1 Programme in Cell Biology, Hospital for Sick Children, Toronto M5G 1X8; 2 Department of Biology, York University, Toronto, Ontario, Canada M3J 1P3; and 3 Department of Pharmacology, The Milton S. Hershey Medical Center, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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Insulin stimulates K+ uptake and
Na+ efflux via the Na+-K+ pump in
kidney, skeletal muscle, and brain. The mechanism of insulin action in
these tissues differs, in part, because of differences in the isoform
complement of the catalytic -subunit of the
Na+-K+ pump. To analyze specifically the effect
of insulin on the
1-isoform of the pump, we have studied
human embryonic kidney (HEK)-293 cells stably transfected with the rat
Na+-K+ pump
1-isoform tagged on
its first exofacial loop with a hemagglutinin (HA) epitope. The plasma
membrane content of
1-subunits was quantitated by
binding a specific HA antibody to intact cells. Insulin rapidly increased the number of
1-subunits at the cell surface.
This gain was sensitive to the phosphatidylinositol (PI) 3-kinase
inhibitor wortmannin and to the protein kinase C (PKC) inhibitor
bisindolylmaleimide. Furthermore, the insulin-stimulated gain in
surface
-subunits correlated with an increase in the binding of an
antibody that recognizes only the nonphosphorylated form of
1 (at serine-18). These results suggest that insulin
regulates the Na+-K+ pump in HEK-293 cells, at
least in part, by decreasing serine phosphorylation and increasing
plasma membrane content of
1-subunits via a signaling
pathway involving PI 3-kinase and PKC.
ouabain; phosphatidylinositol 3-kinase; protein kinase C; hemagglutinin
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INTRODUCTION |
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THE SODIUM-POTASSIUM PUMP (Na+-K+-ATPase) expels three Na+ ions from the cytosol in exchange for two extracellular K+ ions (28). This electrogenic process uses ATP and represents the only means of Na+ extrusion from cells. Changes in pump activity directly impact on processes dependent on Na+ and K+ gradients (e.g., the resting membrane potential, Na+/Ca2+ exchange, and coupled transport of glucose, vitamins, and amino acids).
The mature functional Na+-K+ pump
requires association of its - and
-subunits (28).
The 110-kDa
-subunit is the catalytic component, whereas the highly
glycosylated
-subunit (apparent molecular weight on SDS-PAGE of
~55 kDa) is involved in maturation and assembly of functional pump
dimers in the plasma membrane (32), in allowing
K+ occlusion (29), and in modulation of
Na+ and K+ affinity of the enzyme (15,
16). Four isoforms of
-subunits and three of
-subunits
exist (6). In kidney, the pump also comprises a
-subunit, a small (8-14 kDa) hydrophobic peptide that may
regulate affinity of the
-subunit for ATP, Na+, and
K+ (1, 2, 39). The
- and
-isoforms
exhibit a tissue-specific distribution, and their heterogeneity
contributes to the adaptability of the Na+-K+
pump to respond to hormones in a tissue-specific fashion (6, 19).
A diverse range of hormones can regulate Na+-K+
pump activity acutely or chronically (5, 19). Short-term
regulation may be achieved by several mechanisms including changes in
ion affinity and regulation of availability of pump subunits at the
cell surface through exocytosis or endocytosis. It has been proposed
that changes in phosphorylation of the -subunit may contribute to
regulation of Na+-K+ pump localization by
dopamine in kidney proximal tubule (11, 12). On the other
hand, several studies have established negative correlations between
the phosphorylation of a serine residue in the NH2 terminus
on the
1-subunit and its level of catalytic activity
(4, 24, 33).
Regulation of the Na+-K+ pump by insulin also
occurs through diverse, tissue-specific mechanisms (36).
In skeletal muscle, insulin causes translocation of the
2-isoform to the cell surface (25, 31). In
contrast, no change in
1- or
2-subunit
exposure was noted in 3T3-L1 fibroblasts or adipocytes, respectively,
where instead the pump was activated secondarily to a rise in
intracellular Na+ concentration (30, 35, 37).
In the proximal convoluted tubule of the kidney, insulin activates the
Na+-K+ pump by elevating the affinity for
Na+ of the
1-subunit (21).
