E-cadherin
affects
cell| cycle
Acta BiochimN-glycosylation
Biophys Sin (2008):
140-148
© 2008progression
Institute of Biochemistry and Cell Biology, SIBS, CAS | All Rights Reserved 1672-9145
http://www.abbs.info; www.blackwellpublishing.com/abbs | 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
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 Nglycosylation 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.
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 Edu ca tion (No. 200 30 24 60 42 ), 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]
Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 2 | Page 140
Keywords
E-cadherin; N-glycosylation; β-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 Nglycosylation 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 MDAMB-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
E-cadherin N-glycosylation affects cell cycle progression
signal-regulated protein kinase (ERK) signaling pathway,
instead of β-catenin, might be involved in the effect of Ecadherin 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'-GACCTTCCTCCCCAAACATCTCCCTTCA3' (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
huma n E-cadherin cDNA plasmid. Mutations were
confirmed by automatic DNA sequencing. Wild-type and
mutant E-cadherin cDNAs with one individual Nglycosylation 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 3×105 MDA-MB-435 cells using Lipofectamine
2000 reagent (Invitrogen, Carlsbad, USA) according to
the manufacturer’s recommendations. Cell lines stably
expressing wild-type or mutant E-cadherin cDNAs were
selected by G418 (800 µg/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-Eca d-435 (M1-Ecad stably
transfected MDA-MB-435), M2-Ecad-435 (M2-Ecad
stably transfected MDA-MB-435), M3-Ecad-435 (M3Ecad stably transfected MDA-MB-435), M4-Ecad-435
(M4-Ecad stably transfected MDA-MB-435), M123-Ecad435 (M123-Ecad stably transfected MDA-MB-435), and
M1234-Ecad-435 (M1234-Ecad stably transfected MDAMB-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 β-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 a nd 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% pa raforma ldehyde 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
Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 2 | Page 141
E-cadherin N-glycosylation affects cell cycle progression
(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 µg/ml PI and 250 µg/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 SpectraPhysics 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.
Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 2 | Page 142
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 2×104
cells/well in 200 µl 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 µl MTT was added, and these plates
continued to culture for 4 h. After incubation, the culture
medium was discarded and 150 µl 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-MB435 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-
E-cadherin N-glycosylation affects cell cycle progression
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 growthstimulatory 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 cyclindependent 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-Ecad435 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-
Fig. 1 Effect of E-cadherin N-glycosylation on cell cycle progression and cell proliferation (A) Western blot analysis of the expression
of cyclin-D1 and cyclin-E in mock, wild-type E-cadherin stably transfected human breast carcinoma MDA-MB-435 (wtEcad-435) cells, and M123Ecad stably transfected MDA-MB-435 (M123-Ecad-435) cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading
control. (B) Quantitative analyses of the expression of cyclin-D1 based on densitometry in the above cell lines. The results (mean±SD) of three
independent experiments are shown. (C) Flow cytometric analysis of the percentage of the above three cell lines in G1 phase. Representative results
from one of three experiments are shown. (D) Cell proliferation of the above cell lines was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide assay. Data are presented as mean±SD of three independent experiments.
Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 2 | Page 143
E-cadherin N-glycosylation affects cell cycle progression
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-Ecad435 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-100Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 2 | Page 144
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 β-catenin
signaling pathways [24]. We detected which signaling
pathway might be involved in the effect of E-cadherin Nglycosylation on cell cycle progression.
β-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 α-catenin and
vinculin, through which the adherens complex is linked to
the actin cytoskeleton [25−28]. On activation of the Wnt
cascade, β-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 β-catenin accumulation and its
subsequent translocation into the nucleus [24]. Here we
detected nuclear β-catenin in mock, wtEcad-435, and
M123-Ecad-435 cells by subcellular fractionation analysis.
As shown in Fig. 2(C), the levels of nuclear β-catenin in
wtEcad-435 and M123-Ecad-435 cells were identical,
implying that removal of N-glycosylation on E-cadherin
did not affect β-catenin activity.
Earlier reports showed that E-cadherin could downregulate 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-pERK 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.
