From the Departments of Surgery and ¶ Medicine,
Memorial Sloan-Kettering Cancer Center,
New York, New York 10021
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ABSTRACT |
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Alterations in the expression or function of
molecules that affect cellular adhesion and proliferation are thought
to be critical events for tumor progression. Loss of expression of the
cell adhesion molecule E-cadherin and increased expression of the
epidermal growth factor receptor are two prominent molecular events
that are associated with tumorigenesis. The regulation of
E-cadherin-dependent cell adhesion by epidermal growth
factor (EGF) was therefore examined in the human breast cancer cell
line, MDA-MB-468. In this study, changes were observed in the
subcellular distribution of components that mediate the cytoplasmic
connection between E-cadherin and the actin-based cytoskeleton in
response to activation of the EGF receptor. Serum withdrawal activated
E-cadherin-dependent cell-cell aggregation in MDA-MB-468
cells, and this treatment stimulated the interaction of actin,
-actinin, and vinculin with E-cadherin complexes, despite the
absence of
-catenin in these cells. By contrast, the
co-precipitation of actin with E-cadherin was not detected in several
-catenin positive epithelial cell lines. Treatment with EGF
inhibited cellular aggregation but did not affect either the levels of
E-cadherin or catenin expression nor the association of catenins
(
-catenin, plakoglobin/
-catenin, or p120cas) with
E-cadherin. However, EGF treatment of the MDA-MB-468 cell line
dissociated actin,
-actinin, and vinculin from the
E-cadherin-catenin complex, and this coincided with a robust
phosphorylation of
-catenin, plakoglobin/
-catenin, and
p120cas on tyrosine residues. Furthermore, inactivation of the
EGF receptor in serum-treated MDA-MB-468 cells with either a
function-blocking antibody or EGF receptor kinase inhibitors mimicked
the effects of serum starvation by stimulating both cellular
aggregation and assembly of E-cadherin complexes with vinculin and
actin. These results demonstrate that the EGF receptor directly
regulates cell-cell adhesion through modulation of the interaction of
E-cadherin with the actin cytoskeleton and thus substantiates the
coordinate role of both of these molecules in tumor progression and
metastasis.
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INTRODUCTION |
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Tumor progression is associated with changes in the expression or
activity of cell surface molecules from the growth factor receptor
family (1-3) and the cadherin family of cell adhesion molecules
(reviewed in Refs. 4 and 5). Abnormally high levels of the epidermal
growth factor receptor
(EGFR)1 and one of its
ligands, transforming growth factor , have frequently been observed
in human tumors and in tumor cell lines, and this is thought to play a
critical role in malignant progression by increasing the transduction
of mitogenic signals (6-13). Similarly, down-regulation of the
calcium-dependent cell adhesion molecule, E-cadherin, has
been observed in poorly differentiated tumors and in highly invasive
tumor cell lines (4, 5, 14-17), suggesting that cell adhesion promoted
by this molecule may be important for the maintenance of an epithelial
phenotype and for the suppression of tumor invasion.
The roles of EGFR and E-cadherin in tumor progression have been explored using specific inhibitors and cDNA transfections. Perturbation of EGFR function with specific antibodies or kinase inhibitors was shown to inhibit proliferation of both tumor cell lines in vitro and tumor xenografts in vivo (reviewed in Ref. 13). Inactivation of E-cadherin function with neutralizing antibodies resulted in increased cellular proliferation and invasiveness (14, 18). Recovery of E-cadherin function by cDNA transfection into E-cadherin-deficient tumor cell lines reversed the invasive phenotype and restored an epithelial morphology (19, 20). Other transfection studies have demonstrated that changes in the expression levels of growth factor receptor tyrosine kinases and the cell adhesion molecule E-cadherin may be coordinated. For example, transfection with the Her2/neu oncogene down-regulated E-cadherin expression in a mammary cell line, and inhibition of Her2/neu autophosphorylation reversed this effect (21). Conversely, EGFR expression was shown to be down-regulated in a cervical carcinoma cell line after transfection with E-cadherin (22). It appears therefore that increased receptor tyrosine kinase activity and loss of E-cadherin function may be related cellular events that are associated with tumor progression.
