Original Paper |
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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2008.00380.x |
N-glycosylation at Asn residues 554 and
566 of E-cadherin affects cell cycle progression through extracellular
signal-regulated protein kinase signaling pathway
Hongbo Zhao, Lidong Sun,
Liying Wang, Zhibin Xu, Feng Zhou, Jianmin Su, Jiawei Jin, Yong Yang, Yali Hu,
and Xiliang Zha*
Key Laboratory
of Glycoconjugate Research, Ministry of Health, Department of Biochemistry and
Molecular Biology, Shanghai Medical College, Fudan University, Shanghai 200032,
China
Received: September
3, 2007
Accepted: November
15, 2007
This work was
supported by the grants from the National Natural Science Foundation of
China (No. 30670468), the Research Fund for Doctoral Program of Higher Education
(No. 20030246042), the Foundation of Shanghai Municipal Health Bureau (No.
044087), and the Shanghai Leading Academic Discipline Project (No. B110)
*Corresponding
author: Tel, 86-21-54237696; Fax, 86-21-64179832; E-mail, [email protected]
E-cadherin,
which has a widely acknowledged role in mediating calcium-dependent cell-cell
adhesion between epithelial cells, also functions as a tumor suppressor. The
ectodomain of human E-cadherin contains four potential N-glycosylation
sites at Asn residues 554, 566, 618, and 633. We investigated the role of
E-cadherin N-glycosylation in cell cycle progression by site-directed
mutagenesis. We showed previously that all four potential N-glycosylation
sites of E-cadherin were N-glycosylated in human breast carcinoma
MDA-MB-435 cells. Removal of N-glycan at Asn633 dramatically affected
E-cadherin stability. In this study we showed that E-cadherin mutant missing N-glycans
at Asn554, Asn566 and Asn618 failed to induce cell cycle arrest in G1 phase and to suppress cell proliferation in comparison with
wild-type E-cadherin. Moreover, N-glycans at Asn554 and Asn566, but not
at Asn618, seemed to be indispensable for E-cadherin-mediated suppression of
cell cycle progression. Removal of N-glycans at either Asn554 or Asn566
of E-cadherin was accompanied with the activation of the extracellular
signal-regulated protein kinase signaling pathway. After treatment with
PD98059, an inhibitor of the extracellular signal-regulated protein kinase
signaling pathway, wild-type E-cadherin transfected MDA-MB-435 and E-cadherin N-glycosylation-deficient
mutant transfected MDA-MB-435 cells had equivalent numbers of cells in G1 phase. These findings implied that N-glycosylation
might be crucial for E-cadherin-mediated suppression of cell cycle progression.
Keywords E-cadherin;
N-glycosylation; b-catenin; ERK signaling
pathway; cell cycle progression
E-cadherin is a
well-characterized cell surface molecule expressed in epithelial cells that
plays a major role in mediating cell-cell adhesion through the establishment
of calcium-dependent homophilic interactions. The misregulated expression of
E-cadherin can alter the growth, differentiation, and proliferation of
epithelial cells. Transfection of E-cadherin into several tumor cell lines
caused decreased cell proliferation [1–3].
Previous studies also showed that E-cadherin initiated cell cycle arrest in G1 phase
in prostate and mammary epithelial cells [4].
The ectodomain of human
E-cadherin contains four potential N-glycosylation sites at Asn residues
554, 566, 618, and 633 based on amino acid sequence analysis (GenBank Accession
No. L08599) [5]. Protein N-glycosylation usually possesses a wide
variety of functions for many proteins, as it affects protein folding, quality
control, sorting, degradation, and secretion [6,7]. Nevertheless, the function of
E-cadherin N-glycosylation remains elusive. To further elucidate this
problem, we obliterated each consensus sequence of human E-cadherin by
substituting Gln for Asn, either individually or in combination, and expressed
mutated cDNAs in human breast carcinoma cell line MDA-MB-435 that lacks expression of E-cadherin at
both the mRNA and protein levels [8]. Previously, we found that all four
potential N-glycosylation sites of E-cadherin were N-glycosylated
in MDA-MB-435 cells and Asn633-linked N-glycan seemed to be required for
E-cadherin stability, whereas N-glycans at the other three sites
contributed slightly to protein stability [9].
