1 Centre for Cardiovascular Biology and Medicine, King's College London, London, United Kingdom; and 2 Department of Medicine, University of California Los Angeles School of Medicine and Molecular Biology Institute, Los Angeles, California
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ABSTRACT |
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The regulation of plasma membrane Na+/H+ exchanger (NHE) activity by protein kinase D (PKD), a novel protein kinase C- and phorbol ester-regulated kinase, was investigated. To determine the effect of PKD on NHE activity in vivo, intracellular pH (pHi) measurements were made in COS-7 cells by microepifluorescence using the pH indicator cSNARF-1. Cells were transfected with empty vector (control), wild-type PKD, or its kinase-deficient mutant PKD-K618M, together with green fluorescent protein (GFP). NHE activity, as reflected by the rate of acid efflux (JH), was determined in single GFP-positive cells following intracellular acidification. Overexpression of wild-type PKD had no significant effect on JH (3.48 ± 0.25 vs. 3.78 ± 0.24 mM/min in control at pHi 7.0). In contrast, overexpression of PKD-K618M increased JH (5.31 ± 0.57 mM/min at pHi 7.0; P < 0.05 vs. control). Transfection with these constructs produced similar effects also in A-10 cells, indicating that native PKD may have an inhibitory effect on NHE in both cell types, which is relieved by a dominant-negative action of PKD-K618M. Exposure of COS-7 cells to phorbol ester significantly increased JH in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M (because basal JH was already near maximal). A fusion protein containing the cytosolic regulatory domain (amino acids 637-815) of NHE1 (the ubiquitous NHE isoform) was phosphorylated in vitro by wild-type PKD, but with low stoichiometry. These data suggest that PKD inhibits NHE activity, probably through an indirect mechanism, and represents a novel pathway in the regulation of the exchanger.
pH regulation; COS-7; A-10; green fluorescent protein; sodium/hydrogen exchanger type 1; protein kinase Cµ
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INTRODUCTION |
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IN RECENT YEARS, the type 1 isoform of the Na+/H+ exchanger (NHE1) has been the focus of considerable investigative effort as an important modulator of cellular function. Indeed, altered activity of this exchanger has been implicated in such diverse pathologies as cancer (17), epilepsy (5), hypertension (20), arterial restenosis (18), myocardial hypertrophy (40), and postischemic cardiac dysfunction (31). NHE1 is predicted to consist of two domains: an NH2-terminal membrane-bound transport domain and a COOH-terminal cytoplasmic regulatory domain (25, 37). The exchanger is quiescent at physiological intracellular pH (pHi) but is activated rapidly in response to intracellular acidosis via an allosteric modifier site in its transport domain (36). Several stimuli, including growth factors, hormones, hypertonic stress, and mechanical stretch, also increase NHE1 activity through various signaling pathways (25, 37). These include direct phosphorylation of the cytoplasmic regulatory domain (28), binding of calmodulin (2), and interaction with accessory proteins [e.g., CHP (23)]. Although NHE1-regulatory roles have been suggested for several kinase pathways (4, 22, 26, 33), the most extensively studied kinase-mediated mechanism of NHE1 activation involves protein kinase C (PKC). Thus activation of PKC, either directly by phorbol esters (8, 38) or indirectly via cell surface receptors (29, 38, 41), leads to increased exchanger activity in a variety of cell types, and putative PKC inhibitors inhibit such effects (38, 41). There is evidence that negative regulators of NHE1 activity also exist (21, 24), but less is known about inhibitory signaling pathways.
The recently identified protein kinase D [PKD; also known as PKCµ (16)] is a novel serine/threonine kinase (34) that has distinct structural and enzymatic properties and is found in most tissues [see review by Rozengurt et al. (27)]. The catalytic domain of PKD is distantly related to Ca2+-regulated kinases and shows little similarity to the highly conserved regions of the kinase subdomains of the PKC family. Consistent with this, PKD does not phosphorylate a variety of substrates utilized by PKCs (34, 35), indicating that PKD is a protein kinase with distinct substrate specificity. The NH2-terminal region of PKD contains a tandem repeat of cysteine-rich motifs that bind phorbol esters with high affinity (34, 35), and immunopurified PKD is stimulated in vitro by either diacylglycerol or biologically active phorbol esters in the presence of phosphatidylserine (35). More recently, a second mechanism of PKD activation has been identified, which involves phosphorylation of PKD via a PKC-dependent pathway (43).
