From the Department of Biochemistry, Osaka University
Graduate School of Medicine, B1, 2-2 Yamadaoka, Suita, Osaka 565-0871, the § Department of Obstetrics & Gynecology, Asahikawa
Medical College, 2-1 Midorigaoka higashi, Asahikawa 078-8510, and the
¶ Department of Internal Medicine and Molecular Science, Graduate
School of Medicine, Osaka University, 2-2 Yamadaoka, Suita,
Osaka 565-0871, Japan
Received for publication, September 19, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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N-Acetylglucosaminyltransferase
III (GnT-III) is a key enzyme that inhibits the extension of
N-glycans by introducing a bisecting N-acetylglucosamine residue. In this study we investigated
the effect of GnT-III on epidermal growth factor (EGF) signaling in HeLaS3 cells. Although the binding of EGF to the epidermal growth factor receptor (EGFR) was decreased in GnT-III transfectants to a
level of about 60% of control cells, the EGF-induced activation of
extracellular signal-regulated kinase (ERK) in GnT-III transfectants was enhanced to ~1.4-fold that of the control cells. A binding analysis revealed that only low affinity binding of EGF was decreased in the GnT-III transfectants, whereas high affinity binding, which is
considered to be responsible for the downstream signaling, was not
altered. EGF-induced autophosphorylation and dimerization of the EGFR
in the GnT-III transfectants were the same levels as found in the
controls. The internalization rate of EGFR was, however, enhanced in
the GnT-III transfectants as judged by the uptake of
125I-EGF and Oregon Green-labeled EGF. When the EGFR
internalization was delayed by dansylcadaverine, the up-regulation of
ERK phosphorylation in GnT-III transfectants was completely suppressed
to the same level as control cells. These results suggest that GnT-III
overexpression in HeLaS3 cells resulted in an enhancement of
EGF-induced ERK phosphorylation at least in part by the up-regulation
of the endocytosis of EGFR.
It is a generally accepted fact that N-glycans play an
important role in the folding, stability, and sorting of glycoproteins (1, 2). They have a common core structure, and their branching patterns
are determined by glycosyltransferases (3-6). Epidermal growth factor receptor (EGFR) is one of membrane
glycoproteins and the oligosaccharide side chains linked to the extracellular domain are essential for its function (16-18). However, the relevance of the composition and structure of N-glycans
to epidermal growth factor (EGF) signaling is not fully understood. It
was previously reported that the EGF binding to its receptor is
significantly decreased in GnT-III-transfected human glioma cells U373
MG (15). In this paper, we report on a study involving the downstream
EGF signaling in GnT-III-transfected HeLaS3 cells to further elucidate
the mechanism by which this enzyme affects EGF signaling. Our findings
show that the internalization rate of EGFR was up-regulated and that
EGF-induced extracellular signal-regulated kinase (ERK) phosphorylation
was enhanced in the GnT-III transfectants.
Cell Lines, Culture, and Transfection--
HeLaS3 cells were
obtained from the American Type Culture Collection (Rockville, MD) and
were maintained in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum. A GnT-III expression vector
was constructed by inserting the cDNA, which encodes for the open
reading frame of human GnT-III into a mammalian expression vector
pCXN2, which was regulated by the Immunoprecipitation and Western Blotting--
Cell cultures
(60-80% confluent) were rinsed twice with ice-cold phosphate-buffered
saline (PBS), harvested in lysis buffer (20 mM Tris-HCl, pH
7.4, 150 mM NaCl, 5 mM EDTA, 1% (w/v) Nonidet P-40, 10% (w/v) glycerol, 5 mM sodium pyrophosphate, 10 mM NaF, 1 mM sodium orthovanadate, 10 mM GnT-III Enzyme Assay--
GnT-III activity was determined by
high liquid performance chromatography using the fluorescence-labeled
sugar chain,
GlcNAc Cell Surface Biotinylation and Immunoprecipitation of
EGFR--
Cell surface biotinylation was performed as described
previously (21). Briefly, cells were rinsed twice with PBS supplemented with 0.1 mM CaCl2 and 1 mM
MgCl2 and then incubated with freshly prepared
sulfosuccinimidobiotin (s-NHS-biotin; Pierce) diluted in the same
solution (1 mg/ml) for 30 min on ice. The reaction was quenched with 50 mM NH4Cl. The resulting cell lysate was
immunoprecipitated with anti-EGFR antibody as described as above and
subjected to 10% SDS-PAGE, after which it was transferred to a
nitrocellulose membrane. After the membranes were blocked with 3%
(w/v) bovine serum albumin (BSA) in Tris-buffered saline containing
0.1% (v/v) Tween 20 (TBST, pH 7.5), the biotinylated proteins were
visualized using Vectastain ABC kit (Vector Laboratories, Burlingame,
CA) and an ECL kit.