Recently, tyrosine phosphorylation of the
1-subunit was
shown to correlate positively with increased pump activity in response
to insulin in kidney proximal tubule (20). Although the
insulin receptor is itself a tyrosine kinase, once activated it engages
signaling cascades involving the lipid kinase phosphatidylinositol (PI)
3-kinase and the serine/threonine kinases Akt and atypical protein
kinase C (PKC), as well as serine/threonine and tyrosine phosphatases
(38).
Clearly, there is a need to identify the mechanism of regulation of
each Na+-K+ pump isoform by specific stimuli.
The objective of this study was to explore whether insulin can cause
changes in the surface exposure of the Na+-K+
pump 1-subunit. For this purpose, we used human
embryonic kidney (HEK)-293 cells stably transfected with an exofacially
epitope-tagged rat Na+-K+ pump
1-isoform in combination with an intact cell assay to
examine its presence at the plasma membrane. Furthermore, we
measured changes in
1-subunit phosphorylation and
examined the possible signaling pathways involved in the response to insulin.
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MATERIALS AND METHODS |
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Materials.
All cell culture solutions and supplements were obtained from GIBCO BRL
(Burlington, ON, Canada). HEK cells stably overexpressing the
hemagglutinin (HA)-tagged rat Na+-K+ pump
1-isoform protein [HA (2x) 119 I] were generated as
previously described (8). Human insulin (Humulin) was
obtained from Eli Lilly Canada (Toronto, ON, Canada). Protein A- and
protein G-Sepharose were from Pharmacia (Uppsala, Sweden). Enhanced
chemiluminescence (ECL) reagent was purchased from Amersham (Oakville,
ON, Canada). The Na+-K+ pump
1-subunit antibody McK1 (22) was a generous
gift from Dr. K. Sweadner (Massachusetts General Hospital, Boston, MA). Anti-HA antibody (12CA5) was purchased from Roche Diagnostics (Quebec,
Canada). Horseradish peroxidase (HRP)-conjugated sheep anti-mouse and
donkey anti-mouse antiserum were from Jackson ImmunoResearch (Baltimore
Pike, PA). o-Phenylenediamine dihydrochloride (OPD reagent)
and wortmannin were from Sigma (St. Louis, MO), and bisindolylmaleimide (BIM) was from Calbiochem (San Diego, CA). All electrophoresis and
immunoblotting reagents were purchased from Bio-Rad (Mississauga, ON,
Canada). All other reagents were of the highest analytical grade.
Cell culture.
HEK-293 cells were grown in monolayer culture in Dulbecco's modified
Eagle's medium supplemented with 10% (vol/vol) fetal bovine serum and
1% (vol/vol) antibiotic solution (10,000 U/ml penicillin and 10 mg/ml
streptomycin) in an atmosphere of 5% CO2 at 37°C.
Transfection of the HA (2x) 119 I mutant into HEK-293 cells conferred
ouabain resistance; hence, cells were grown in the presence of 0.5 µM
ouabain. For 1-subunit translocation assays, cells were
seeded in 12-well plates (2.5-cm-diameter well). Cells were maintained
under the same conditions in six-well plates for preparation of whole
cell lysates and subsequent immunoprecipitation.
Immunodetection of
Na+-K+
pump 1-subunit translocation to the plasma membrane.
Stably transfected HEK cells were seeded on 12-well plates at a high
density and used 1-2 days postconfluence. Before each experiment,
cells were depleted of serum for 2 h. Cells were then incubated
with inhibitors and/or insulin for the desired time and rinsed quickly
in PBS, and then 300 µl of 3% paraformaldehyde were added per well
for 3 min at 4°C. In initial experiments, similar results were
obtained when paraformaldehyde was added before or after incubation
with antibody, suggesting that permeabilization of cells with
paraformaldehyde was not significant. Paraformaldehyde was aspirated,
and 500 µl of 1% glycine in PBS were added for 10 min. Each well was
then rinsed once with 1 ml PBS before 300 µl of 5% goat serum plus
3% BSA in PBS were added for 30 min. After this period, 300 µl of
anti-HA monoclonal antibody (12CA5) were added (1:5,000 dilution in 5%
goat serum + 3% BSA) for 30 min, followed by extensive washing (3 times with 1 ml of PBS per well). Peroxidase (300 µl)-conjugated
donkey anti-mouse IgG (1:5,000 dilution in 5% goat serum + 3%
BSA) was added for 30 min, again followed by extensive washing.