In light of the evidence that PKD can be activated in parallel with or downstream of PKC, we determined whether PKD may be involved in the regulation of NHE1 activity. The results presented here suggest that PKD mediates a novel inhibitory pathway in the regulation of the exchanger and counteracts the stimulatory effects of the PKC pathway.
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EXPERIMENTAL PROCEDURES |
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Cell culture. Stock cultures of COS-7 (African Green monkey kidney) and A-10 (rat aortic smooth muscle) cells were maintained at 37°C in DMEM supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO2. For experimental purposes, cells were plated in 90-mm dishes, with or without acid-washed 15-mm diameter glass coverslips, at 6 × 105 cells/dish.
Transfection of cultured cells. The
wild-type PKD cDNA fragment spanning bases 125 to 3179 was
inserted into the mammalian expression vector pcDNA3, as described
earlier (35). The kinase-deficient mutant PKD-K618M was also inserted
into pcDNA3 (43). cDNA for green fluorescent protein (GFP), in the
mammalian expression vector pCAGGS, was a kind gift from Drs L. Wightman and M. Marber (Department of Cardiology, St. Thomas'
Hospital, London, UK).
Cultured cells (40-50% confluent) were transfected with the various plasmids using Lipofectin (Life Technologies), as recommended by the manufacturer. A total of 12 µg DNA was used per 90-mm dish, consisting of 6 µg pCAGGS-GFP and 6 µg pcDNA3, pcDNA3-PKD, or pcDNA3-PKD-K618M. Briefly, DNA was mixed with 1 ml Opti-MEM I (Life Technologies) and then mixed with Lipofectin (20 µl in 1 ml Opti-MEM I). After 20 min, the Lipofectin/DNA complex was diluted to 5 ml with Opti-MEM I and overlaid onto the cells. After overnight incubation, the procedure was completed by the addition of 5 ml of Opti-MEM I containing 20% FBS. Cells were used 24-48 h later.
Western blots. Cultured cells were washed three times with ice-cold PBS and lysed in 1 ml of lysis buffer, which contained 50 mM Tris · HCl (pH 7.5), 2 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol (DTT), 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM 4-(2'-aminoethyl)-benzenesulfonyl fluoride hydrochloride, and 1% Triton X-100. Samples of cell lysate (15 µg protein) were then subjected to SDS-PAGE and transferred to polyvinylidene difluoride membrane (Amersham, Bucks, UK), using a Pharmacia LKB Multiphor II transfer apparatus. Membranes were probed with commercial antibody that recognizes both wild-type PKD and its kinase-deficient mutant (sc-935; Autogen Bioclear UK, Calne, UK). Donkey anti-rabbit horseradish peroxidase-conjugated antibody (Amersham) was used at a dilution of 1:2,000 to label bound antibody, and the antibody complex was detected using the Amersham enhanced chemiluminescence system, as recommended by the manufacturer.
NHE activity measurement.
pHi was measured in single cells
using an adaptation of the microepifluorescence method that we have
used previously in neonatal (9) and adult (9, 31, 41, 42) rat cardiac
myocytes. Cultured cells grown on coverslips were mounted in a 150-µl
volume imaging chamber (Warner RC-25F; Clark Electromedical Intruments,
Reading, UK) and visualized on a Nikon Diaphot 300 inverted microscope,
with a ×40 oil-immersion objective (numerical aperture 1.3). The
cells were loaded with the fluorescent indicator
carboxy-seminaphthorhodofluor-1 (cSNARF-1) by immersion in 5 µM of
its acetoxymethyl ester for 20 min at room temperature, after which
they were superfused with Tyrode solution (in mM: 137 NaCl, 5.4 KCl,
1.8 CaCl2, 0.5 MgCl2, 10 HEPES; pH 7.4 at
34°C) at a flow rate of 2-3 ml/min. To select successfully transfected cells for pHi
measurement, GFP fluorescence was initially visualized following
excitation of cells at 486 nm. A single cell emitting green light was
then selected and framed with adjustable shutters for cSNARF-1
fluorescence measurements. Fluorescence emission at 580 and 640 nm was
detected during excitation at 540 nm, using a dual emission photometer
system [model D-104; Photon Technology International (PTI),
Surbiton, UK] with photon-counting photomultipliers (model 710;
PTI). Emission intensity data were acquired at 2 Hz and stored on disk
using PTI Felix software (version 1.1). The 580:640 emission intensity
ratios were converted to pHi
values by reference to calibration curves constructed using nigericin,
and cellular intrinsic buffering capacity
(i) measurements were made
during progressive washout of extracellular
NH4Cl, as described previously (9,
41). Because experiments were performed in bicarbonate-free medium, the
rates of acid efflux
(JH) estimated during recovery from intracellular acidosis (induced by transient exposure to 20 mM NH4Cl) could be
used as indicators of plasma membrane NHE activity (9, 31, 41, 42). As
described previously (9, 41),
JH was calculated
at various pHi during recovery from acidosis, from the equation
JH = dpHi/dt ·
i,
in which
dpHi/dt is the rate of change of pHi.