Lectin Blot Analysis--
The immunoprecipitated EGFR was
electrophoresed on 10% SDS-PAGE and transferred to nitrocellulose
membranes as described above. The membrane was blocked with 3% BSA
(w/v) in TBST and then incubated with 2 µg/ml biotinylated
erythroagglutinating phytohemagglutinin (E-PHA) (Seikagaku Corp.,
Japan) in TBST for 30 min at room temperature. After washing with TBST
three times, lectin-reactive proteins were detected using a Vectastain
ABC kit and an ECL kit.
EGF Binding Assays--
For the cell surface binding assay, the
cells were seeded at a density of 8 × 104 cell/well
in 24-well plates and incubated overnight. The medium was then replaced
with DMEM, which contained 0.1% (w/v) BSA (M-BSA) and incubated for 20 min at 37 °C. After the medium was replaced with ice-cold M-BSA,
125I-EGF was added in the presence of unlabeled EGF over a
concentration range of 0-2.0 ng/ml. Nonspecific binding was determined
by adding 1 µM cold EGF. After incubation for 2 h at
4 °C, the cells were washed three times with ice-cold PBS and
hydrolyzed in 0.5 ml of 1 M NaOH. The radioactivity of the
cell lysates was counted with a Dimerization Assay--
Subconfluent cells, grown in 100-mm
dishes, were rinsed with ice-cold PBS and incubated with or without 50 ng/ml EGF in DMEM for 2 h at 4 °C. After rinsing with ice-cold
M-BSA, the cells were incubated with or without 15 mM
1-ethyl-3-(3-dimethylaminopropyl)carbodimide (EDAC) (Sigma) for 20 min at room temperature. Cells were washed, harvested in the lysis
buffer, and centrifuged to remove debris. Lysate samples were
immunoprecipitated with an anti-EGFR antibody as described above.
Immunocomplexes were resolved by 7% SDS-PAGE and then transferred to
nitrocellulose membranes. The membranes were probed with anti-EGFR
antibody as described above.
Internalization of EGFR and Transferrin Receptor--
To monitor
EGFR and transferrin receptor internalization, a previously described
protocol (23, 24) was followed. Cells were seeded at a density of
8 × 104 cells/well in a 12-well plate and then
cultured overnight. 125I-EGF (final concentration 1 ng/ml)
or 125I-transferrin (final concentration, 1 µg/ml) in
M-BSA was added to each well and incubated at 37 °C for 1.5, 3.0, 4.5, and 6.0 min. At the indicated times, cells were rapidly rinsed
three times with ice-cold M-BSA to remove unbound ligand. Subsequently,
the cells were incubated for 5 min with 0.2 M acetic acid
(pH 2.8), which contained 0.5 M sodium chloride at 4 °C.