In this study, we showed that
removal of N-glycans at Asn554 and Asn566 impaired the tumor-suppressive
role of E-cadherin in cell cycle progression. The extracellular
signal-regulated protein kinase (ERK) signaling pathway, instead of b-catenin, might be involved in the effect
of E-cadherin N-glycosylation on cell cycle progression.
Materials and Methods
Plasmid construction,
site-directed mutagenesis, and transfections
The plasmid pcDNA3.0-Ecad
containing human full-length E-cadherin cDNA was kindly supplied by Dr. Cara J. Gottardi
(Memorial Sloan-Kettering Cancer Center, New York, USA). To create either
individual or combined mutations of N-glycosylation sites of
E-cadherin, a polymerase chain reaction (PCR)-based site-directed mutagenesis
was carried out using a three-round method. In the first-round PCR, the forward
primer was 5‘-AGTGACGAATGTGGTACCTTTTGA-3‘ (for N554Q, N566Q,
N618Q, and N633Q), and the reverse primers were 5‘-TTAGGGCTGTGTACGTGCTTTGCTTCA-3‘
(for N554Q), 5‘-AGCAACTGGAGAACCTTGGTCTGTAGCTAT-3‘ (for
N566Q), 5‘-TGAAGGGAGATGTTTGGGGAGGAAGGTC-3‘ (for N618Q), and 5‘-TACTGAATGGTCCATTGGGGCACTCGCC-3‘
(for N633Q). In the second round, the forward primers were 5‘-TGAAGCAAAGCACGTACACAGCCCTAA-3‘
(for N554Q), 5‘-ATAGCTACAGACCAAGGTTCTCCAGTTGCT-3‘ (for N566Q),
5‘-GACCTTCCTCCCCAAACATCTCCCTTCA-3‘ (for N618Q), 5‘-GGCGAGTGCCCCAATGGACCATTCAGTA-3‘
(for N633Q), and the reverse primer was 5‘-GCTCTAGATCTCGAGTCCCCTAGTGGTCC-3‘
(for N554Q, N566Q, N618Q, and N633Q). In the last round, PCR products from the
first two steps were purified, ligated, and used to replace the similar
fragment of human E-cadherin cDNA plasmid. Mutations were confirmed by
automatic DNA sequencing. Wild-type and mutant E-cadherin cDNAs with one
individual N-glycosylation site abrogated (M1-Ecad, N554Q; M2-Ecad,
N566Q; M3-Ecad, N618Q; M4-Ecad, N633Q) and several N-glycosylation
sites abrogated in combinations (M123-Ecad, N554QN566QN618Q; M1234-Ecad,
N554QN566QN618QN633Q) were purified and transfected into 3105
MDA-MB-435 cells using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA)
according to the manufacturer抯 recommendations. Cell lines
stably expressing wild-type or mutant E-cadherin cDNAs were selected by G418
(800 mg/ml) and screened by reverse
transcription-PCR and Western blot analysis.
Cell lines, antibodies, and
reagents
Human breast carcinoma cell
line MDA-MB-435, mock (empty plasmid stably transfected MDA-MB-435), wtEcad-435
(wild-type E-cadherin stably transfected MDA-MB-435), M1-Ecad-435 (M1-Ecad
stably transfected MDA-MB-435), M2-Ecad-435 (M2-Ecad stably transfected
MDA-MB-435), M3-Ecad-435 (M3-Ecad stably transfected MDA-MB-435), M4-Ecad-435
(M4-Ecad stably transfected MDA-MB-435), M123-Ecad-435 (M123-Ecad stably
transfected MDA-MB-435), and M1234-Ecad-435 (M1234-Ecad stably transfected
MDA-MB-435) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)
(Gibco BRL, Grand Island, USA) supplemented with 10% fetal bovine serum
(HyClone Laboratories, Logan, USA) and 1% penicillin/streptomycin (Life
Technologies, Grand Island, USA).