Finally, each well was incubated with 1 ml of OPD reagent for 12 min,
at which time 0.25 ml of 3 M HCl was added, and the solution was
transferred to a cuvette for absorbance reading at 492 nm. Background
values were calculated by using the same procedure but omitting primary
antibody, and basal values were calculated by subtracting this
background value from those obtained with transfected but untreated
cells. To estimate the percentage of total cellular HA-tagged
1-subunits expressed at the cell surface, we employed a
similar method with the addition of a permeabilization step, using
0.1% Triton X-100, after cells were fixed by paraformaldehyde. We
found that approximately one-third of these subunits were inserted in
the plasma membrane under resting conditions.
Assay of PI 3-kinase activity associated with anti-phosphotyrosine. PI 3-kinase activity was measured on phosphotyrosine immunoprecipitates as described previously (41). Briefly, the ability of PI 3-kinase associated with phosphotyrosine to convert phosphatidylinositol to phosphatidylinositol-3-phosphate (PI3P) was detected by separation of these lipids by thin-layer chromatography (TLC). Detection and quantitation of [32P]PI3P on the TLC plates were done using a Molecular Dynamics PhosphorImager system (Sunnyvale, CA).
Assay of atypical PKC activity.
Immunoprecipitation of atypical PKC isoforms was performed with
antibody PKC- (C-20; Santa Cruz, CA), which recognizes both PKC-
and PKC-
. Kinase assays were performed by lysing cells in buffer
containing 50 mM HEPES, pH 7.6, 150 mM NaCl, 10% glycerol (vol/vol),
1% Triton X-100 (vol/vol), 30 mM
Na4P2O7, 10 mM NaF, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine, 1 mM
Na3VO4, 1 mM dithiothreitol (DTT), and 100 nM
okadaic acid. Antibody (2 µg per condition) precoupled to protein
A-/protein G-Sepharose [20 µl (100 mg/ml) each per condition] beads
was added to 200 µg of total protein from cell lysates.
Antibody-coupled beads were washed twice with ice-cold PBS and once
with ice-cold lysis buffer before use. PKC-
/
was
immunoprecipitated by incubation with the antibody-bead complex for
2- 3 h under constant rotation (4°C). Immunocomplexes were
isolated and washed four times with 1 ml of wash buffer [25 mM HEPES,
pH 7.8, 10% glycerol (vol/vol), 1% Triton X-100 (vol/vol), 0.1% BSA,
1 M NaCl, 1 mM DTT, 1 mM PMSF, 1 µM microcystin, and 100 nM okadaic
acid] and twice with 1 ml of kinase buffer (50 mM Tris · HCl,
pH 7.5, 10 mM MgCl2, 10 nM okadaic acid, and 1 mM DTT). The
complexes were then incubated under constant agitation for 30 min at
30°C with 30 µl of reaction mixture (kinase buffer containing 5 µM ATP, 2 µCi [
-32P]ATP, and 5 µg of myelin
basic protein). After the reaction, 30 µl of the supernatant were
transferred onto Whatman p81 filter paper and washed four times for 10 min with 3 ml of 175 mM phosphoric acid and once with distilled water
for 5 min. Filters were air-dried and then subjected to liquid
scintillation counting.
Immunoprecipitation of HA-tagged
Na+-K+
pump 1-subunits.
Cell monolayers in six-well plates were serum starved for 2 h
before treatment with insulin. After stimulation, cells were lysed in 1 ml of lysis buffer [50 mM HEPES, pH 7.6, 150 mM NaCl, 10% glycerol
(vol/vol), 1% Triton X-100 (vol/vol), 30 mM
Na4P2O7, 10 mM NaF, 1 mM EDTA, 1 mM
PMSF, 1 mM benzamidine, 1 mM Na3VO4, 1 mM DTT,
and 100 nM okadaic acid] and passed five times through a 25-gauge
syringe needle. To each lysate, we added 2 µg of HA antibody and then
incubated overnight with constant rotation, followed by 25 µl each of
protein A- and protein G-Sepharose (10% wt/vol) for 1 h.
