705A
(A). Cells were stimulated with 20% FCS for the indicated
times and used in a GST-14-3-3 pull-down assay. Precipitated
(PPT) proteins were separated by SDS-PAGE,
transferred to polyvinylidene difluoride, and immunoblotted
(IB) with anti-NHE1 antibody. GST-14-3-3 bound only wild
type NHE1 from serum-stimulated cells. B, specificity of
interaction of 14-3-3 with Ser703 and Pro705
was confirmed using PS 120 cells (NHE1 null), PS 127 cells, or PS 120 cells stably transfected with NHE1 S703A, NHE1 P705A, NHE1 S648A, NHE1
S723A, NHE1 S726A, or NHE1 S729A and stimulated for 5 min with 20%
FCS. From each cell lysate, 500 µg of protein were used in a
GST-14-3-3 pull-down assay, and 20 µg protein were assayed directly
by Western blot using an anti-NHE1 antibody. GST-14-3-3 pulled down all
NHE1 mutants from serum-stimulated cells except S703A and P705A,
proving specificity of interaction of 14-3-3 with the NHE1 motif
containing Ser703 and Pro705. Results are
mean ± S.E., n = 4. **, p < 0.01 versus control (0 min).
|
|
Some recent publications report that 14-3-3 binds to proteins within a
motif bearing not only a phosphorylated serine but also a proline in
the +2-position (24). Based on those data, we postulated that mutating
proline 705 to alanine would also prevent binding of 14-3-3 to the
exchanger. Indeed, in PS120 cells stably transfected with NHE1 P705A,
14-3-3 could no longer pull down NHE1 despite serum stimulation (Fig.
3).
The specificity of the interaction of 14-3-3 with the motif
encompassing Ser703 and Pro705 was further
confirmed using stable transfectants of four other serine to alanine
mutants of NHE1 in the same region of the cytoplasmic tail (amino acids
625-747) that we identified as potential kinase substrates. Fig.
3B illustrates that in cells stimulated with 20% serum for
5 min, the S648A, S723A, S726A, and S729A mutants of NHE1 were
successfully pulled down by 14-3-3, unlike the S703A or P705A mutants,
despite equivalent expression levels of all mutants in PS120 cells.
Hence, our combined data prove that the RIGSDP sequence at amino acids
700-705 is the key motif for binding of 14-3-3 to NHE1 and that other
neighboring serines are not necessary for this association.
The K49Q Mutant of 14-3-3 No Longer Binds
NHE1--
14-3-3 primarily binds ligands via a phosphorylated serine.
In the 14-3-3
isoform, several critical amino acids lying within its
ligand binding groove that make contact with phosphoserine have been
identified including lysine 49, which when mutated reduces the
interaction of 14-3-3
with its binding partners Bcr, Raf (27), and
Cbl (28). Therefore, we constructed a K49Q mutant of
GST-14-3-3
and tested whether it would pull down NHE1 from serum-stimulated cells. As depicted in Fig.
4, lysine 49 is essential for binding of
14-3-3 to NHE1, since replacement of this single amino acid abolished
serum-stimulated association of 14-3-3 with NHE1.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 4.
The 14-3-3 K49Q mutant does not bind
NHE1. PS 127 cells were stimulated with serum for 0-40 min and
lysed for use in a pull-down assay with GST-14-3-3 or GST-14-3-3 K49Q.
Precipitated (PPT) proteins were resolved by SDS-PAGE,
transferred to polyvinylidene difluoride, and probed with anti-NHE1
antibodies. IB, immunoblot. The single point mutation at
Lys49 completely prevented interaction of 14-3-3 with NHE1.
Results are mean ± S.E., n = 3. **,
p < 0.01.
|
|
14-3-3 Protects NHE1 from Dephosphorylation--
14-3-3 proteins
are known to bind to phosphoserine, and one of their key roles may be
prevention of dephosphorylation of the serines by phosphatases such as
PP1 and PP2. PP1 has been proposed to be a NHE1 phosphatase, based on
findings that okadaic acid prevents dephosphorylation of NHE1 (29). To
determine the ability of 14-3-3 binding to protect the exchanger from
dephosphorylation, we developed a biochemical assay in which
GST-NHE1-(625-747) phosphorylated by RSK was exposed to PP1
for
differing times in the presence of GST or GST-14-3-3. Dephosphorylation
of GST-NHE1-(625-747) incubated with GST alone was marked within 5 min
of PP1
addition and progressed steadily thereafter (to 65, 41, and
27% of initial phosphorylation at 15, 30, and 60 min, respectively;
Fig. 5A). In contrast,
dephosphorylation of GST-NHE1-(625-747) was significantly slower when
incubated with GST-14-3-3 before exposure to PP1
(89, 87, and 66%,
respectively of initial phosphorylation at the same times;
p < 0.05 compared with GST alone; Fig. 5A).