E-cadherin N-glycosylation affects cell cycle progression
Fig. 2 Effect of N-glycosylation on localization of E-cadherin and extracellular signal-regulated protein kinase (ERK) signaling
pathway (A) Immunofluorescence analysis of the localization of M123-Ecad mutant on the cell surface. Mock, wild-type E-cadherin stably
transfected breast carcinoma MDA-MB-435 (wtEcad-435) cells, and M123-Ecad stably transfected MDA-MB-435 (M123-Ecad-435) cells were
stained with anti-E-cadherin antibody, followed by fluorescein-isothiocyanate-conjugated anti mouse secondary antibody. Bar=10 µm. (B)
Western blot analysis of the distribution of M123-Ecad mutant at adherens junctions. Triton X-100-insoluble cytoskeletal fractions derived
from wtEcad-435 and M123-Ecad-435 cells under different conditions [D, dense conditions (cells grown to >90% confluence); S, sparse
conditions (cells grown to <30% confluence)] were prepared, subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and
probed with antibodies against E-cadherin and tubulin. Tubulin was used as a loading control. Representative results from one of three experiments are shown. (C) Western blot analysis of nuclear β-catenin in mock, wtEcad-435, and M123-Ecad-435 cells. The cytoplasmic protein
tubulin and nuclear protein sp1 were used as loading controls. Representative results from one of three experiments are shown. C, cytoplasm; N,
nucleus. (D) Western blot analysis of phosphorylated ERK (p-ERK) levels in the above cell lines. The results represent one of three independent
experiments.
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 Nglycosylation 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 G 1
phase, suggesting that E-cadherin N-glycosylation might
affect cell cycle progression through the ERK signaling
pathway.
Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 2 | Page 145
E-cadherin N-glycosylation affects cell cycle progression
Fig. 3 E-cadherin N-glycosylation impacts on cell cycle progression through extracellular signal-regulated protein kinase (ERK) signaling pathway
(A) Western blot analysis of
phosphorylated-ERK (p-ERK) levels in mock, wild-type E-cadherin
stably transfected breast carcinoma MDA-MB-435 (wtEcad-435) cells,
and M123-Ecad stably transfected MDA-MB-435 (M123-Ecad-435)
cells. The cell lines were treated with 0.5 µg/ml tunicamycin (TM) for
24 h then subjected to Western blot analysis. Representative results
from one of three experiments are shown. +, TM; –, control. (B)
Western blot analysis of p-ERK levels in wtEcad-435 cells transiently
transfected with M123-Ecad plasmid. C, empty plasmid control. (C)
Flow cytometric analysis of the percentage of the above cell lines in
G1 phase after treatment with 20 µM PD98059, a specific inhibitor of
the ERK signaling pathway, for 1 h.
Fig. 4 Phosphorylated extracellular signal-regulated protein
kinase (p-ERK) levels and the percentage of G1 phase cells in
six human breast carcinoma cell lines (A) Western blot analysis
of p-ERK levels in empty plasmid stably transfected MDA-MB-435
(mock), wild-type E-cadherin stably transfected MDA-MB-435
(wtEcad-435), M1-Ecad stably transfected MDA-MB-435 (M1-Ecad435), M2-Ecad stably transfected MDA-MB-435 (M2-Ecad-435), M3Ecad stably transfected MDA-MB-435 (M3-Ecad-435), and M123Ecad stably transfected MDA-MB-435 (M123-Ecad-435). Representative results from one of three experiments are shown. (B) Flow
cytometric analysis of the percentage of the six cell lines in G1 phase.
Data are presented as mean±SD of three independent experiments.
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
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, M2Ecad-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,
Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 2 | Page 146
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, Ecadherin is also viewed as a tumor suppressor because it
is frequently down-regulated in ca rcinomas a nd
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
E-cadherin N-glycosylation affects cell cycle progression
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 Nglycosylation 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 β-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. β-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 β-catenin tyrosine phosphorylation and reduced
β-catenin and α-catenin at AJs [9]. These findings strongly
implied that E-cadherin N-glycosylation might affect the
organization of β-catenin at AJs. Therefore, we speculated
that removal of E-cadherin N-glycosylation could impact
on the organization of β-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 β-catenin
at AJs, and antagonizes the nuclear β-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 β-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.
