Membrane Biology Group, University of Toronto, Toronto, Ontario, Canada M5S 1A8
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ABSTRACT |
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We have used the recombinant
NH2-terminal
myc-tagged rabbit
Na+-glucose transporter (SGLT1) to
study the regulation of this carrier expressed in COS-7 cells.
Treatment of cells with a protein kinase C (PKC) agonist, phorbol
12-myristate 13-acetate (PMA), caused a significant decrease (38.03 ± 0.05%) in methyl
-D-glucopyranoside transport
activity that could not be emulated by 4
-phorbol 12,13-didecanoate. The decrease in sugar uptake stimulated by PMA was reversed by the PKC
inhibitor bisindolylmaleimide I. The maximal rate of
Na+-glucose cotransport activity
(Vmax) was
decreased from 1.29 ± 0.09 to 0.85 ± 0.04 nmol · min
1 · mg
protein
1 after PMA
exposure. However, measurement of high-affinity
Na+-dependent phloridzin binding
revealed that there was no difference in the number of cell surface
transporters after PMA treatment; maximal binding capacities were 1.54 ± 0.34 and 1.64 ± 0.21 pmol/mg protein for untreated and
treated cells, respectively. The apparent sugar binding affinity
(Michaelis-Menten constant) and phloridzin binding affinity
(dissociation constant) were not affected by PMA. Because PKC reduced
Vmax without
affecting the number of cell surface SGLT1 transporters, we conclude
that PKC has a direct effect on the carrier, resulting in a lowering of
the transporter turnover rate by a factor of two.
rabbit SGLT1; protein kinase C; regulation
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INTRODUCTION |
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THE MECHANISMS GOVERNING the regulation of many membrane transport proteins are increasingly becoming the focus of investigation. Protein kinases can alter the activity of a protein either directly or indirectly. Direct effects involve altering the kinetics of the transporter, the apparent substrate binding affinity, or the turnover number of the carrier. Indirect effects of protein kinases involve altering the rate at which the protein is retrieved or inserted into the plasma membrane. Protein kinase A (PKA) and protein kinase C (PKC), both serine/threonine kinases, have been shown to be involved in the regulation of the Na+-glucose transporter (SGLT1) in polarized epithelial cell lines (9, 20-22), when expressed in Xenopus oocytes (13, 27), and also in rat intestinal brush-border membrane vesicles (14).
Measurement of sugar-induced Na+ currents using the two-electrode voltage-clamp technique in Xenopus oocytes expressing rabbit SGLT1 (rSGLT1) showed that exposure to a membrane-permeant activator of PKC phorbol ester, 1,2-dioctanoyl-sn-glycerol, decreased rabbit Na+-glucose cotransport activity by 51%. When the number of transporters was measured in the same oocyte by determination of Qmax, the maximal number of charges translocated across the oolemma in response to voltage pulses, there was also a concomitant decrease in rSGLT1 protein from the plasma membrane (27). Conversely, stimulation of PKA using 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) resulted in a 28% increase in SGLT1 transport activity and an increase in Qmax as well as in the plasma membrane surface area.
In this expression system, the activation of PKA or PKC did not have
any measurable effect on the apparent sugar binding affinity [Michaelis-Menten constant
(Km)],
the inhibition constant for phloridzin, or the turnover number for
rSGLT1 (27). These observations indicated that rSGLT1 transport
activity was regulated indirectly by protein kinases and was linked to
mechanisms governing the trafficking of rSGLT1 to and from the oocyte
plasma membrane by regulated endo- or exocytosis. Similar observations
have been made for the development of SGLT1 transport activity in the
differentiating human cell clone HT-29-D4 (9) and also for brush-border
membrane vesicles prepared from perfused rat intestine exposed to the
-adrenergic agonist epinephrine (14). The cloned rat brain
-aminobutyric acid transporter 1 (GAT1) expressed in
Xenopus oocytes has also been shown to
be indirectly modulated by protein kinases by regulated protein
trafficking (8). Alternatively, the activity of the dopamine
transporter (DAT1) and the serotonin transporter are directly affected
by protein kinases, since the observed changes in their
Vmax were
independent of transporter number (1, 2, 15).