The protective effect of GST-14-3-3 was completely lost when incubated
with the GST-NHE1 P705A mutant that cannot bind 14-3-3 (Fig.
5B). Similarly, the GST-14-3-3 K49Q mutant, which does not
associate with NHE1, was unable to protect wild-type GST-NHE1 from
dephosphorylation by PP1
more efficiently than GST alone (Fig.
5C). Hence, binding of 14-3-3 to NHE1 protects phosphoserine
703 from phosphatases and may contribute to maintaining the exchanger
in a phosphorylated, active state.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 5.
14-3-3 protects GST-NHE1-(625-747) from
dephosphorylation by PP1 .
GST-NHE1-(625-747) or GST-NHE1-(625-747) P705A was phosphorylated by
RSK with [ -32P]ATP and then incubated with GST
or GST-14-3-3. PP1 was added for 0 (control) or 5-60 min.
Thereafter, proteins were boiled in Laemmli buffer and separated by
SDS-PAGE, and GST-NHE1 phosphorylation was quantified by
autoradiography. A, incubation with GST-14-3-3 reduced
dephosphorylation of GST-NHE1 by PP1 significantly compared with
incubation with GST. B, the protective effect of GST-14-3-3
was lost on the GST-NHE1 P705A mutant, which does not bind 14-3-3. Similarly, the GST-14-3-3 K49Q mutant, which fails to interact with
NHE1, could no longer protect GST-NHE1 from dephosphorylation by PP1
(C). Results are mean ± S.E., n = 3-5. *, p < 0.05; **, p < 0.01.
|
|
Binding of 14-3-3 Is Essential for Activation of NHE1 by
Serum--
We have previously demonstrated that serine 703 plays a
major role in NHE1 function, since mutation to alanine prevents
serum-stimulated NHE1 activation (20). We hypothesized that association
of 14-3-3 with NHE1 contributes to activation of the exchanger by
serum, since phosphorylation of serine 703 correlates with enhanced
exchanger activity (20) and binding of 14-3-3 to NHE1 maintains
Ser(P)703 in a phosphorylated state. To test this
hypothesis, we evaluated pHi recovery from acid loading in
cells expressing wild type NHE1 or the NHE1 P705A mutant. Fig.
6A depicts typical pHi recovery curves of acid-loaded cells in the presence and absence of
serum. PS120 cells stably expressing wild type NHE1 rapidly recovered
pHi and reached pHi = 6.98 ± 0.09 by 150 s.
Treatment with 20% serum accelerated pH recovery in these cells, such
that pHi reached 7.30 ± 0.05 at 150 s
(p < 0.01 versus untreated). PS120 cells
stably expressing the NHE1 P705A mutant demonstrated pHi
recovery in unstimulated cells similar to that found in cells with
wild-type NHE1, reaching pHi = 6.86 ± 0.11 at 150 s.
However, serum failed to stimulate exchanger activity in these cells,
since pHi recovered only to 6.97 ± 0.09 at 150 s
(Fig. 6B), similar to our previous observations with the
NHE1 S703A mutant (20).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 6.
Serum stimulation does not accelerate pH
recovery in cells transfected with the NHE1 P705A mutant. PS 120 cells stably transfected with wild type NHE1 (NHEwt) or NHE1
P705A were acid-loaded by incubation with 20 mM
NH4Cl followed by 0 mM NH4Cl in 130 mM NaCl buffer at pH 7.4. pH recovery curves were plotted
from the point of minimum pH (0 s). A, the rate of pH
recovery was moderate in PS 120 cells transfected with wild type NHE1
under control conditions. Treatment with 20% FCS accelerated pH
recovery in these cells. In contrast, the rate on pH recovery in PS 120 cells transfected with NHE1 P705A was equivalent under control and 20%
FCS conditions (B). C, the rate of proton flux
(JH) during pH recovery was calculated for PS 120 cells transfected with wild type NHE1 or NHE1 P705A. At pH 6.8 (D) and throughout pH recovery, only serum-stimulated cells
transfected with wild type NHE1 showed increased JH.