The acid wash was combined with another short rinse with the same
acidic solution to determine the amount of surface-bound
125I-ligand. Finally, the cells were solubilized in 1 M sodium hydroxide for the quantitation of the internalized
125I-ligand. Nonspecific binding was measured in the
presence of a 200-fold molar excess of unlabeled ligand and was not
more than 5% of the total cell-associated radioactivity. Specific
binding is calculated for all data points. For Oregon Green-labeled EGF internalization assay, GnT-III transfectants and mock transfectants cultured on glass coverslips were serum-starved for 2 h at
37 °C. These transfectants were then chilled to 4 °C and
incubated with 200 ng/ml of Oregon Green 514-conjugated EGF (Molecular
Probes, Eugene, OR) for 1 h at 4 °C. Cells were warmed to
37 °C for 10 min, transferred to ice, and stripped of the cell
surface-bound ligand by incubation in ice-cold 0.2 M acetic
acid (pH 2.5), which contained 0.5 M NaCl for 5 min. They
were fixed in 4% paraformaldehyde (buffered at pH 7.4 with 0.1 M phosphate buffer) for 10 min at 4 °C, and mounted with
Permafluor aqueous mounting medium (Immunon, Pittsburgh, PA), and
observed on a fluorescent microscope (Provis AX80) (Olympus, Japan).
Nuclei were visualized with 1:5000 diluting of
4',6-diaminido-2-phenylindole (Molecular Probes).
Establishment of HeLaS3 Clones Stably Expressing
GnT-III--
HeLaS3 cells were transfected with pCXN2/GnT-III or pCXN2
alone and neomycin-resistant clones were selected as described under "Experimental Procedures." Transfection was verified by Western blotting using an anti-GnT-III antibody in conjunction with an enzyme
activity assay. The selected clones showed high expression of GnT-III
(Fig. 1) and an elevated enzymatic
activity (Table I). We consider the bands
seen near 220 kDa in Fig. 1 are nonspecific, since these are observed
in mock transfectants. All other bands were considered to be derived
from GnT-III. Following experiments were performed with both clones and
similar results were observed for all data. The data with clone 1 are
shown in subsequent figures.
Analysis of EGFR of GnT-III Transfectants--
Changes in EGFR
levels on GnT-III-transfected cells were then determined. We examined
the issue of whether the transfection affected the cell surface
expression of EGFR. Using cell surface biotinylation and precipitation,
it was shown that the two cell types expressed nearly the same amount
of EGFR on cell surface (Fig.
2A). Subsequently, lectin
blotting was performed to analyze the alterations in the carbohydrate
structures associated with EGFR. E-PHA binds preferentially to
bisecting GlcNAc residues in N-glycans. The upper
panel of Fig. 2B shows that bisecting GlcNAc increased
significantly on EGFR in the case of the GnT-III transfectants. As a
control, the same blot was probed with anti-EGFR antibody, the results
of which confirmed that the amount of immunoprecipitated EGFR was
nearly the same (lower panel of Fig. 2B). From
this result, it was also indicated that the molecular mass of
immunoprecipitated EGFR in the GnT-III transfectants was lower than
that of the mock transfectants. These data suggest that the
transfection of GnT-III into HelaS3 cells increased the amount of
bisecting oligosaccharide structures and shortened the
N-glycans associated with EGFR without affecting their cell
surface expression.
Enhancement of ERK Activation in GnT-III Transfectants--
To
determine the effect of GnT-III transfection on EGF signaling,
EGF-induced phosphorylation of ERK was investigated. GnT-III transfectants and mock transfectants were treated with 50 ng/ml EGF,
and cell lysate samples were analyzed by Western blotting. Although no
difference in the level of tyrosine phosphorylation between whole cell
lysates of GnT-III transfectants and mock transfectants was observed
(Fig. 3A), ERK phosphorylation
was up-regulated in the case of the GnT-III transfectants by 140% over
that of the mock transfectants (upper panel of Fig.
3B). As a control, the same blot was probed with anti-ERK2
antibody, thus confirming that the amount of ERK2 was nearly the same
(lower panel of Fig. 3B).