It is known that the rabbit Na+-glucose carrier contains four putative PKC serine/threonine phosphorylation sites and one PKA site (27). However, a direct regulatory effect of either PKC or PKA on Na+-glucose transport activity has not been reported, and this may largely be due to the nature of the cells in which the transporter has been expressed. We have previously used a mammalian expression system, the COS-7 cell line, to characterize both a recombinant NH2-terminal myc-tagged rSGLT1 isoform and an rSGLT1 A166C mutant (25). To further exploit this cell system, we decided to investigate how rSGLT1 was regulated in this cell line. We used the myc-tagged rSGLT1 isoform 1) because it was phenotypically identical to wild-type rSGLT1 and 2) because the incorporation of the myc epitope sequence greatly facilitated immunodetection of SGLT1 with the anti-c-myc monoclonal antibody (9E10).
After exposure of cells to the PKC agonist phorbol 12-myristate
13-acetate (PMA), methyl
-D-glucopyranoside (
-MG)
uptake into transiently transfected COS-7 cells was significantly
reduced compared with controls, and this effect could be totally
reversed by the specific PKC inhibitor bisindolylmaleimide I. Kinetic
analysis revealed that the maximal rate of rSGLT1 transport activity
(Vmax) was
decreased as a result of PKC activity without any effect on the
apparent sugar binding affinity
(Km).
Interestingly, we found that the number of surface-expressed rSGLT1
transporters, measured using high-affinity
Na+-dependent phloridzin binding,
was not different before and after PMA treatment, i.e., there was no
change in the maximal binding capacity
(Bmax) values, and the
dissociation constant
(Kd) for phloridzin binding was unaltered. We conclude from these data that PKC
regulation of rSGLT1 expressed in COS-7 cells is controlled by direct
alteration of the carrier by means of a lowering of the turnover rate
and that this expression system may prove useful in furthering our
understanding of the mechanism(s) underlying phosphorylation-dephosphorylation of SGLT1.
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MATERIALS AND METHODS |
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Cell culture and transfection. COS-7 cells were grown in complete RPMI 1640 medium (GIBCO, Burlington, ON, Canada) that was supplemented with 21 mM NaHCO3, 25 mM HEPES-NaOH (pH 7.4), 10% (vol/vol) FCS, and 50 U/ml antibiotic solution containing penicillin and streptomycin. Cells were maintained at 37°C in 5% CO2. Stock cultures were grown in 75-cm2 flasks (Corning, Cambridge, MA) and were fed every 3-4 days. For uptake experiments or for immunodetection of SGLT1, cells were seeded into 12-well plates or 35-mm culture dishes (Corning), respectively. Cells reaching 50-70% confluency were transiently transfected using the polycationic diethylaminoethyl ether of dextran (DEAE dextran) at a DNA-to-DEAE dextran ratio of 1:40 as described previously (25). Experiments were carried out 24-48 h after transfection.
Molecular biology.
The c-myc epitope sequence was
introduced onto the NH2-terminal
region of the rabbit intestinal SGLT1 protein as described previously
(25). Myc-tagged rSGLT1 cDNA was
cloned into the eukaryotic expression vector pMT4 containing the simian
virus 40 origin of replication suitable for expression in the COS-7 cells (25). The DNA used for the COS-7 transfections was prepared from
Escherichia coli DH5 cells
harboring either pMT4, pMT4-SGLT1 wild type, or
pMT4-myc-SGLT1 wild type using the
Qiagen plasmid midi kit (Qiagen, Chatsworth, CA). COS-7 cells
transfected with vector pMT4 lacking cloned SGLT1 cDNA served as a control.
-MG uptake.
The uptake of 14C-labeled
-MG
(sp act 293 mCi/mmol) into COS-7 cells was measured at room temperature
(20°C) as described in Ref. 28. In brief, the medium was aspirated
from the cells, and reactions were started by the addition of 500 µl
of incubation medium that contained (except as stated otherwise) 140 mM
NaCl, 20 mM mannitol, 10 mM HEPES-Tris (pH 7.4), and 1 mM
14C-labeled
-MG. After
incubation for the desired time, the incubation medium was removed and
the cells were washed three times in 3-ml volumes of ice-cold stop
buffer that contained 140 mM KCl, 20 mM mannitol, 10 mM HEPES-Tris (pH
7.4), and 0.2 mM phloridzin. The termination and washing procedure took
<20 s/well. Any remaining solution was aspirated, and 500 µl of PBS
containing 0.1% (wt/vol) SDS were added to solubilize the cells. After
20 min, this solution was removed and processed for liquid
scintillation counting. When indicated, measurements of transport rates
(10 min) were performed. Uptake was directly proportional to time over
a period of 20 min.