Results are mean ± S.E., n = 4-6. *,
p < 0.05; **, p < 0.01.
|
|
Since serum activates NHE1 by increasing affinity of the exchanger for
intracellular H+ (30), we evaluated the kinetics of
H+ flux in the PS120 transfectants. As shown in Fig. 6,
C and D, serum stimulation of wild-type
NHE1-expressing cells stimulated the rate of H+ flux
characterized by an increase in the JH from
0.22 ± 0.03 to 0.48 ± 0.05 H+/min/liter of
cells at pH 6.8 (p < 0.01). Note that the
JH values were lower than those we obtained
previously (20), since experiments were performed at 25 °C. In
contrast, JH was not increased by serum in cells
expressing NHE1 P705A (0.13 ± 0.02 and 0.14 ± 0.04 H+/min/liter for cells with and without serum, respectively).
Our combined observations support a model in which agonist-induced
phosphorylation of NHE1 at Ser703 creates a binding site
for 14-3-3 (Fig. 7). In fact, a critical role is indicated for 14-3-3, since mutation of either amino acid required for 14-3-3 binding (Ser703 and Pro705)
prevents serum stimulation of Na+/H+ exchange.
It would have been interesting to assay NHE1 activity in cells
transfected with a dominant negative form of 14-3-3. However, these
experiments would be confounded by the fact that dominant negative
14-3-3 mutants also block activation of ERK1/2 (31), which is required
for phosphorylation of NHE1 Ser703.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 7.
Schematic representation of interaction
between NHE1 and 14-3-3. In unstimulated cells, 14-3-3 does not
bind to NHE1. Upon serum or growth factor stimulation of cells, the MAP
kinase cascade is activated, RSK phosphorylates NHE1 at
Ser703, and 14-3-3 binds to the exchanger at the RIGpSDP
motif. Both Ser703 and Pro705 are necessary for
binding of 14-3-3 to NHE1 and for activation of the exchanger by serum,
and binding of 14-3-3 and/or another
Ser703/Pro705-interacting protein is essential
for Na+/H+ exchange activation.
|
|
 |
DISCUSSION |
The major finding of the present study is that 14-3-3 interacts
with NHE1 in serum-stimulated cells, and our data establish a new
functional role for 14-3-3 proteins in the regulation of intracellular
pH. We previously found that p90 RSK phosphorylated NHE1
Ser703 in response to serum and that mutation of serine 703 to alanine inhibited serum stimulation of the exchanger. The present
study extends these observations and shows that 14-3-3 binds NHE1 at phospho-Ser703 and is a key determinant of exchanger
activation by serum. Our results provide the first evidence for
interaction of 14-3-3 with the Na+/H+ exchanger
and indicate a new role for 14-3-3 proteins as regulators of
intracellular pH.
While several pathways have been proposed for serum regulation of NHE1,
it is clear that phosphorylation is an essential mechanism (7, 8, 20).
The present study defines a critical role for 14-3-3 and serine 703 of
NHE1. Previously, deletion of the carboxyl-terminal 180 amino acids of
NHE1 was found to reduce agonist-induced activation of the exchanger by
~50% (32). It should be noted that removal of such a large portion
of the cytoplasmic tail may have altered its conformation and modified
exchanger properties, producing ambiguous results. In comparison,
single point mutations of all serines in the 635-670 range did not
alter the extent of growth factor-induced NHE1 activation (32). We recently found that phosphorylation of Ser703 is crucial
for activation of NHE1 by serum, since a S703A mutation abolishes
serum-stimulated activation of the exchanger (20). Consistent with
these data, the present study shows that phosphorylation of
Ser703 creates a binding site for 14-3-3. The association
of 14-3-3 with the exchanger via Ser(P)703 provides a
mechanistic explanation for the effects of the S703A mutation on
stimulation of NHE1 activity by serum.