Monoclonal E-cadherin antibody
was purchased from BD Transduction Laboratories (San Diego, USA). Monoclonal
antibodies to b-catenin, ERK1/2, and
phosphorylated ERK (p-ERK)1/2 were obtained from Santa Cruz Biotechnology
(Santa Cruz, USA). Monoclonal antibody to glyceraldehyde-3-phosphate
dehydrogenase and secondary antibodies conjugated with horseradish peroxidase
were from Kang-Chen Biotech (Shanghai, China). fluorescein-isothiocyanate-conjugated secondary antibody, fluorescent-mounting
medium, tunicamycin, and dimethylsulfoxide were purchased from Sigma-Aldrich
(St. Louis, USA).
Western blot analysis
Cells were lysed in 1 sodium
dodecyl sulfate (SDS) lysis buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10%
glycerol) supplemented with 1 mM phenylmethylsulphonyl fluoride (PMSF) and 1 mM
Na3VO4, and
carried out as described previously
[9]. Equal
amounts of protein were loaded on an SDS-polyacrylamide gel and transferred to
a polyvinylidene difluoride membrane. After blocking with 5% bovine serum
albumin in phosphate-buffered saline (PBS; containing 0.05% Tween 20), the
membranes were incubated with specific primary antibodies, followed by
incubation with horseradish peroxidase-conjugated secondary antibodies.
Proteins were visualized by fluorography using an enhanced chemiluminescence
system (Shanghai Perfect Biotech, Shanghai, China).
Immunofluorescence
Confluent cells were grown on
glass coverslips and fixed with 4% paraformaldehyde in PBS for 20 min,
permeabilized with 0.1% Triton X-100 in PBS for 10 min, and non-specific
binding sites were blocked with 3% bovine serum albumin in PBS for 30 min.
Specimens were incubated with mouse anti-E-cadherin monoclonal antibody (1:50
dilution in blocking solution) at 37 ºC for 3 h, followed by
fluorescein-isothiocyanate-conjugated anti-mouse secondary antibody (1:50
dilution in blocking solution) at 37 ºC for 1 h. After washing with PBS, the
coverslips were mounted upside-down on object slides using fluorescent-mounting
medium. Immunofluorescence was visualized by a Leica TCS SP2 confocal
microscope (Leica, Wetzlar, Germany) and subjected to image analysis (Leika TCS
SP2 confocal software).
Flow cytometric (FCM) analysis
Cells
were G0-synchronized
by replacing growth medium with starvation medium (DMEM, 1% fetal bovine serum)
for 24 h. Synchronization was confirmed by FCM analysis of cells stained with
propidium iodide (PI; Molecular Probes; Sigma-Chemical, St. Louis, USA) (data
not shown). Cells were then cultured in DMEM containing 10% fetal bovine serum
for 12 h, then digested, washed, fixed in 75% ethanol, and stored at –20 ºC. For PI staining, cells were
washed in 1% fetal
bovine serum in PBS prior to incubation at 37 ºC for 30 min in the same
solution containing
40 mg/ml PI and 250 mg/ml
RNase A
(Roche Diagnostics, Mannheim, Germany). Data were collected using a Coulter EPICS
Elite ESP flow cytometer (Beckman Coulter, High Wycombe, UK) equipped with a
Spectra-Physics argon-ion laser (Spectra-Physics, High Wycombe, UK) and
analyzed using the WinMDI program (version 2.8; Joseph Trotter, Scripps
Research Institute, La Jolla, USA). Results represent a minimum of 20,000 cells
assayed for each sample.