Phloridzin binding assay.
The methodology for the measurement of
[3H]phloridzin binding
(sp act 55 Ci/mmol) was essentially similar to that for the -MG uptake assay. The incubation medium contained a specified concentration of phloridzin: 0.01, 0.05, 0.1, 0.3, 0.4, 0.5, or 1.0 µM.
Measurements of phloridzin binding were carried out after 1 min. There
was no significant increase in the level of phloridzin binding after this period. The stop solution did not contain any phloridzin. Time
zero binding was not measured.
Estimation of protein. Protein determination was carried out as described by Lowry et al. (17), using the Bio-Rad DC micro-protein assay kit. BSA was used as the standard.
Statistical analysis.
In Table 1, data are expressed as means ± SD of three individual experiments in which mean values were
determined in triplicate. Tests of significance of difference between
mean values were made using ANOVA and a Bonferroni method for
multiple-comparison t-tests between
data pairs. The SDs reported (see also Figs. 1 and 2) were calculated
from the triplicate measurements made at each experimental point. When
appropriate, curve fitting was made using a nonlinear least squares fit
program (Microcal Origin 4.00, Microcal Software, Northampton, MA).
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Materials.
14C-labeled -MG and
[3H]phloridzin were
purchased from Amersham International (Oakville, ON, Canada) and
DuPont-NEN (Boston, MA), respectively. PMA, 4
-phorbol
12,13-didecanoate (4
-PDD), bisindolylmaleimide I HCl,
bisindolylmaleimide V, calphostin C, and chelerythrine chloride were
from Calbiochem (Hornby, ON, Canada). The PKC inhibitor compound
(bisindolylmaleimide I HCl, calphostin C, or chelerythrine chloride) or
the inactive PKC inhibitor analog bisindolylmaleimide V was added to
the cells just before the addition of PMA, without preincubation. For
experiments involving phorbol ester or PKC inhibitor(s), the final
concentration of solvent (DMSO) did not exceed 0.1% (vol/vol) and did
not affect
-MG uptake into
myc-tagged rSGLT1-transfected COS-7
cells. Mouse anti-c-myc monoclonal
antibody (9E10) was from Berkeley Antibody (Richmond, CA). Goat
anti-mouse antibody conjugated to horseradish peroxidase was from
Jackson Immunoresearch Laboratories (West Grove, PA). All other
chemicals were of the highest quality available from Sigma (St. Louis, MO).
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RESULTS |
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Time course for the expression of rSGLT1 in transfected COS-7 cells.
Before investigating the effect of PMA on rSGLT1 transport activity in
transfected COS-7 cells, we first decided to determine the time frame
in which rSGLT1 was functionally expressed at the cell surface. This
information would be useful in determining a time period when rSGLT1
might be more susceptible to either direct or indirect regulation by
PKC. The results in Fig.
1A
show the initial rate of 1 mM -MG uptake into
transfected cells, in the presence of external NaCl, at 6-h intervals
after transfection.
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PMA-mediated decrease in -MG transport activity.
We have previously shown that the recombinant
NH2-terminal
myc-tagged rSGLT1 protein has
-MG
transport characteristics identical to those of wild-type rSGLT1 (25).
However, before using it to study the regulation of SGLT1 by PKC in the
COS-7 cells, we wanted to make sure that addition of the epitope tag
did not interfere with a PMA-mediated response. We first tested the
effect of PMA on
-MG transport activity into cells expressing
wild-type rSGLT1. In the absence of PMA,
-MG uptake into cells
expressing wild-type rSGLT1 was 1.40 ± 0.13 nmol · min
1 · mg
protein
1; it was decreased
by 30% after exposure to the phorbol ester. Because this response was
comparable to that of cells transfected with
myc-tagged rSGLT1 (see Fig. 2), the
epitope tag did not appear to affect the PMA-induced regulation of the
transporter and we could reliably use it to further study PKC
regulation of rSGLT1.
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Effect of PKC inhibitor bisindolylmaleimide I on PMA-mediated
decrease in -MG transport activity.
To determine that the decrease in
-MG transport activity induced by
PMA was linked to the stimulation of PKC activity, we investigated the
effects of the inactive phorbol ester analog 4
-PDD and the specific
PKC inhibitor bisindolylmaleimide I on
-MG uptake into transfected
cells. The results are shown in Fig. 3.