Generation of glutathione-S-transferase (GST)-NHE
fusion protein. The regulatory domain of
rabbit NHE1 was expressed as a fusion protein using the vector pGEX-3X
(Pharmacia; see Ref. 32). DNA for the COOH-terminal 178 amino acids of
NHE1 was amplified from rabbit heart cDNA by PCR using the primers
5'-GCG GAT CCT GCA GAA GAC CCG GCA GCG GCT-3' and
5'-AAG AAT TCT ACT GCC CTT TGG GGA TGA-3'. The product
generated was digested with EcoR I and
BamH I and ligated into pGEX-3X.
Fusion protein expression in several strains of
Escherichia coli was tested, as
problems with degradation were encountered using DH5 (32). Fusion
protein expressed in the protease-deficient strain BL21
(ompT
,
lon
) was largely intact
after purification using a glutathione-Sepharose 4B column, as
recommended by the manufacturer (Pharmacia). Attempts to separate GST
from the NHE1 fragment with Factor Xa led to degradation of the NHE1
portion, so intact fusion protein was used for in vitro phosphorylation
assays. GST alone, expressed and purified in the same system, was used
as control.
NHE1 phosphorylation assay. NHE1
phosphorylation by PKD was assessed following immunoprecipitation of
the kinase as described previously (34, 35, 43). Cultured COS-7 cells
were washed three times with ice-cold PBS and lysed in 1 ml of lysis
buffer. PKD was immunoprecipitated from the cell lysate at 4°C for
3 h with the PA-1 anti-peptide serum [1:100 dilution (34)].
Immune complexes were recovered by the addition of 50 µl protein
A-Sepharose (Pharmacia; 100 mg/ml), and pellets were washed three times
with lysis buffer and three times with assay buffer [30 mM
Tris · HCl (pH 7.5), 10 mM
MgCl2 and 1 mM DTT]. The
final pellet was resuspended to a total volume of 40 µl with assay
buffer. To initiate the phosphorylation reaction, 10 µl of
phosphorylation mix [assay buffer containing GST-NHE fusion
protein or GST (10 µg/ml) and 100 µM
[-32P]ATP;
400-600
counts · min
1 · pmol
1]
were added. The mixture was incubated at 30°C for 5 min, and the
reaction was terminated by the addition of hot SDS-PAGE sample buffer.
After boiling for 5 min, the samples were subjected to SDS-PAGE
followed by autoradiography.
Statistical analysis. Data are expressed as means ± SE. Intragroup comparisons were by Student's paired t-test, whereas intergroup comparisons were by ANOVA followed by Bonferroni t-test. P < 0.05 was considered significant.
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RESULTS |
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Characterization of NHE activity in COS-7
cells. To determine plasma membrane NHE activity
selectively in transfected cells, we measured such activity in
individual cells expressing the transfection marker GFP. The
fluorescence characteristics of GFP and cSNARF-1 and the emission
filters used precluded signal interference between the two
fluorophores. To confirm the absence of either such interference or a
GFP-induced change in the pHi
sensitivity of cSNARF-1, we compared the
pHi calibration curves in
nontransfected COS-7 cells and those transfected with GFP.
As illustrated in Fig. 1, the calibration curves from these populations of cells were superimposed, indicating that expression of GFP does not affect the utility of
cSNARF-1 as a pHi indicator.