Subcellular fractionation
Subcellular fractionation was
carried out as described previously [10]. Cells were lysed in an ice-cold
solution containing 0.02% digitonin, 5 mM sodium phosphate (pH 7.4), 50 mM
NaCl, 150 mM sucrose, 5 mM KCl, 2 mM dithiothreitol, 1 mM MgCl2, 0.5 mM
CaCl2,
and 0.1 mM PMSF. The cytoplasmic fraction was collected after centrifugation
at 1000 g for 10 min at 4 ºC. The final pellet was resuspended in the
lysis solution without digitonin and loaded onto a cushion of a solution
containing 30% (w/v) sucrose, 2.5 mM Tris-HCl (pH
7.4), and 10 mM NaCl. After centrifugation at 1000 g for 10 min at 4 ºC,
nuclei were collected and extracted for 30 min at 4 ºC with an ice-cold
solution containing 0.5% Triton X-100, 50 mM Tris-HCl (pH 7.5), and 300 mM
NaCl. After centrifugation at 10,000 g for 10 min at 4 ºC, the
supernatant was collected as the nuclear fraction. The cytoplasmic protein
tubulin and nuclear protein sp1 were used as loading controls.
Preparation of triton X-100-insoluble cytoskeletal
fraction
All procedures were carried
out as described previously [11]. Briefly, cells were lysed in a buffer
containing 10 mM Tris-HCl (pH 6.8), 1 mM EDTA, 150 mM NaCl, 0.25% Nonidet P-40,
1% Triton X-100, 1 mM PMSF, and 1 mM Na3VO4, followed by centrifugation
at 16,000 g. For Triton X-100-insoluble cytoskeletal fraction, the
remaining pellet was re-extracted twice with lysis buffer to ensure that all
detergent-soluble material was removed. The final pellet after centrifugation
at 16,000 g was extracted with SDS-containing buffer (10 mM Tris-HCl,
pH 6.8, 2 mM EDTA, 150 mM NaCl, and 1% SDS). Equal amounts of protein were
loaded on a 10% SDS-polyacrylamide gel, then analyzed using Western blot
assay.
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) assay
Cells were seeded in 96-well
plates at a density of 2104 cells/well in 200 ml DMEM medium, and grown for 0, 24, 48,
or 72 h. Meanwhile, an equal amount of fresh medium was added into wells
without cells, and used as the control. For each assay, 20 ml MTT was added, and these plates
continued to culture for 4 h. After incubation, the culture medium was
discarded and 150 ml dimethylsulfoxide was added
to each well. These plates were vibrated gently for 10 min, followed by
detection in the Elx800 universal microplate reader (Bio-TEK instruments,
Highland Park, USA) at 490 nm.
Results
Occupation of four potential N-glycosylation
sites of human E-cadherin
The ectodomain of human
E-cadherin contains four potential N-glycosylation sites at Asn residues
554, 566, 618, and 633 based on amino acid sequence analysis (GenBank Accession
No. L08599) [5]. Here we generated N-glycosylation-deficient mutants of
E-cadherin by substituting Gln for Asn in each N-glycosylation
consensus sequence NXS/T, either individually or in combination, by
site-directed mutagenesis. The E-cadherin mutants with one individual N-glycosylation
site abrogated and several N-glycosylation sites abrogated in
combination were stably expressed in human breast carcinoma cell line
MDA-MB-435 that lacks E-cadherin expression at both the mRNA and protein levels
[8]. Previously, our findings showed that the four potential N-glycosylation
sites of E-cadherin were N-glycosylated in MDA-MB-435 cells and
Asn633-linked N-glycan seemed to be required for E-cadherin stability,
whereas N-glycans at the other three sites contributed slightly to
protein stability [9].
Effect of E-cadherin N-glycosylation
on cell cycle progression
The eukaryotic cell cycle is
composed of four phases (G1, S, G2, and M) as well as an
out-of-cycle quiescent phase designated G0. In normal mammary epithelial
cells, an intricate network of growth-inhibitory and growth-stimulatory
signals is transduced from the extracellular environment and stringently regulates
cell cycle progression [12,13]. The final targets of these extracellular
growth signaling pathways are specific sets of cyclin-dependent kinase (CDK)
protein. In the G1 phase of the cell cycle, cyclin-E and
especially cyclin-D1 are necessary for the activation of CDKs (CDK2, CDK4, and
CDK6) and the regulation of G1 phase cell cycle progression [14,15].