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Effect of PMA on Cl-driven
-MG uptake,
K+ channels, and
Na+/H+
exchange.
Cl
channels have been shown
to be regulated by phosphorylation-dephosphorylation reactions (3, 5,
11, 16, 23). To eliminate the possible effect of PMA stimulating a
PKC-regulated Cl
conductance pathway that might decrease electrogenic
Na+-
-MG uptake, we measured
substrate uptake into COS-7 cells expressing myc-tagged rSGLT1 in the presence of
external Cl
or in the
presence of the membrane-impermeant anion gluconate.
Effect of PMA on transporter kinetics and phloridzin binding.
We next set out to determine the effects of PMA on
1) the kinetics of -MG uptake and
2) the number of cell surface
transporters, as measured by high-affinity
Na+-dependent phloridzin binding.
The results for both the kinetic parameters and phloridzin binding,
measured at 24 and 48 h after transfection, are shown in Table 1.
Transfected cells exposed to PMA showed a significant decrease in
Vmax at both 24- and 48-h time periods, compared with untreated cells (34.51 ± 1.43 and 26.46 ± 0.03%, respectively). There was no
change in the apparent sugar binding affinity
(Km) for
myc-tagged rSGLT1 after treatment with
PMA. Of significance, the
Na+-dependent phloridzin binding
data (Table 1) indicate that PMA treatment did not affect the maximal
number of transporters (Bmax) at
the cell surface compared with untreated cells. In addition, the
binding affinity
(Kd) for
phloridzin was also unaltered by PMA treatment and was the same for
each time point.
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DISCUSSION |
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We investigated the regulation of rSGLT1 expressed in the mammalian
cell line COS-7. Addition of PMA to transfected cells expressing
myc-tagged rSGLT1 caused a significant
decrease (38%) in -MG transport activity that was not elicited with
4
-PDD, indicating that the response stimulated by PMA was due
specifically to the phorbol ester. Interestingly, exposure to PMA at 24 h after transfection resulted in a greater decrease in
Na+-glucose uptake, almost twice
as much as when PMA was added 48 h after transfection. Because we did
not measure PKC levels over the 48-h period, we do not know whether
there is a change in the expression level of this enzyme. However, if
we assume that the intracellular concentration of PKC does not increase
concomitantly with rSGLT1 biosynthesis, then at 24 h posttransfection
the relative ratio of PKC to rSGLT1 would be expected to be high, and
consequently the regulatory effect of the enzyme on rSGLT1 transport
activity would be more pronounced. The level of
-MG uptake 48 h
after transfection was found to be double that at 24 h (Fig.
1A and Vmax Table 1),
and this was reflected in a twofold increase in the number of
surface-expressed transporters
(Bmax; Table 1). Noticeably,
however, the PMA-mediated decrease in sugar transport activity was
almost halved (20%) from that at 24 h. It is interesting that the
PMA-mediated decrease in
-MG transport activity rarely exceeded
40%. Whether this is correlated with the level of PKC expression or
the proportion of cytosolic PKC that can translocate to the plasma
membrane on stimulation to regulate rSGLT1 is highly speculative at
this point and is beyond the scope of this report.
The highly specific PKC inhibitor bisindolylmaleimide I inhibited the
PMA effect on -MG transport activity. Thus we were able to
demonstrate in COS-7 cells that PKC plays an important role in the
regulation of rSGLT1. Although calphostin C and chelerythrine chloride
have been shown to inhibit PKC activity, neither compound was able to
reverse the effects of PMA exposure on
-MG transport in COS-7 cells,
and the reasons for this are not yet fully understood.
Regulation of the
Na+/H+
exchanger expressed in PS120 fibroblast cells was shown to be
influenced by serum, fibroblast growth factor, and phorbol ester (29).
Because COS-7 cells are derived from the simian kidney fibroblast cell
line, we wanted to make sure that growth factors in the serum were not
a contributing factor to the regulation of rSGLT1. This was proved not
to be the case, since growth-arrested cells cultured in serum-free
minimal medium, although they showed one-half as much rSGLT1 transport activity as cells in normal medium, exhibited an identical reduction in
-MG uptake in response to PMA exposure (23 and 29% decreases for
cells in serum and serum-free medium, respectively).