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Because i is determined largely
by intracellular protein composition and is a critical factor in
calculating JH
(3), we tested whether overexpression of PKD affects
i. To this end,
i was determined at various
pHi in COS-7 cells transfected
with pcDNA3 (control) wild-type PKD, or PKD-K618M, all concomitantly with GFP. Over the pHi range
6.90-7.30 (mean 7.07 ± 0.01), there was no significant
difference between the three groups in
i (30.2 ± 1.5, 32.8 ± 2.0, and 31.8 ± 1.5 mM, respectively;
n = 10 cells/group), indicating that
overexpression of PKD has no effect on
i in these cells.
To exclude an involvement of bicarbonate-dependent mechanisms in pHi regulation, all microepifluorescence experiments were carried out in bicarbonate-free medium; under these conditions NHE represents the primary route of acid extrusion in other cell types (9, 17, 41). To verify that this is the case in COS-7 cells, we tested the effect of the NHE inhibitor HOE-642 (30) on pHi recovery from acidosis. At 3 µM, HOE-642 completely inhibited pHi recovery from acidosis after transient exposure to NH4Cl (data not shown). This substantiates that, under the experimental conditions used, NHE activity represents the primary route of acid extrusion. Furthermore, it confirms that such activity arises from the NHE1 isoform, which is the only isoform that has been shown to be expressed in COS-7 cells (24), since at 3 µM HOE-642 is NHE1 selective (30).
Overexpression of PKD-K618M increases NHE activity in
COS-7 cells. To determine whether PKD had any effect on
NHE activity in COS-7 cells, these were transfected with pcDNA3
(control), wild-type PKD, or the kinase-deficient mutant PKD-K618M
before being exposed to intracellular acidosis. Figure
2A shows a
representative Western blot of protein samples from COS-7 cells and
illustrates the presence of native PKD in control cells as well as the
equivalent overexpression of wild-type PKD and PKD-K618M following
transfection. There was no significant difference between the groups in
resting pHi measured before
exposure to NH4Cl (control, 7.31 ± 0.01; wild-type PKD, 7.31 ± 0.01; PKD-K618M, 7.34 ± 0.01;
n = 36-37 cells/group), and a
similar extent of intracellular acidification was achieved in each
group following NH4Cl washout.
Figure 2B shows representative pHi recordings from a control cell
and cells transfected with wild-type PKD or PKD-K618M. As illustrated,
the rates of pHi recovery from
acidosis were similar in the cell transfected with wild-type PKD and
the control cell transfected with empty vector; the salient feature in
Fig. 2 is that recovery from intracellular acidosis was markedly faster
in the cell transfected with PKD-K618M. Figure 2C shows quantitative data, in the
form of
JH-vs.-pHi
curves, from several such experiments
(n = 36-37 cells/group). In cells transfected with wild-type PKD,
JH was slightly
lower than control at pHi < 7.10, although this difference did not reach statistical significance.
In marked contrast, in cells transfected with PKD-K618M, JH was
significantly greater than control over the
pHi range 6.90-7.15. This
indicates that overexpression of the kinase-deficient PKD mutant
increases NHE activity in COS-7 cells.
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Overexpression of PKD-K618M increases NHE activity in
A-10 cells. To determine whether NHE activity is
regulated in a similar manner in cell types other than COS-7, A-10
cells were also transfected with pcDNA3 (control), wild-type PKD, or
the kinase-deficient mutant PKD-K618M. Figure
3A shows a
representative Western blot of protein samples from A-10 cells,
demonstrating once again both the presence of native PKD and the
equivalent overexpression of wild-type PKD and PKD-K618M following
transfection. Over the pHi range
6.60-7.20 (mean 6.96 ± 0.03), there was no significant
difference between the three groups of A-10 cells in
i (19.0 ± 1.2, 19.4 ± 1.8, and 15.9 ± 1.3 mM for control, wild-type PKD, and PKD-K618M groups, respectively; n = 8 cells/group), indicating that, as in COS-7 cells, overexpression of PKD
had no effect on
i.
Nevertheless, mean
i in the
overall population was significantly lower in A-10 cells than in COS-7
cells (18.1 ± 0.8 vs. 31.5 ± 0.9 mM;
P < 0.05).