References
1 Gottardi CJ, Wong E, Gumbiner BM. E-cadherin suppresses cellular
transformation by inhibiting beta-catenin signaling in an adhesionindependent manner. J Cell Biol 2001, 153: 1049−1060
2 St Croix B, Sheehan C, Rak JW, Florenes VA, Slingerland JM, Kerbel
RS. E-cadherin-dependent growth suppression is mediated by the
cyclin-dependent kinase inhibitor p27KIP1. J Cell Biol 1998, 142:
557−571
3 Navarro P, Gomez M, Pizarro A, Gamallo C, Quintanilla M, Cano
A. A role for the E-cadherin cell-cell adhesion molecule during
tumor progression of mouse epidermal carcinogenesis. J Cell Biol
1991, 115: 517−533
4 Day ML, Zhao X, Vallorosi CJ, Putzi M, Powell CT, Lin C, Day
KC. E-cadherin mediates aggregation-dependent survival of prostate
and mammary epithelial cells through the retinoblastoma cell cycle
control pathway. J Biol Chem 1999, 274: 9656−9664
5 Rimm D, Morrow JS. Molecular cloning of human E-cadherin
suggests a novel subdivision of the cadherin superfamily. Biochem
Biophys Res Commun 1994, 200: 1754−1761
6 Rudd PM, Dwek RA. Glycosylation: Heterogeneity and the 3D
structure of proteins. Crit Rev Biochem Mol Biol 1997, 32: 1−100
7 Helenius A, Aebi M. Roles of N-linked glycans in the endoplasmic
reticulum. Annu Rev Biochem 2004, 73: 1019−1049
8 Liu Z, Brattain MG, Appert H. Differential display of reticulocalbin
in the highly invasive cell line, MDA-MB-435, versus the poorly
invasive cell line, MCF-7. Biochem Biophys Res Commun 1997,
231: 283−289
9 Zhao HB, Liang YL, Xu ZB, Wang LY, Zhou F, Li ZX, Jin JW et al.
N-glycosylation affects the adhesive function of E-cadherin through
modifying the composition of adherens junctions (AJs) in human
breast carcinoma cell line MDA-MB-435. J Cell Biochem 2007,
Nov 2 (Epub ahead of print)
1 0 Ishida N, Hara T, Kamura T, Yoshida M, Nakayama K, Nakayama
KI. Phosphorylation of p27Kip1 on serine 10 is required for its
binding to CRM1 and nuclear export. J Biol Chem 2002, 277:
14355−14358
1 1 Kim DY, Ingano LA, Kovacs DM. Nectin-1 alpha, an immunoglobulin-like receptor involved in the formation of synapses, is a
substrate for presenilin/gammasecretase-like cleavage. J Biol Chem
2002, 277: 49976−49981
Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 2 | Page 147
E-cadherin N-glycosylation affects cell cycle progression
1 2 Stillman B. Cell cycle control of DNA replication. Science 1996,
274: 1659−1664
1 3 Cover CM, Hsieh SJ, Tran SH, Hallden G, Kim GS, Bjeldanes LF,
Firestone GL. Indole-3-carbinol inhibits the expression of cyclindependent kinase-6 and induces a G 1 cell cycle arrest of human
breast cancer cells independent of estrogen receptor signaling. J
Biol Chem 1998, 273: 3838−3847
1 4 Coleman ML, Marshall CJ, Olson MF. RAS and RHO GTPases in
G 1 -phase cell-cycle regulation. Nat Rev Mol Cell Biol 2004, 5:
355−366
1 5 Sherr CJ. Cancer cell cycle. Science 1996, 274: 1672−1677
1 6 Hermiston ML, Wong MH, Gordon JI. Forced expression of Ecadherin in the mouse intestinal epithelium slows cell migration
and provides evidence for nonautonomous regulation of cell fate in
a self-renewing system. Genes Dev 1996, 10: 985−996
1 7 Zhong X, Malhotra R, Woodruff R, Guidotti G. Mammalian plasma
membrane ecto-nucleoside triphosphate diphosphohydrolase 1,
CD39, is not active intracellularly. The N-glycosylation state of
CD39 correlates with surface activity and localization. J Biol Chem
2001, 276: 41518−41525
1 8 Martinez-Maza R, Poyatos I, López-Corcuera B, Núñez E, Giménez
C, Zafra F, Aragón C. The role of N-glycosylation in transport to
the plasma membrane and sorting of the neuronal glycine transporter
GLYT2. J Biol Chem 2001, 276: 2168−2173
1 9 Bryant DM, Stow JL. The ins and outs of E-cadherin trafficking.