To determine whether the action of PKC on rSGLT1 transport activity
could be linked to a protein kinase/phosphatase cascade system, we
tested the effect of okadaic acid (100 nM), a specific inhibitor of
protein phosphatases 1 and 2A, on -MG uptake before and after
exposure to PMA. Similarly, we also tested the effect of the
broad-range tyrosine kinase inhibitor genistein (5 µM). Neither
compound had any effect on the action of PMA-mediated decrease in
-MG transport activity. This suggests that these enzymes may not be
directly involved in the PKC regulation of
-MG transport activity.
In oocytes expressing human SGLT1, the protein phosphatase 1 and 2A
inhibitor calyculin A produced effects similar to those of 8-BrcAMP and
1,2-dioctanoyl-sn-glycerol on the
maximal rate of transport activity
(Vmax), the
number of SGLT1 transporters
(Qmax), and oocyte surface area
(13, 27).
If electrogenic Na+-glucose
transport activity were regulated via PKC-dependent
Cl channels, we would
expect the addition of PMA to cause a decrease in
-MG uptake in the
presence of Cl
medium and
have no effect on
-MG uptake in the presence of external gluconate
medium. This was not the case, since
-MG uptake in the presence of
external gluconate medium was reduced equally to that in
Cl
medium after PMA
exposure. Therefore, we can eliminate the possibility that the
decreased transport activity was due to a PKC-regulated anion
conductance pathway. Similarly, the broad-spectrum
K+ channel blocker TEA did not
diminish the PMA-mediated decrease in
-MG transport activity, and
therefore it is unlikely that the PKC agonist caused a collapse in the
membrane potential. We could also rule out the possibility of the PKC
agonist stimulating Na+/H+
exchange systems, leading to increased intracellular
Na+ and indirectly affecting
Na+-coupled transport (6, 26),
since neither amiloride nor
5-(N,N-dimethyl)-amiloride could prevent the PMA-mediated decrease in
-MG transport activity.
The ability to demonstrate diminished rabbit
Na+-glucose transport activity via
a PKC-stimulated pathway in COS-7 cells is identical to that observed
for both rabbit and rat SGLT1 isoforms in oocytes. However, in oocytes,
rSGLT1 transport activity was regulated indirectly by PKC via a
mechanism(s) that controlled rSGLT1 protein trafficking to and from the
plasma membrane. When rSGLT1 was expressed in a nonpolarized cell such
as the COS-7 cells, however, we observed a different effect of PKC on
rSGLT1 transport activity. Kinetic analysis revealed that
the maximal rate of transport activity
(Vmax) after
PMA exposure was decreased, without any effect on the apparent sugar
binding affinity
(Km). When the
number of cell surface transporters was measured under the same
conditions, using high-affinity
Na+-dependent
[3H]phloridzin
binding, the Bmax was unaltered,
as was the Kd for phloridzin. The validity of phloridzin binding as a measure of the
number of surface-expressed rSGLT1 carriers has previously been
discussed (25). From the ratio
Vmax/Bmax,
the turnover rates before and after PMA exposure were 13.9 and 8.6 s1, respectively.
The observation that PKC activation by PMA causes a decrease in the turnover rate of the transporter by nearly twofold implies that PKC has a direct effect on the carrier. The differences observed in PKC regulation of rSGLT1 in COS-7 cells compared with oocytes are intriguing and require further investigation. It should also be pointed out that, in oocytes, PKC regulation of SGLT1 appears to exhibit isoform specificity, PKC activation causing reduced transporter activity of rSGLT1 but increased transporter activity of the human isoform.
The main conclusion of the present study, that PKC activation has a direct effect on rSGLT1 transporter activity, raises interesting questions about the interaction of PKC with SGLT1. One possibility is that this effect is mediated by direct phosphorylation on one or more of the four PKC phosphorylation consensus sites. Another explanation is that PKC activation results in phosphorylation of another intracellular or membrane-bound protein that then interacts with the Na+-glucose carrier, influencing its activity through a nonphosphorylation pathway. These different possibilities require investigation.
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FOOTNOTES |
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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. Silverman, Dept. of Medicine, University of Toronto, Medical Sciences Bldg., Rm. 7207, 1 King's College Circle, Toronto, ON, Canada M5S 1A8 (E-mail: melvin.silverman{at}utoronto.ca).
Received 9 September 1998; accepted in final form 29 January 1999.
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