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There was again no significant difference between the groups in resting pHi measured before exposure to NH4Cl (control, 7.09 ± 0.03; wild-type PKD, 7.09 ± 0.02; PKD-K618M, 7.07 ± 0.02; n = 30-35 cells/group), which was ~0.2 pH unit lower than that observed in COS-7 cells. As in COS-7 cells, pHi recovery from acidosis was inhibited by 3 µM HOE-642 (data not shown), confirming that, in A-10 cells also, such recovery arose from NHE1 activity. Figure 3B shows that JH values were similar in control cells and cells transfected with wild-type PKD but were increased in cells transfected with PKD-K618M over the pHi range 6.85-6.95 (n = 30-35 cells/group). These data indicate that, in common with our finding in COS-7 cells, overexpression of the kinase-deficient PKD mutant markedly increases NHE activity in A-10 cells. This common observation in two distinct cell types suggests that PKD may mediate a novel inhibitory pathway in the regulation of NHE, such that overexpression of its kinase-deficient mutant stimulates exchanger activity by attenuating the inhibitory action of native PKD.
PKD inhibits PKC-induced stimulation of NHE activity
in COS-7 cells. Direct activation of PKC is known to
increase NHE1 activity in many cell types. Consistent with this, we
have verified that in nontransfected COS-7 cells phorbol 12-myristate
13-acetate (PMA) increases plasma membrane NHE activity
{as reflected by increases in
JH at
pHi 7.00 [JH(7.0)],
with an EC50 of 24 nM (Fig. 4A)}.
The data illustrated in Figs. 2 and 3 and the regulatory association
between PKC and PKD led us to hypothesize that cellular PKD activity
may oppose PKC-mediated stimulation of the exchanger. To test this
hypothesis, we determined the response to PMA of NHE activity in COS-7
cells transfected with pcDNA3 (control), wild-type PKD, or the
kinase-deficient mutant PKD-K618M. As would be expected from the data
shown in Fig. 4A, 100 nM PMA
significantly increased
JH(7.0) in
control cells transfected with empty vector (Fig.
4B). In striking contrast, PMA
failed to increase
JH(7.0) in cells
transfected with wild-type PKD, although basal
JH(7.0) was
similar to that observed in control cells (Fig.
4B). This is consistent with our
hypothesis that PKD mediates a novel inhibitory pathway, such that
overexpression of wild-type PKD counteracts the NHE-stimulatory effect
of the PKC pathway.
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Figure 4B additionally shows that PMA failed to increase JH(7.0) also in cells transfected with PKD-K618M. It should be noted, however, that basal JH(7.0) in these cells was greater than that in control cells, which is consistent with the findings reported above (Fig. 2C). Indeed, basal JH(7.0) in cells overexpressing PKD-K618M (7.34 ± 1.55 mM/min) was greater than the JH(7.0) obtained in response to PMA in control cells (5.45 ± 0.53 mM/min). Thus it is likely that PMA did not increase NHE activity in cells transfected with the kinase-deficient PKD mutant, because NHE activity in these cells was already near maximal.
PKD phosphorylates NHE1 in vitro. To
establish whether the observed inhibitory effect of PKD on NHE activity
could arise from direct phosphorylation of the exchanger, we determined
whether the regulatory region of NHE1, corresponding to the
COOH-terminal 178 amino acids, was a substrate for PKD. COS-7 cells,
transiently transfected with wild-type PKD, were treated with 200 nM
PDB (to activate PKD) or vehicle for 10 min and lysed. PKD was
immunoprecipitated from cell extracts with the PA-1 antibody, and the
immune complexes were incubated with
[-32P]ATP in the
absence or presence of the GST-NHE fusion protein (50 kDa). As shown in
Fig. 5A,
and in agreement with previous results (35, 43), PKD activity (as
reflected by PKD autophosphorylation) was markedly increased by PDB
stimulation of intact cells (lanes 1 and 2; ~10-fold increase). GST-NHE
incubated with wild-type PKD showed some phosphorylation under basal
conditions (lane 5), and this was
markedly increased by PDB stimulation, concomitantly with PKD
autophosphorylation (lane 6). These
results indicate that PKD phosphorylates the COOH-terminal regulatory
domain of NHE1 in vitro.
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To confirm that phosphorylation of the NHE fusion protein requires
active PKD, we also transfected COS-7 cells with the kinase-deficient mutant PKD-K618M and incubated PKD immunoprecipitates from these cells
with GST-NHE in the presence of
[-32P]ATP. In
contrast to the results obtained with wild-type PKD, immune complexes
from cells transfected with PKD-K618M did not catalyze GST-NHE
phosphorylation under basal conditions or after PDB stimulation
(lanes 7 and
8). Indeed, consistent with earlier findings (43), PDB stimulation did not increase PKD activity in cells
transfected with the kinase-deficient mutant (lanes
3 and 4). These
results verify that the PDB-inducible NHE1 kinase activity observed in
PA-1 immunoprecipitates was dependent on the kinase activity of PKD.