Examination of Subtype of EGF Receptors--
To elucidate the
mechanism by which GnT-III transfection affected EGF signaling, a
binding analysis of 125I-EGF to EGFR in GnT-III
transfectants and mock transfectants was performed. As shown in Fig.
4A, the binding of
125I-EGF to EGFR was significantly reduced in the GnT-III
transfectants, compared with the mock transfectants. A Scatchard
analysis revealed that both high and low affinity EGFR were present in
GnT-III and mock transfectants, and Kd or the number
of EGF binding sites per cell of the high affinity EGFR did not change
significantly, but the binding sites of the low affinity class were
decreased by about 40% in GnT-III transfectants (Fig. 4B,
Table II). These data suggest that
modulation of N-glycans with the bisecting GlcNAc by GnT-III
on EGFR may disturb the function of the low affinity of EGFR but not
that of the high affinity of EGFR.
Examination of EGFR Dimerization and
Autophosphorylation--
To further clarify EGFR function in
GnT-III transfectants, EGF-induced EGFR dimerization and
autophosphorylation were examined. After cells were treated with 50 ng/ml EGF, cell surface proteins were cross-linked by using EDAC, and
EGFR was then immunoprecipitated as described under "Experimental
Procedures." In the presence of EGF and EDAC, the approximate
molecular mass of the 340-kDa proteins were detected that corresponded
to a dimer of EGFR (Fig. 5A).
The dimerization level of EGFR was not significantly different between
GnT-III transfectants and the mock transfectants. After the cells were
treated with 50 ng/ml EGF, EGFR was immunoprecipitated with the
anti-EGFR antibody and subjected to blotting analysis with
anti-phosphotyrosine antibody. As shown in Fig. 5B, the
autophosphorylation of EGFR was not significantly changed in GnT-III
transfectants compared with mock transfectants. Collectively, these
results indicate that the levels of dimerization and
autophosphorylation of EGFR were not changed significantly in the two
cell types.
Enhancement of Internalization Rate of EGFR in GnT-III
Transfectants--
To understand how ERK phosphorylation is
up-regulated in GnT-III transfectants, we performed an internalization
assay, since endocytosis is generally thought to affect ERK activation
(25, 26). As shown in Fig. 6A,
the rate of 125I-EGF induced EGFR internalization was
increased by about 40% in the GnT-III transfectants compared with mock
transfectants. In Fig. 6B, the internalized Oregon
Green-labeled EGF in the cytoplasm was clearly detected showing dotlike
fluorescence. EGF endocytosis in the GnT-III transfectants (upper
left panel) was appeared to be increased compared with mock
transfectants (upper right panel). GnT-III transfectants and
mock transfectants were then treated with the internalization chemical
inhibitor, dansylcadaverine and hypertonic medium.
Dansylcadaverine delays receptor internalization at steps that are
proximal to the formation of early endosomes. Cells were serum-starved
for 24 h and incubated with dansylcadaverine, and the EGFR
internalization rate and ERK phosphorylation were examined. When the
internalization of EGFR in two cell types was suppressed to the same
level by dansylcadaverine (upper panel of Fig.
7A), the difference in ERK
phosphorylation diminished (lower panel of Fig.
7A). A hypertonic medium, which contained 0.45 M
sucrose, also inhibits clathrin-mediated endocytosis. The internalization of EGFR in the two cell types was completely inhibited in the hypertonic medium (upper panel of Fig. 7B)
and ERK phosphorylation in two cell types nearly vanished (lower
panel of Fig. 7B). These data, therefore, suggest that
the up-regulation of EGFR internalization could account for the
increase in ERK activation in the GnT-III transfectants. We performed
internalization assay by using CHO-HER cells, and it was observed that
endocytosis of EGFR was also up-regulated in CHO-HER cells, which had
been transfected with GnT-III (Fig. 8).
To see the specificity of effects of GnT-III overexpression, internalization rate of transferrin receptor was examined. As shown in
Fig. 9, there was no difference in
internalization rate of transferrin receptor between GnT-III- and
mock-transfected HeLaS3 cells.