In addition to 14-3-3, four other NHE1-interacting proteins have been
identified that may modulate exchanger activity: calmodulin (33),
70-kDa heat-shock protein (HSP70) (34), a calcineurin analogue protein
(CHP) (35), and a 24-kDa protein yet to be identified (36). Recently,
phosphatidylinositol 4,5-bisphosphate (37) was shown to interact with
NHE1 as well. Although a role for HSP70 in the negative regulation of
the exchanger remains to be demonstrated directly, its interaction with
NHE1 is hindered in the presence of ATP, the depletion of which reduces
NHE1 activity (34). CHP may also modulate NHE1 activity, since
transient overexpression of this protein inhibits serum- and
GTPase-stimulated NHE1 activity (35). In comparison, there is
compelling evidence that interaction of calcium-calmodulin with NHE1
relieves the exchanger from a negative constraint (33, 38). Finally,
Aharonovitz et al. (37) found that elimination of the
putative phosphatidylinositol 4,5-bisphosphate-binding sites on NHE1
greatly reduced the ATP-sensitive fraction of
Na+/H+ exchange and that an additional,
phosphoinositide-independent mechanism also contributes to the effect
of ATP on NHE1.
We suggest that the interaction of 14-3-3 with NHE1
modulates exchanger activity by two mechanisms: preventing
dephosphorylation and stabilizing an active conformation. It is clear
that binding of 14-3-3 to the exchanger prolongs the phosphorylation of
Ser703. Our results agree with an earlier report describing
increased phosphorylation state of NHE1 accompanied by enhanced
exchanger activity in cells treated with okadaic acid, which inhibits
PP1 and PP2 (29). The effects of okadaic acid and thrombin were additive and corresponded to phosphorylation of the same NHE1 phosphopeptide, which we expect contains Ser(P)703.
Likewise, expression of SV-40 small-t antigen, which binds and inhibits
protein phosphatase 2A, increased phosphorylation of NHE1 (39). In
addition to this dephosphorylation, 14-3-3 probably exerts other
effects such as stabilization of an active conformation, as has been
demonstrated for interaction of 14-3-3 with Raf1 (40). Specifically,
14-3-3 may promote dimer formation at Ser703 (24, 28), or
assembly of additional signal transduction proteins via a scaffold
function (41).
Binding of 14-3-3 to NHE1 is likely to have broad implications for cell
pH regulation, since 14-3-3 proteins are ubiquitously expressed and the
MEK-ERK-RSK pathway is activated by many stimuli. The present study
shows that MEK1 inhibition completely prevents binding of 14-3-3 to
NHE1 in cells stimulated with serum. Previous reports show that
inhibition of MEK1 or use of a dominant negative ERK1 construct
prevents activation of NHE1 by serum, platelet-derived growth factor,
angiotensin II, thrombin, phorbol esters, vasopressin,
1-adrenergic receptor agonists, and
H2O2 (10, 13-16, 26, 37, 42).
Association with 14-3-3 probably participates in the activation of NHE1
by all stimuli acting through the MEK-ERK-RSK pathway, since RSK
regulates phosphorylation of Ser703 (20), which we found
here to be essential for its binding to 14-3-3.
Our laboratory has focused on mechanisms that regulate NHE1 activity,
because enhanced NHE1 activity is a ubiquitous feature of hypertension
in humans and several animal models, but how this occurs remains
undefined. The present study suggests that alterations in 14-3-3 expression and/or function may be an important component in the
pleiotropic alterations in signal transduction observed in
hypertension. Increased NHE1 activity in hypertension is best explained
by post-translational modification of the exchanger, and indeed
phosphorylation of the exchanger is augmented in cells from
hypertensive patients (43) and the SHR (7, 44). The present data
suggest that increased binding of 14-3-3 to the exchanger, possibly
subsequent to increased activity of kinases that phosphorylate Ser703, could contribute to activation of the exchanger in
hypertension. Upstream differences in regulation of MAP kinase
signaling distinguishing SHR VSMC from WKY VSMC have already been
established (18, 19), and our group has characterized a 90-kDa NHE1
kinase, now proven to be RSK, which shows greater activity in SHR than
WKY VSMC (10). Furthermore, another NHE1 kinase, p160 ROCK (9), has
been implicated in the pathophysiology of hypertension (45). A
mechanistic link between these upstream kinases and 14-3-3 is suggested
by evidence that 14-3-3 interacts with Raf-1 and several MAP kinase
kinases (MEKK1, MEKK2, and MEKK3), although this interaction has not
been shown to influence MEKK activity (41). We suggest therefore that a
change in the function of 14-3-3 proteins (increased expression and/or
altered binding) that promotes increased interaction with 14-3-3 binding partners such as NHE1 (and altered binding partner activity)
may be a pathophysiologic feature of hypertension.