Additional experiments confirmed that GST alone (30 kDa) was not
significantly phosphorylated by activated PKD, suggesting that
phosphorylation of the GST-NHE fusion protein occurred in the NHE
domain (Fig. 5B).
To establish the potential functional importance of the observed
phosphorylation by PKD of the NHE fusion protein, we determined the
stoichiometry of this phosphorylation. Time course experiments showed
that maximal 32P incorporation into the NHE fusion protein
occurred at 30 min and was maintained for up to 5 h (data not shown).
Subsequent phosphorylation reactions were performed for 2 h, using
known amounts of the GST-NHE fusion protein and the protocol described earlier. After autoradiography, the band corresponding to the fusion
protein was excised from the gel and subjected to liquid scintillation
counting. The stoichiometry of phosphorylation was calculated from the
radioactive count, the specific activity of the
[-32P]ATP used,
and the GST-NHE content of the reaction mix, as 0.068 ± 0.004 mol phosphate/mol fusion protein
(n = 10).
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DISCUSSION |
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The data presented above, which show the effects of changing the cellular PKD composition in intact cells on plasma membrane NHE activity, strongly suggest that PKD mediates a novel NHE-inhibitory pathway, through a mechanism that is dependent on its kinase activity. This conclusion is based to a significant extent on the ability of transfection with the kinase-deficient mutant PKD-K618M to increase plasma membrane NHE activity, in two distinct cell types. Kinase-deficient mutants of an extensive range of protein kinases [e.g., Raf (11), MEK (1), and Akt/PKB (19)] have been shown to exert dominant-negative effects in a variety of systems. Of more direct relevance to our work, recent studies have shown that kinase-deficient PKD/PKCµ mutants can also act in a dominant-negative fashion (14, 15). Thus, although an indirect effect of PKD-K618M cannot be completely discounted, the most likely mechanism underlying our findings is that native PKD exerts an inhibitory effect on NHE activity and that this inhibition is abolished by the dominant-negative action of the kinase-deficient PKD mutant.
Inhibition of NHE by a kinase that is downstream of PKC may at first appear paradoxical, since PKC activation is known to stimulate NHE1 activity in many cell types, as confirmed by our present findings in nontransfected COS-7 cells exposed to PMA. However, our results may be readily reconciled with a hypothetical scheme whereby PKC stimulates NHE activity via a PKD-independent pathway, as well as activating PKD. Indeed, activation of PKD may curb PKC-mediated stimulation of the exchanger (which is supported by our observation that PMA cannot stimulate NHE activity in COS-7 cells that overexpress wild-type PKD), thereby allowing tighter control over NHE activity. In this regard, it would be informative to determine the time course of activation in response to PMA of the NHE-stimulatory pathway and to compare this with the time course of PKD activation. Unfortunately, such a comparison is not possible at present, since the identity of the NHE-stimulatory pathway that is downstream of PKC is unknown. In the context of the hypothesis proposed above, it is also notable that a parallel activation of both NHE-stimulatory and NHE-inhibitory pathways (mediated by ERK1/2 and p38 MAPK, respectively) has recently been reported in cultured rat vascular smooth muscle cells exposed to angiotensin II (21). Thus the PKD pathway may not be unique as an NHE-inhibitory pathway that is activated in parallel with an NHE-stimulatory pathway, in response to a given stimulus.
Despite our observation that transfection with PKD-K618M increases NHE
activity in both COS-7 and A-10 cells, basal
pHi was not affected in either
system by transfection with this kinase-deficient mutant. This is
likely to arise from the absence of an increase in NHE activity at
basal pHi, since at
pHi 7.20 and 7.00 in the COS-7
and A-10 systems, respectively,
JH was almost
identical in cells transfected with PKD-K618M and those transfected
with empty vector. Thus PKD-mediated inhibition of the exchanger may be
of greater physiological impact under conditions of intracellular acidosis (which is commonly encountered in disease conditions such as
ischemia) or during exposure to stimuli that increase cellular
PKC activity (which, as noted above, are associated with stimulation of
NHE activity).