In this paper, we report on a study of the effect of GnT-III
transfection on EGF signaling in HeLaS3 cells. Consistent with our
previous report (15), EGF binding to its receptor was significantly decreased in GnT-III transfectants. However, our findings showed that
EGF-induced ERK phosphorylation was up-regulated in the GnT-III transfectants. To know the mechanism by which ERK phosphorylation is
up-regulated, we first examined EGF binding to EGFR in GnT-III transfectants. A Scatchard analysis of the binding assay data revealed
that only low affinity binding was decreased in the GnT-III transfectants, whereas the high affinity binding was not changed. The
fact that the cell surface expression of EGFR was not altered in
GnT-III transfectants suggests that about 40% of the low affinity receptors lost their function, whereas high affinity receptors remained
intact. EGFR dimerization and autophosphorylation were not
down-regulated in the GnT-III transfectants, which is consistent with the fact that the high affinity class, which constitutes 5-10%
of the EGFR population, is required, and sufficient for EGF-induced
responses (27, 28).
The difference between high affinity and low affinity EGFR has not yet
been fully elucidated. It has been shown that the intracellular part of
the EGFR regulates its affinity for EGF. It was observed that the
activation of PKC converted high affinity EGFR to low affinity EGFR
(29), a deletion of the entire intracellular domain (30) or domain from
amino acids 921 to 940 (31) diminished the high affinity EGFR, and HeLa
cells that overexpress a mutant dynamin (K44A) lost the ability for
high affinity binding of EGFR (32). It has been also demonstrated that
high affinity EGFR represents the cytoskeleton-associated population
(33-36). One possible mechanism by which GnT-III overexpression
affected the EGF binding of low affinity receptor is that GnT-III
transfection alters the status of the intracellular domain of EGFR. We
observed that GnT-III overexpression in CHO-HER cells changed the
reactivity of EGFR toward the antibody which recognized a part of
intracellular domain of EGFR (residues
996-1022).2 Therefore, the
introduction of GnT-III might result in some modification of the
intracellular domain of EGFR. Another possible mechanism is that
GnT-III affects an affinity-modulating protein, the presence of which
has been suggested by van der Heyden et al. (31).
In this study, we demonstrated that the overexpression of GnT-III
affected the internalization rate of EGFR. This effect seems specific
to EGFR, since internalization rate of transferrin receptor was not
affected, which is internalized by different mechanisms (24). The
internalization of the EGFR is generally thought to be essential for
ERK activation (25, 26), as well as the internalization of other
receptors such as insulin receptor (37), insulin-like growth factor
receptor (38), G protein-coupled receptor (39), and serotonin
5-HT1A receptor (40). Several studies have also demonstrated that down-stream signaling molecules locate in endosomes with activated EGFR in response to EGF stimulation and support a role
for membrane trafficking in EGF signaling (41-43). Thus, the
up-regulation of the internalization rate of EGFR observed in GnT-III
transfectants could be involved in the enhancement of downstream
signaling. Quite recently, Tong et al. (44) demonstrated that endocytosis of EGFR affects downstream of Ras. We observed that
activation levels of Ras were not changed in GnT-III transfectants compared with mock transfectants (data not shown). This observation also supports the hypothesis that the enhancement of EGF-stimulated ERK
activation in GnT-III transfectants is due to the increase of
internalization of EGFR.
The mechanisms by which GnT-III overexpression affects endocytosis is
now under investigation. Recently, Altschuler et al. (45)
reported that the internalization of MUC1 is affected by its
O-glycosylation state and that MUC1, which is expressed in glycosylation-defective cells, accumulates in intracellular
compartments. They hypothesized that more of the underglycosylated MUC1
can fit into a clathrin-coated pit because of less steric hindrance and
that this enhances the recruitment of cytoplasmic proteins regulating
endocytosis such as dynamin. Our results are consistent with this
hypothesis since GnT-III overexpression suppresses the processing and
elongation of N-glycans. Another potential cause of
enhancement of endocytosis is the molecular changes observed in the
intracellular domain of EGFR (residues 996-1022) as stated above. The
residues 996-1022 are located in the CaIn domain, so-called because it
mediates the EGF-induced calcium response and internalization (46), and
modification within or near the domain could cause a change of status
or the physicochemical constitution of the molecule such as charges or
steric hindrance, which could, in turn, affect the receptor
internalization rate.