Immunoprecipitates were then washed three times with PBS containing
0.1% Nonidet P-40 and 100 µM Na3VO4,
solubilized in 30 µl of 2× Laemmli sample buffer, and incubated at
40°C for 20 min before separation by 10% SDS-PAGE. Monoclonal
1-subunit antibodies 6H (1:1,000 dilution) or McK1
[1:4,000 dilution, which recognizes only rat
Na+-K+ pump
1 protein with
nonphosphorylated serine-18 (9, 23)] were used for
immunoblotting, followed by sheep anti-mouse immunoglobulin conjugated
to HRP (1:5,000 dilution), and protein was visualized by the ECL method.
Statistical analysis. Statistical analysis was performed by using either unpaired Student's t-test or analysis of variance (Fischer, multiple comparisons) where appropriate, as indicated.
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RESULTS |
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The HEK-293 cells transfected with HA-tagged
Na+-K+ pump 1-isoform were
developed to localize cytoplasmic and extracellular domains of the
Na+-K+ pump (8). In that study,
analysis of cells expressing the HA (2x) 119 I rat
Na+-K+ pump
1-subunit mutant
constructs showed reactivity with HA antibody in both permeabilized and
intact cells, indicating an extracellular location of the HA epitope.
These cells are thus amenable to analysis of exposure of
Na+-K+ pump
1-isoform at the
cell surface by detecting HA epitope availability in intact cells. We
have developed an assay where cell monolayers are fixed before
incubation with HA antibody and then exposed to an appropriate
HRP-coupled secondary antibody. Peroxidase activity is detected in a
colorimetric assay using the OPD reagent (42). Figure
1A shows that insulin elicited
a rapid, yet transient increase in the amount of HA-tagged
1-subunits in the plasma membrane. The effect of insulin
was apparent within minutes, peaked at around 10 min to achieve an
~1.4-fold stimulation above basal levels, and returned to basal
levels after 20 min. The magnitude of this response is typical of
increases in ion transport via the Na+-K+ pump
seen in response to insulin and other hormones (35, 37). In keeping with most studies in the literature in which
insulin-responsive cells in culture such as 3T3-L1 adipocytes and L6
myotubes were used, most other experiments performed here were done
using 100 nM insulin. However, treatment with increasing concentrations of insulin for 10 min showed that 0.1 nM insulin elevated
1-subunit surface exposure significantly and that 1 nM
caused maximal stimulation (Fig. 1B). This high sensitivity
of HEK-293 cells suggests that they are indeed a good in vitro model of
insulin action and that they retain the high affinity for the hormone
displayed by mature tissues.
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The signaling pathways engaged by insulin to stimulate the increase in
1-subunits to the plasma membrane in these cells was investigated next. Previous studies by us and others (13, 14, 34,
37) have suggested the participation of PI 3-kinase in mediating
the stimulation of Na+-K+ pump activity in
fibroblasts and epithelial cells. Therefore, we examined the effect of
the PI 3-kinase inhibitor wortmannin on the gain in
1-subunits at the plasma membrane of HEK-293 cells. Figure 2A shows that
preincubation of cells with 100 nM wortmannin prevented the
insulin-induced gain in
1-subunits at the cell surface
without significantly affecting basal levels. Figure 2B demonstrates that insulin stimulated phosphotyrosine-associated PI
3-kinase activity. This response occurred with a time course similar to
that observed for stimulation of
1 translocation.
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Atypical PKC isoforms are activated by the lipid products of PI
3-kinase (40). We also have suggested previously that
atypical PKC contributes to the stimulation of the
Na+-K+ pump by insulin in fibroblasts
(37) and insulin-like growth factor (IGF)-I in smooth
muscle cells (27) on the basis of the sensitivity of the
response to micromolar concentrations of the PKC inhibitor BIM. Here we
show that insulin stimulates PKC-/
activity in HEK-293 cells as
measured by the ability of immunoprecipitates to phosphorylate myelin
basic protein; this activity was inhibited by 10 µM BIM (Fig.