In addition to the coordinate regulation of EGFR and E-cadherin
expression levels, activation of EGFR as well as other tyrosine kinases
has been shown to directly affect the adhesive function of E-cadherin
via regulatory proteins known as catenins. Three catenins, -catenin,
plakoglobin/
-catenin, and p120cas, bind to the cytoplasmic
domain of cadherins (23-26) and, except for p120cas, associate
with the cortical actin cytoskeleton through
-catenin (27, 28).
Phosphorylation of catenins on tyrosine strongly correlates with
modulation of cell adhesion. In particular, tyrosine phosphorylation of
-catenin was shown to be associated with loss of epithelial
differentiation and increased cellular migration which was concomitant
with decreased cadherin-dependent adhesion (29-36). One
study, however, has shown that tyrosine phosphorylation of
-catenin
may not be required for modulation of cell adhesion as chimeric
cadherin/
-catenin molecules were sufficient to mediate the shift
from strong to weak adhesion in the presence of v-src kinase
(37). On the other hand, another study has shown that mutations in the
region of
-catenin that is tyrosine-phosphorylated by
erbB-2 results in suppression of the invasive phenotype
(38). Therefore, it remains to be determined whether a direct causality exists between tyrosine phosphorylation of
-catenin and the loss of
an adhesive phenotype. Tyrosine phosphorylation, however, did not
change the levels of catenins that were associated with the cadherin
cytoplasmic domain, implying that tyrosine phosphorylation may affect
the interaction of catenins with adhesion components other than
cadherins (26, 31, 34).
Several studies have implicated -catenin as being particularly
important in maintaining intercellular adhesion. Aberrant
-catenin
expression has been observed frequently in human cancer cell lines with
low adhesive activity (39-43), and a mutated form of
-catenin
lacking the
-catenin binding site has been detected in cancer cell
lines (44). These observations suggested that the adhesive activity of
cadherins is tightly regulated by
-catenin, possibly due to its role
in linking cadherins to the cytoskeleton. In a recent study, the
-catenin-deficient cell line MDA-MB-468 was found under conditions
of serum starvation to regain E-cadherin-dependent adhesion
(46). This adhesion was suggested to be mediated by vinculin which is a
protein of adheren junctions sharing sequence similarity with
-catenin (47, 48). This study implied that vinculin may connect the
cadherin cytoplasmic domain to the cortical cytoskeleton in the absence
of
-catenin by directly interacting with
-catenin.
In the present study, the relationship between tyrosine phosphorylation
of the adhesion complex, mediated by EGFR, and the connection of
E-cadherin with the actin-based cytoskeleton in the
-catenin-deficient MDA-MB-468 cell line were examined. The absence
of
-catenin in this cell line appears to facilitate the isolation of
detergent-soluble complexes of cadherins that contain components of the
cortical cytoskeleton. Furthermore, the high levels of EGFR expression
in this cell line (49) enable study of the modulation of cadherin
interactions with components of the actin cytoskeleton by activation of
EGFR. Here it is shown that, under serum-free conditions, actin,
-actinin, and vinculin co-precipitated with E-cadherin. The
association of actin with E-cadherin was inhibited by cytochalasin D
treatment, and this interaction was only detected in MDA-MB-468 cells
but not in the MCF-10A, MDA-MB-361, or A431 cell lines, which all
express
-catenin. E-cadherin-mediated cell-cell aggregation in
MDA-MB-468 cells was dramatically inhibited by EGF treatment, which
correlated with prominent tyrosine phosphorylation of catenins
(
-catenin, plakoglobin/
-catenin, and p120cas) and with
the dissociation of actin,
-actinin, and vinculin from E-cadherin
complexes. Furthermore, inactivation of EGFR in serum-treated
MDA-MB-468 cells with a function-blocking antibody or with EGFR kinase
inhibitors resulted in induction of E-cadherin-dependent adhesion and in the association of the E-cadherin complex with actin
and vinculin. These findings demonstrate an interaction between
E-cadherin and the cortical cytoskeleton which is sensitive to the
activity of the EGF receptor. Furthermore, these results establish a
molecular link between EGFR activity, cadherin-based adhesion, and the
actin-cytoskeleton.