It was reported that the progression of hepatic neoplasms is retarded
in GnT-III
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-4
N-Acetylglucosaminyltransferase III
(GnT-III)1 catalyzes the
addition of N-acetylglucosamine (GlcNAc) to the
-mannoside of the tri-mannose core, to produce a bisecting GlcNAc (7, 8). The introduction of a bisecting GlcNAc results in the
suppression of further processing and elongation of
N-glycans, since other glycosyltransferases are not able to
act on the resulting bianntennary sugar chains. To elucidate the
biological function of bisecting GlcNAc, GnT-III-overexpressing
transfectants have been examined from many aspects (9-15). For
example, we previously reported that the introduction of GnT-III in B16
mouse melanoma cells suppresses lung metastasis (9), increases
E-cadherin-mediated homotypic adhesion (10), and enhances cell adhesion
to hyaluronate via the modulation of N-glycans of CD44 (11).
GnT-III transfection also reduces the susceptibility of human leukemia
K562 cells to the cytotoxicity of natural killer cells (12) and
suppresses the expression of hepatitis B virus in a hepatoma cell line
(13). It has been reported that GnT-III overexpression affects signal transduction such as nerve growth factor signaling by suppressing the
dimerization of Trk A on PC12 cells (14).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin promoter. HeLaS3 cells were
transfected with pCXN2/GnT-III or pCXN2 using LipofectAMINE reagent
(Life Technologies, Inc.) following the manufacturer's instructions.
Selection was performed in the medium, which contained 1.4 mg/ml
neomycin and after a 2-week incubation, neomycin-resistant colonies
were isolated and recloned by serial dilution to ensure clonality.
Positive clones were selected by Western blotting. CHO cells were
transfected with pTJ/human EGFR (19), which was kindly provided by Dr.
Masabumi Shibuya (Institute of Medical Science, University of Tokyo,
Tokyo, Japan) to produce CHO-HER cells and cloned. CHO-HER cells were then transfected with pCXN2/GnT-III or pCXN2 as described above.
-glycerophosphate, 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 5 µg/ml
leupeptin, and 1 mM dithiothreitol). Cell lysates were
centrifuged at 15,000 × g for 10 min at 4 °C, the
supernatants were collected, and the protein concentrations were
determined using a protein assay CBB kit (Nacalai tesque, Japan). For
the immunoprecipitation of EGFR, whole cell lysates (500 µg) were
incubated with 4 µg of sheep anti-human EGFR antibody (06-129, Upstate Biotechnology, Lake Placid, NY) for 2 h at 4 °C, and
then with 15 µl of Protein G-Sepharose 4 Fast Flow (Amersham Pharmacia Biotech) for 2 h at 4 °C. For Western blot analysis, whole cell lysates or immunoprecipitates were subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE), and the resulting proteins were transferred to nitrocellulose membranes (Schleicher & Schuell). The blots were probed with anti-GnT-III antibody (Fujirebio Inc., Japan), sheep anti-EGFR antibody (06-129, Upstate Biotechnology) or anti-phosphotyrosine antibody (4G10, Upstate Biotechnology). For
ERK1/2 activation analysis, the blots were probed with anti-ACTIVE MAPK
polyclonal antibody (Promega, Madison, WI) and anti-MAPK (ERK2)
monoclonal antibody (05-157, Upstate Biotechnology). After the blots
were incubated with peroxidase-conjugated secondary antibody,
immunoreactive bands were visualized using an ECL kit (Amersham
Pharmacia Biotech).