3B). At this concentration BIM
also caused inhibition of insulin-stimulated
1-subunit
content in the plasma membrane (Fig. 3A). A lower
concentration of BIM (1 µM), which spares atypical PKCs but inhibits
conventional and novel PKCs, partially inhibited the insulin-stimulated
increase in cell surface
1-subunits [insulin (10 min):
100 ± 3%; insulin + 1 µM BIM: 66 ± 5%; and
insulin + 10 µM BIM: 12 ± 9%]. Collectively, these
results suggest that PKC may participate in relaying the insulin signal to the Na+-K+ pump in HEK cells.
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To explore whether the phosphorylation status of the
1-subunit is affected by insulin, we made use of an
antibody (McK1) that preferentially recognizes the nonphosphorylated
form of this subunit (23). The use of McK1 as a tool to
investigate phosphorylation of the
1-subunit has been
validated several times (9, 26). The antibody specifically
recognizes the nonphosphorylated form of the enzyme on serine-18,
a residue that is a target for phosphorylation by PKC (23,
26). This sequence is unique to the rat
1-isoform (22), and the HA-tagged
1-subunit expressed
in HEK cells used here is of rat origin; therefore, it is possible to
detect changes in its
1-subunit phosphorylation using
McK1 antibody. Serine-18 phosphorylation of HA-tagged
1-subunit of HEK-293 cells was determined by
immunoprecipitating the protein with anti-HA antibody, followed by
immunoblotting with McK1. Insulin caused a 30% increase in McK1
immunoreactivity (Fig. 4). The presence
of equivalent total amounts of
1-subunit protein content
in all samples was confirmed with the use of a monoclonal antibody (6H)
that does not discriminate between phosphorylated and nonphosphorylated
protein (Fig. 4, inset). The effect of insulin was
attenuated by both wortmannin and BIM (Fig. 4). In experiments similar
to that illustrated in Fig. 4, we were unable to detect any
insulin-dependent tyrosine phosphorylation of the
1-subunit by immunoblotting HA immunoprecipitates with
anti-phosphotyrosine antibody (results not shown).
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DISCUSSION |
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The studies described in the introduction and the results
presented in this study document a very versatile regulation of the
Na+-K+ pump. It is clear that changes in
cellular localization of specific isoforms of the -subunit, along
with changes in its phosphorylation status, contribute to the ultimate
ability of the cells to regulate Na+ efflux and
K+ influx. Until now, other studies had made use of
subcellular fractionation and morphological analysis by immunochemistry
to analyze changes in cellular localization. Neither of these
techniques resolve the issue of whether or how many of the pump
subunits are truly inserted into the plasma membrane or are in docked
but not fused subplasmalemmal vesicles. Direct approaches to detect pump exposure at the exofacial face of the membrane were lacking. The
present study utilized an approach to assess the number of Na+-K+ pump
1-subunits inserted
in the plasma membrane. HEK cells were transfected to overexpress the
rat Na+-K+ pump
1-subunit tagged
with an HA epitope such that the epitope would be located on a region
of the
1 protein exposed to the extracellular
environment when the protein is incorporated in the cell membrane.
Immunodetection of exposure of this epitope in cell monolayers with
anti-HA antibody, followed by HRP-coupled secondary antibody and then
detection of peroxidase activity, allowed quantitative analysis of
Na+-K+ pump
1-isoform content at
the cell surface. Our results therefore show that insulin regulates
Na+-K+ pump activity in HEK-293 cells, at least
in part, by increasing the amount of
1-subunits inserted
in the cell membrane. It will be interesting in the future to assess
the magnitude of contribution made by changes in subunit localization
vs. changes in parameters such as Na+, K+, and
ATP affinity to ion transport.
PI 3-kinase is a central signaling molecule in insulin action, and
atypical PKCs are activated by the lipid products of PI 3-kinase.