E-cadherin has been shown to
down-regulate the expression of cyclin-D1, induce cell cycle arrest at G1 and
inhibit cell proliferation [1–4,16]. In this study, we
investigated the effect of E-cadherin N-glycosylation on the endogenous
expression of cyclin-D1. Total protein lysates derived from mock, wtEcad-435,
and M123-Ecad-435 cells were subjected to Western blot analysis. As shown in Fig.
1(A,B), cyclin-E was expressed at similar levels in the above three
cell lines. Nevertheless, cyclin-D1 expression in wtEcad-435 cells was
decreased by approximately 30% compared with mock cells. In contrast, cyclin-D1
expression in M123-Ecad-435 cells was elevated, similar to that in mock cells.
As mentioned, cyclin-D1 promotes the G1/S phase transition by
regulating the activity of CDKs, therefore we examined the percentage of the
above cell lines in G1 phase using FCM analysis. Consistent with
cyclin-D1 expression, the percentage of M123-Ecad-435 cells in G1 phase
was decreased by 20% in comparison with that of wtEcad-435 cells [Fig. 1(C)].
As cell proliferation is frequently regulated by cell cycle machinery, we next
determined the proliferation capacity of the above cell lines using MTT assay.
These data showed that the proliferation capacity of M123-Ecad-435 cells was
significantly increased compared to that of wtEcad-435 cells [Fig. 1(D)].
These observations revealed
that wild-type E-cadherin could down-regulate cyclin-D1 expression, induce G1 phase
arrest, and inhibit cell proliferation in MDA-MB-435 cells. Once N-glycosylation
on E-cadherin was obliterated, the tumor-suppressive function of E-cadherin was
impaired. These findings indicated that N-glycosylation could be
essential for the inhibitory effect of E-cadherin on cell cycle progression and
cell proliferation.
Effect of N-glycosylation
on localization of E-cadherin
N-glycosylation has been known to ensure
correct localization of many glycoproteins [6,7,17,18]. In the case of
E-cadherin, localization on the cell surface is indispensable for its normal
function. E-cadherin is not always at adherens junctions (AJs), and it spends
variable amounts of time in vesicles trafficking to and from the cell surface.
E-cadherin on the cell surface, instead of those in intracellular vesicles and
compartments, plays a crucial role in mediating cell-cell adhesion [19]. Next,
we wanted to know whether N-glycosylation affects the localization of
E-cadherin to the cell surface, and ultimately impacts on protein biological
function. To this end, the localization of M123-Ecad mutant was determined
using immunofluorescence analysis. As illustrated in Fig. 2(A), mock
cells had
undetectable immunofluorescence. However, the immunofluorescence staining of
M123-Ecad-435 cells and wtEcad-435 cells was almost identical, and most immunofluorescent
signals were localized at the cell surface.
E-cadherin in the Triton
X-100-insoluble cytoskeletal fraction has been shown to reach AJs and anchor to
the actin cytoskeleton [20,21]. To further determine whether N-glycosylation
affects the localization of E-cadherin at AJs, we investigated the distribution
of M123-Ecad mutant and wild-type E-cadherin in the Triton X-100-insoluble
cytoskeletal fraction. Because cell confluence influences the recruitment of
E-cadherin at AJs [22], Triton X-100-insoluble cytoskeletal fraction derived
from wtEcad-435 and M123-Ecad-435 cells were grown and maintained under
different densities (dense conditions: cells were grown to >90% confluence;
sparse conditions: cells were grown to <30% confluence) and extracted. The
distribution of E-cadherin at AJs was substantially increased in cells cultured under
dense conditions compared with those cultured under sparse conditions,
both in wtEcad-435 and M123-Ecad-435 cells, however, the two cell lines had
equivalent amounts of E-cadherin at AJs under the same conditions [Fig. 2(B)].
These data suggested N-glycans at Asn554, Asn566, and Asn618 of
E-cadherin did not affect the localization of E-cadherin to the cell surface
or AJs.