-1,2-Man
-1,6-[GlcNAc
-1,2-Man
-1,3-]Man
-1,4-GlcNAc
1,4-GlcNAc-pyridylamino as a substrate, as described previously (20).
counter. The total number of EGF
binding sites was determined from the specific bound counts by the
method of Scatchard (22).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of transfected GnT-III gene in
HeLaS3 cells. Figure shows Western blot (WB) analysis
using an anti-GnT-III antibody. A whole cell lysate (20 µg) was
electrophoresed and transferred to a nitrocellulose membrane. GnT-III
was detected by Western blot with a GnT-III antibody.
Enzyme activities of GnT-III in transfectants
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Fig. 2.
Analysis of EGFR of GnT-III
transfectants. A, cell surface biotinylation and
immunoprecipitation (IP) of EGFR from the GnT-III
transfectants and mock transfectants. Cells were biotinylated, and
whole cell lysates were immunoprecipitated with an anti-EGFR antibody.
The samples were subjected to 10% SDS-PAGE and transferred to a
nitrocellulose membrane, and the biotinylated proteins were detected as
described under "Experimental Procedures." B, lectin
blot analysis of the immunoprecipitated EGFR from the GnT-III
transfectants and the mock transfectants. EGFR was immunoprecipitated
from 500 µg of proteins of a whole cell lysate and transferred to
nitrocellulose membranes, which were probed by E-PHA (upper
panel) or anti-EGFR antibody (lower panel).
WB, Western blot.
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Fig. 3.
The time course of tyrosine phosphorylation
and ERK phosphorylation in mock- and GnT-III-transfected HeLaS3
cells. Cells were stimulated with 50 ng/ml EGF and harvested at
the indicated times. Whole cell lysates of mock and GnT-III
transfectants were subjected to 10% SDS-PAGE and transferred to a
nitrocellulose membrane. The blots were probed with
anti-phosphotyrosine (4G10) (panel A), anti-phospho ERK
(panel B, upper) and anti-ERK2 (panel
B, lower) as described under "Experimental
Procedures." WB, Western blot;
-pTyr, anti-phosphotyrosine.
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Fig. 4.
125I-EGF binding to mock- and
GnT-III-transfected HeLaS3 cells. A, competition
binding curves of 125I-EGF binding to mock- and
GnT-III-transfected cells. Binding of different dilutions of
125I-EGF to the cells were evaluated using a -counter
after a 2-h incubation of cells at 4 °C with 125I-EGF as
described under "Experimental Procedures." B, Scatcard
analysis of the binding experiments. The figure shows one
representative experiment of three, all of which gave similar
results.
Analysis of 125I-EGF binding
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Fig. 5.
Dimerization and autophosphorylation of EGFR
from mock- and GnT-III-transfected HeLaS3 cells. A,
dimerization analysis of EGFR from mock- and GnT-III-transfected cells.
Cells were treated with 50 ng/ml EGF for 2 h at 4 °C and then
incubated with 15 mM EDAC for 20 min at room temperature.
Cells were harvested and subjected to 7% SDS-PAGE and transferred to a
nitrocellulose membrane. EGFR were detected with anti-EGFR antibody.
IP, immunoprecipitation; WB, Western blot.
B, autophosphorylation of EGFR from mock- and
GnT-III-transfected cells. Cells were stimulated with 50 ng/ml EGF and
harvested at the indicated times. Whole cell lysates were
immunoprecipitated with anti-EGFR. They were subjected to 10% SDS-PAGE
and the blots were probed with anti-phosphotyrosine
( -pTyr, upper panel) and anti-EGFR
antibody (lower panel) as described under "Experimental
Procedures."
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Fig. 6.