Inhibitors of PI 3-kinase or atypical PKC prevented the
insulin-stimulated Na+-K+ pump activity in
3T3-L1 fibroblasts (37) and L6 muscle cells (34) and prevented the IGF-I-stimulated activity in smooth
muscle cells (27). Inhibition of PI 3-kinase also
prevented insulin-stimulated Na+-K+ pump
activity in porcine glandular endometrial epithelial cells grown in
primary culture (14). Both PI 3-kinase and conventional PKC appear to regulate the endocytosis of
Na+-K+ pump 1-subunits and,
thus, Na+-K+ pump inhibition in response to
dopamine in proximal tubule cells (13). It also has been
shown that activation of PKC-
by dopamine resulted in inhibition of
renal Na+-K+ pump (17). In
contrast, PKC-
activation appears to be responsible for increased
renal pump phosphorylation and activity in response to phorbol
12-myristate 13-acetate (17, 18), suggesting that the role
of PKC in regulating the Na+-K+ pump is complex
and likely isoform and tissue specific. Here we demonstrated that
insulin can stimulate phosphotyrosine-associated PI 3-kinase activity
and atypical PKC activity in HEK-293 cells. We then showed that
inhibitors of these enzymes, i.e., wortmannin (100 nM) and BIM (10 µM), prevented insulin-induced increases in HA-tagged
1-subunit exposure at the cell surface. These results suggest that the insulin-signaling pathway regulating increased Na+-K+ pump function may involve PI 3-kinase
and PKC. Indeed, the temporal activation of these lipid and serine
kinases in these experiments and the transient nature of HA-tagged
1-subunit translocation also provides correlative
evidence for a close association of these phenomena.
Wortmannin and BIM also reduced the ability of insulin to reduce serine
phosphorylation of 1-subunits. The fact that insulin stimulated PKC activity yet ultimately caused a reduction in serine phosphorylation of the
1-subunit suggests that a
phosphatase may lie downstream of PKC in this insulin-signaling
pathway. Phosphatase activation as a distal event in kinase cascades is
common, and indeed, insulin activates several serine phosphatases,
including protein phosphatase-1 (PP1) (3). Notably,
activation of PP1 in muscle cells was wortmannin sensitive (14,
34), but it is not known whether its activation requires PKC
activity. We hypothesize that activation of PI 3-kinase and PKC by
insulin result in increased phosphatase activity and dephosphorylation of the
1-subunit of the Na+-K+
pump in HEK-293 cells. It previously has been shown that the phosphorylation status of the
1-isoform can be altered
as a result of PKA or PKC activation (7, 22), and in some
cases changes in phosphorylation of the pump have been linked to
changes in
1 localization. Notably, dopamine-induced
phosphorylation of
1 by PKC accompanies increased
endocytosis of this subunit in proximal kidney tubules
(10). The clathrin-dependent endocytosis of
1 into endosomes required phosphorylation of
1 at serine-18 (11). These studies suggest
that phosphorylation constitutes a signal for
Na+-K+ pump endocytosis (12). In
the present study, insulin appeared to reduce the phosphorylation of
serine-18. This might explain the increased cell surface content of the
1-subunit if indeed phosphorylation of this residue is a
cause of increased endocytosis of the protein. Consistent with this
scenario, the magnitude of the insulin-dependent dephosphorylation of
the
1-subunit (30%) closely correlated with the gain in
surface exposure of this protein (40%).
In summary, the results of this study suggest that insulin leads to a
gain in Na+-K+ pump at the cell surface that
may be linked to the reduction in phosphorylation of serine-18 on the
1-subunit. The mechanism promoting this
dephosphorylation in response to insulin appears to require activation
of a PI 3-kinase- and PKC-dependent pathway.
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ACKNOWLEDGEMENTS |
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This work was supported by Canadian Institute of Health Research/Medical Research Council (Canada) Grant MT12601 (to A. Klip). W. Niu was supported by an award from The Hospital for Sick Children, Toronto, Ontario, Canada. G. Sweeney was supported by a Banting & Best Diabetes Centre and Novo Nordisk Fellowship.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. Klip, Programme in Cell Biology, Hospital for Sick Children, 555 Univ. Ave., Toronto, Ontario, Canada M5G 1X8 (E-mail: amira{at}sickkids.ca).
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.
Received 7 September 2000; accepted in final form 19 July 2001.
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