Effect of E-cadherin N-glycosylation
on activation of ERK signaling pathway
cyclin-D1 transcription
is usually facilitated by several signaling pathways, including ERK [23] and b-catenin signaling pathways [24]. We
detected which signaling pathway might be involved in the effect of E-cadherin
N-glycosylation on cell cycle progression.
-Catenin, as a key mediator of
E-cadherin-mediated cell-cell adhesion, has been shown to play a dual role in
the Wnt signaling pathway [25,26]. It binds tightly to the cytoplasmic domain
of E-cadherin then to a-catenin and vinculin, through
which the adherens complex is linked to the actin cytoskeleton [25–28]. On activation of the Wnt cascade, b-catenin translocates into the nucleus,
where it can bind to transcription factors of the lymphocyte enhancer factor-1
(LEF-1) family and regulate the expression of cyclin-D1 [24]. The activation of
the Wnt signaling pathway depends on b-catenin
accumulation and its subsequent translocation into the nucleus [24]. Here we
detected nuclear b-catenin in mock, wtEcad-435,
and M123-Ecad-435 cells by subcellular fractionation analysis. As shown in Fig.
2(C), the levels of nuclear b-catenin
in wtEcad-435 and M123-Ecad-435 cells were identical, implying that removal of N-glycosylation
on E-cadherin did not affect b-catenin activity.
Earlier reports showed that
E-cadherin could down-regulate the ERK signaling pathway in intestinal
epithelial cells and colon tumor cells [29,30]. In our study, ERK activity in
the above cell lines was investigated using Western blot analysis. Because
p-ERK is an active form of ERK, the activity of ERK was measured with an
anti-p-ERK antibody. As shown in Fig. 2(D), the total
level of ERK in the above three cell lines was equal. However, the level of
p-ERK in wtEcad-435 was reduced significantly so that the protein band was
almost undetectable. Nevertheless, the level of p-ERK in M123-Ecad-435 was
equivalent to that in mock cells.
To further detect the
involvement of E-cadherin N-glycosylation in the activation of the ERK
signaling pathway, we treated wtEcad-435 and M123-Ecad-435 cells with
tunicamycin that inhibits the N-glycosylation of glycoproteins in higher
organisms by blocking the first step in biosynthesis of the lipid-linked
oligosaccharide precursor. These data showed that p-ERK levels in wtEcad-435
and M123-Ecad-435 cells became almost identical after tunicamycin treatment,
indicating that the difference in p-ERK levels in the above two cell lines was
attributed to carbohydrate moiety of E-cadherin [Fig. 3(A)].
Furthermore, wtEcad-435 cells were transiently transfected with M123-Ecad
plasmid. As shown in Fig. 3(B), the expression of M123-Ecad mutant in
wtEcad-435 cells elicited the elevated p-ERK. All of these findings
suggested that E-cadherin N-glycosylation presumably affected the
activation of the ERK signaling pathway.
To confirm whether E-cadherin N-glycosylation
affects cell cycle progression through the ERK signaling pathway, mock,
wtEcad-435, and M123-Ecad-435 cells were treated with PD98059, a specific
inhibitor of the ERK signaling pathway. As illustrated in Fig. 3(C), the
three cell lines showed an equal percentage of cells in G1 phase,
suggesting that E-cadherin N-glycosylation might affect cell cycle
progression through the ERK signaling pathway.
Effect of individual N-glycans
of E-cadherin on cell cycle progression and ERK signaling pathway
We also investigated the role
of N-glycans at Asn554, Asn566, and Asn-618 of E-cadherin in cell cycle
progression. As shown in Fig. 4(A), M1-Ecad-435, M2-Ecad-435, and
M123-Ecad-435 cells showed elevated levels of p-ERK compared with wtEcad-435
cells. Consistent with the up-regulation of the ERK signaling pathway, the
percentage of these three cell lines in G1 phase decreased in comparison
with that in wtEcad-435 cells. Nevertheless, the percentage of G1 phase
cells and the p-ERK level in M3-Ecad-435 cells were similar to that in
wtEcad-435 cells [Fig. 4(B)]. Together, these data indicated that N-glycans
at Asn554 and Asn566, but not N-glycan at Asn618, might be essential for
the inhibitory effect of E-cadherin on the ERK signaling pathway and cell cycle
progression.