EGF-induced internalization of EGFR in mock-
and GnT-III-transfected HeLaS3 cells. A, mock-
and GnT-III-transfected HeLaS3 cells cultured in 12-well dishes were
serum-starved for 24 h, followed by a incubation with M-BSA for 30 min at 37 °C. Cells were subsequently incubated with
125I-EGF in M-BSA and after the indicated times (1.5, 3.0, 4.5, and 6.0 min), surface-bound ligand was extracted with acidic
binding buffer and the internalized radioactivity was determined by
alkaline lysis as described under "Experimental Procedures." Data
were expressed as the rate of internalized radioactivity divided
surface-bound radioactivity. Data represent the averages ± S.E.
of three independent experiments. B, mock- and
GnT-III-transfected HeLaS3 cells were serum-starved and exposed to 200 ng/ml Oregon Green 514-conjugated EGF (Molecular Probes) at 4 °C.
Cells were then warmed for 10 min at 37 °C, after which the
uninternalized cell surface ligand was stripped by an acid wash, and
these transfectants were fixed in 4% paraformaldehyde. The
distribution of Oregon Green-labeled EGF was visualized by fluorescence
microscopy (upper panels). Lower panels show the
nuclei of these cells stained with 4',6-diaminido-2-phenylindole
(scale bar, 10 µm).
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Fig. 7.
Effects of dansylcadaverine and hypertonic
medium on EGFR internalization and ERK phosphorylation in mock- and
GnT-III-transfected HeLaS3 cells. A, mock- and
GnT-III-transfected HeLaS3 cells were serum-starved for 24 h,
followed by a 30-min incubation with or without dansylcadaverine (500 µM) at 37 °C. B, cells were serum-starved
for 24 h followed by a 60-min incubation with sucrose (0.45 M) at 37 °C. Upper panel of A and
B, the internalization rate of EGFR was determined as
described in the figure legend of Fig. 6A and under
"Experimental Procedures." Lower panel of A
and B, cells were incubated with 50 ng/ml EGF and harvested
at the indicated times (0, 2, and 10 min). Each cell lysate sample was
analyzed by Western blotting as described under "Experimental
Procedures." pERK, phospho-ERK; WB, Western
blot.
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Fig. 8.
EGF-induced internalization of EGFR in mock-
and GnT-III-transfected CHO-HER cells. Internalization of EGFR in
mock- and GnT-III-transfected CHO-HER cells was determined as described
for Fig. 6A, and under "Experimental Procedures." Data
represent the averages ± S.E. of three experiments.
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Fig. 9.
Ligand-induced internalization of transferrin
receptor in mock- and GnT-III-transfected HeLaS3 cells.
Internalization of transferrin receptor in mock- and
GnT-III-transfected HeLaS3 cells was determined as described under
"Experimental Procedures." Data were expressed as the rate of
internalized radioactivity divided surface-bound radioactivity. Data
represent the averages ± S.E. of three independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice (47, 48). They suggested
that GnT-III plays an important role in tumor progression in liver. We
offer evidence here that ERK phosphorylation is up-regulated in GnT-III
transfectants, and it might account for the tumor progression activity
of GnT-III. Further investigations are now under way with a goal of
identifying and characterizing the molecules that are involved in
changes of EGF signaling in GnT-III transfectants.
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ACKNOWLEDGEMENT |
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We thank Shoichi Ihara for technical help.
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FOOTNOTES |
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* This work was supported, in part, by Grant-in-aid 05274103 for Scientific Research on Priority Areas from the Ministry of Education, Science, Culture, and Sports, Japan.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.
To whom all correspondence should be addressed. Tel.:
81-6-6879-3421; Fax: 81-6-6879-3429; E-mail:
proftani@biochem.med.osaka-u.ac.jp.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M008551200
2 M. Takahashi, Y. Ikeda, and N. Taniguchi, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: GnT, N-acetylglucosaminyltransferase; BSA, bovine serum albumin; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodimide; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; E-PHA, erythroagglutinating phytohemagglutinin; ERK, extracellular signal-regulated kinase; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; M-BSA, Dulbecco's modified Eagle's medium containing bovine serum albumin; TBST, Tris-buffered saline with Tween 20.
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