Although considerable progress has been made in the understanding of the cellular regulation of PKD activity (13, 27, 35, 43, 44) since the original cloning and characterization of this enzyme (34), little is known about its cellular substrate(s). The present study indicates, for the first time, that PKD phosphorylates the regulatory COOH-terminal domain of NHE1 in vitro. However, the stoichiometry of this phosphorylation was <0.1 mol phosphate/mol fusion protein. For comparison, earlier studies have revealed stoichiometries of 0.5-1, 1, and 3 mol/mol, respectively, for the in vitro phosphorylation of similar NHE fusion proteins by ERK1/2 (39), p90rsk (21), and Ca2+/calmodulin-dependent protein kinase II (6). Thus, relative to PKD, other putative regulators of NHE activity appear to possess greater efficiency as NHE1 kinases, suggesting that direct phosphorylation may not be the primary mechanism underlying PKD-mediated regulation of the exchanger. Nevertheless, such in vitro data may not reflect accurately the efficiency of PKD as an NHE1 kinase in vivo and do not preclude a role for direct phosphorylation in PKD-mediated regulation of NHE activity in the intact cell. In this regard, recent evidence (33) suggests that a COOH-terminal-truncated NHE1 mutant, in which the region that we have shown to be phosphorylated by PKD in vitro has been deleted, exhibits increased basal activity in vivo. This is consistent with the existence of autoinhibitory domain(s) within the PKD-phosphorylatable region of the exchanger.
In our studies, we have used the expression of GFP as the marker for successful transfection, to enable the measurement of pHi (and thereby NHE activity) selectively in cells transfected with a wild-type or mutant PKD construct. Although we have confirmed that GFP expression does not interfere with the measurement of pHi in single cells, we cannot be certain that 100% of the cells that expressed GFP also expressed the cotransfected PKD construct. At present, it is impossible to determine the proportion of GFP-positive cells that also express the cotransfected PKD construct by conventional approaches such as immunocytochemistry, because available antibodies would not differentiate between native and transfected PKD protein. Nevertheless, in cotransfection experiments, it is generally accepted that cells that take up one expression vector are highly likely to also take up cotransfected vector(s), with numerous examples in the literature. Indeed, in a recent study, Ho et al. (10) have used cotransfection with GFP in a manner similar to ours, to monitor intracellular Ca2+ transients selectively in single cells transfected with various regulators of the ERK pathway. On this issue, it is important to stress that, even if the efficiency of cotransfection in our experiments was <100%, this would lead to an underestimation of the effects of wild-type PKD and PKD-K618M on plasma membrane NHE activity and would not invalidate our novel findings.
The majority of the work in the literature on the regulation of NHE
activity has concentrated on the identification of stimuli that
increase exchanger activity, such as a variety of growth factors/mitogens (28, 29) and neurohormonal and other mediators (7, 38,
41, 42), as well as the delineation of their signaling pathways (2, 4,
12, 22, 41). However, NHE-inhibitory pathways, such as those that
involve G12 (24), CHP (23), and
p38 MAPK (21), also exist and are likely to be important in determining
exchanger activity in various cell types. The findings of the present
study strongly suggest that PKD mediates a novel NHE-inhibitory
pathway, regardless of whether this inhibition occurs by direct
phosphorylation of the exchanger or via intermediary proteins. A better
understanding of NHE-inhibitory pathways may lead to the development of
novel approaches to therapy, in diseases in which altered exchanger
activity has been implicated as a causal mechanism.
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ACKNOWLEDGEMENTS |
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We thank Gavin Brooks for helpful discussions in the early stages of this work and Lionel Wightman and Michael Marber for the pCAGGS-GFP construct used in this study.
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FOOTNOTES |
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This work was supported by a project grant from the British Heart Foundation (PG/95159), and the microepifluorescence system used was purchased through equipment grants to M. Avkiran from the Wellcome Trust (048021/Z/96/Z) and the Special Trustees for St. Thomas' Hospital (523). M. Avkiran is the holder of a British Heart Foundation (Basic Science) Senior Lectureship Award (BS/93002).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Avkiran, Cardiovascular Research, The Rayne Institute, St. Thomas' Hospital, Lambeth Palace Rd., London SE1 7EH, UK (E-mail: metin.avkiran{at}kcl.ac.uk).
Received 19 January 1999; accepted in final form 30 July 1999.
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