Discussion
E-cadherin mediates the
formation of AJs between epithelials that serve both as mechanical linkages
between cells and as signaling hubs that relay information from the
extracellular environment. As the key component of AJs, E-cadherin is also viewed
as a tumor suppressor because it is frequently down-regulated in carcinomas and
transfection of E-cadherin into several tumor cell lines causes decreased cell
proliferation [1–4].
The ectodomain of human
E-cadherin contains four potential N-glycosylation sites. N-glycosylation
is a metabolic process that has been highly conserved in evolution. In all
eukaryotes, N-glycosylation is necessary for viability. It functions by
modifying appropriate Asn residues of proteins with oligosaccharide structures,
thus influencing their bioactivities. To date, the structure and function of
E-cadherin N-glycosylation remain largely unknown. In this report we
showed that N-glycosylation could affect the tumor-suppressive role of
E-cadherin in cell cycle progression and cell proliferation, and that the ERK
signaling pathway might be involved in this process.
As all four N-glycosylation
sites are located in the ectodomain of E-cadherin, how does E-cadherin N-glycosylation
affect the activation of the intracellular ERK signaling pathway? Several lines
of evidence have shown that E-cadherin adhesive complexes at AJs are components
of larger complexes that involve b-catenin
[1], growth factor receptors such as hepatocyte growth factor receptor c-Met
and epidermal growth factor receptor [31,32], PI3K/PKB [33], P120 [34–36], Rho GTPase [37], and ERK [38].
However, how these molecules bind to the adhesive complexes remains obscure.
-Catenin appears to play a pivotal role in the formation of adhesive complexes
because it mediates the binding of E-cadherin to the actin cytoskeleton,
epidermal growth factor receptor [31], phosphatase and tensin homology deleted
on chromosome ten (PTEN) and PI3K/PKB [33]. We have found that removal of
E-cadherin N-glycosylation resulted in elevated b-catenin
tyrosine phosphorylation and reduced b-catenin
and a-catenin at AJs [9]. These
findings strongly implied that E-cadherin N-glycosylation might affect
the organization of b-catenin at AJs. Therefore, we
speculated that removal of E-cadherin N-glycosylation could impact on
the organization of b-catenin at AJs, further
inducing the destabilization of adhesive complexes and the dysfunction of the
ERK signaling pathway.
Although the role of E-cadherin as a tumor
suppressor has been well established, the exact molecular mechanisms of its
suppressive
function remain poorly defined. One possible mechanism
is that E-cadherin sequesters b-catenin at AJs, and
antagonizes the nuclear b-catenin/T cell factor (TCF) signaling pathway.
Interestingly, in our present study both wild-type E-cadherin and M123-Ecad
mutant could up-regulate the endogenous expression of b-catenin in MDA-MB-435
cells [Fig. 2(C)]. Similar data have been mentioned in other
reports, however, little has been known about the underlying mechanisms.
There is considerable variation in the number and
composition of terminal chains in the mature complex oligosaccharides, giving
rise to N-glycosylation heterogeneity of glycoproteins. In our study,
E-cadherin individual N-glycans appeared to have distinctive functions;
N-glycan at Asn633 seems to be required for E-cadherin stability [9]. N-glycans
at Asn554 and Asn566 of E-cadherin affected cell cycle progression, whereas N-glycan
at Asn618 contributed slightly to the biological function of E-cadherin. Why do
individual N-glycans play distinctive roles in the bioactivities of
E-cadherin? The four Asn residues within E-cadherin might be modified with different oligosaccharide structures, presumably
resulting in the functional diversity of N-glycans. In addition, the
localization of N-glycan on E-cadherin seems to be another important
factor. A precise understanding of N-glycosylation heterogeneity of
E-cadherin still awaits further investigation.
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