Trends Cell Biol 2004, 14: 427−434
2 0 McNeill H, Ryan TA, Smith SJ, Nelson WJ. Spatial and temporal
dissection of immediate and early events following cadherin-mediated
epithelial cell adhesion. J Cell Biol 1993, 120: 1217−1226
2 1 Hinck L, Nathke IS, Papkoff J, Nelson WJ. Dynamics of cadherin/
ca tenin complex forma tion: Novel protein intera ctions a nd
pathways of complex assembly. J Cell Biol 1994, 125: 1327−1340
2 2 Le TL, Yap AS, Stow JL. Recycling of E-cadherin: a potential
mechanism for regulating cadherin dynamics. J Cell Biol 1999,
146: 219−232
2 3 Weber JD, Raben DM, Phillips PJ, Baldassare JJ. Sustained activation
of extracellular-signal-regulated kinase 1 (ERK1) is required for the
continued expression of cyclin D1 in G 1 phase. Biochem J 1997,
326: 61−68
2 4 Shtutman M, Zhurinsky J, Simcha I, Albanese C, D’Amico M, Pestell
R, Ben Ze’ev A. The cyclin D1 gene is a target of the β-catenin/
LEF-1 pathway. Proc Natl Acad Sci 1999, 96: 5522−5527
2 5 Gottardi CJ, Gumbiner BM. Distinct molecular forms of beta-catenin
Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 2 | Page 148
26
27
28
29
30
31
32
33
34
35
36
37
38
are targeted to adhesive or transcriptional complexes. J Cell Biol
2004, 167: 339−349
Harris TJ, Peifer M. Decisions, decisions: Beta-catenin chooses
between adhesion and transcription. Trends Cell Biol 2005, 15:
234−237
Provost E, Rimm DL. Controversies at the cytoplasmic face of the
cadherin-based adhesion complex. Curr Opin Cell Biol 1999, 11:
567−572
Nagafuchi A. Molecular architecture of adherens junctions. Curr
Opin Cell Biol 2001, 13: 600−603
Aliaga JC, Deschenes C, Beaulieu JF, Calvo EL, Rivard N. Requirement of the MAP kinase cascade for cell cycle progression and
differentiation of human intestinal cells. Am J Physiol 1999, 277:
631−641
Laprise P, Langlois MJ, Boucher MJ, Jobin C, Rivard N. Downregulation of MEK/ERK signaling by E-cadherin-dependent PI3K/
Akt pathway in differentiating intestinal epithelial cells. J Cell
Physiol 2004, 199: 32−39
Hoschuetzky H, Aberle H, Kemler R. β-Catenin media tes the
interaction of the cadherin-catenin complex with epidermal growth
factor receptor. J Cell Biol 1994, 127: 1375−1380
Qian X, Karpova T, Sheppard AM, McNally J, Lowy DR. E-cadherin
mediated adhesion inhibits ligand-dependent activation of diverse
receptor tyrosine kinases. EMBO J 2004, 23: 1739−1748
Pece S, Chiariello M, Murga C, Gutkind JS. Activation of the protein
kinase Akt/PKB by the formation of E-cadherin-mediated cell-cell
junctions. J Biol Chem 1999, 274: 19347−19351
Reynolds AB, Daniel JM, McCrea PD, Wheelock MM, Wu J, Zhang
Z. Identification of a new catenin: The tyrosine kinase substrate
p120cas associates with cadherin complexes. Mol Cell Biol 1994,
14: 8333−8342
Miranda KC, Joseph SR, Yap AS, Teasdale RD, Stow JL. Contextual
binding of p120ctn to E-cadherin at the basolateral plasma membrane
in polarized epithelial. J Biol Chem 2003, 278: 43480−43488
Chen X, Kojima S, Borisy GG, Green KJ. p120 catenin associates
with k inesin and fa cilitates the tra nsport of cadherin-catenin
complexes to intercellular junctions. J Cell Biol 2003, 163: 547−557
Wheelock MJ, Johnson KR. Cadherins as modulators of cellular
phenotype. Annu Rev Cell Dev Biol 2003, 19: 207−235
Pece S, Gutkind JS. Signaling from E-cadherins to the MAPK
pathway by the recruitment and activation of epidermal growth
factor receptors upon cell-cell contact formation. J Biol Chem
2000, 275: 41227−41233