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MATERIALS AND METHODS |
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Cell Lines-- The breast cancer cell lines MDA-MB-468, MDA-MB-361, and BT549, the normal mammary epithelial cell line MCF-10A, and the epidermoid A431 carcinoma cell line were all obtained from the American Type Culture Collection (Rockville, MD). Cells were routinely cultured in Dulbecco's modified Eagle's medium/F12 (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) at 37 °C in a humidified 5% CO2 atmosphere.
Reagents-- Epidermal growth factor was purchased from Collaborative Research (Becton Dickinson, Bedford, MA). Geldanamycin was purchased from Life Technologies, Inc. The EGFR-specific tyrosine kinase inhibitor PD 130305 was a gift from Dr. David Fry (Parke Davis). Cytochalasin D and DMSO were purchased from Sigma.
Antibodies--
Monoclonal anti-human E-cadherin was acquired
from Zymed (San Francisco). Monoclonal antibodies to -,
-,
plakoglobin/
-catenin and p120cas were purchased from
Transduction Laboratories (Lexington, KY). Ascites derived
anti-vinculin or
-actinin and rabbit polyclonal anti-actin
antibodies were purchased from Sigma. The monoclonal anti-phosphotyrosine antibody (G410) was obtained from Upstate Biotechnology. mAb 225, recognizing the extracellular domain of the
EGFR, was a gift from Dr. John Mendelsohn (MD Anderson Cancer Center),
and rabbit antiserum to EGFR was provided by Dr Joseph Schlessinger
(New York University).
Cell Treatments-- Confluent monolayers were either incubated in medium with 10% FBS (serum-treated) or incubated for 36 h in medium with 0% FBS (serum-starved). Serum-starved monolayers were then treated with or without 200 ng/ml EGF for 10 min at 37 °C prior to cell lysis. For Geldanamycin or PD130305 treatments, confluent monolayers were split at 1:10 dilution, allowed to grow for an additional 48 h to reach about 50-70% confluence, and were treated for 2 or 18 h with various concentrations of inhibitors as indicated. Control monolayers were treated with DMSO alone.
Cell Lysis-- Cell monolayers were washed twice in cold phosphate-buffered saline and extracted by homogenization in lysis buffer (1% Triton X-100, 10% glycerol, 50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EGTA, 2 mM MgCl2, 1 mM dithiothreitol) with protease inhibitors (10 µg/ml aprotinin and leupeptin, 5 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride). Phosphatase inhibitors were included in this buffer when cells were harvested for anti-phosphotyrosine detection (50 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 3 mM H2O2). Cell lysates were centrifuged at 12,000 × g for 20 min centrifugation at 4 °C, and supernatants were used immediately for immunoprecipitation.
Immunoblotting-- 30 µg of proteins from each indicated extract, as determined by the Bradford method (Bio-Rad), were boiled in SDS sample buffer for 10 min and loaded onto a 7.5% polyacrylamide minigel (Bio-Rad). Proteins were transferred onto Immobilon membranes (Millipore, Bedford, MA), blocked in 3% BSA/phosphate-buffered saline and incubated for 2 h at 25 °C with dilutions of 1:1000 of primary antibodies. After washes, membranes were probed with a 1:5000 dilution of secondary antibodies coupled to horseradish peroxidase for 1 h at 25 °C and developed with enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia Biotech).
Immunoprecipitation-- Cell lysates (5 mg) were incubated with 10 µg of primary antibodies for 2 h at 4 °C and supplemented with 3 mg of Protein A-Sepharose beads for 1 h, and the beads were subsequently washed with lysis buffer. Bound proteins were eluted by boiling the beads in sample buffer for 10 min, processed by SDS-PAGE, transferred onto membranes, and probed with 1:1000 dilutions of indicated antibodies. Blots were further processed as described above.
Cell-Cell Aggregation Assays-- Cell monolayers grown to 98% confluence were incubated for 36 h in medium containing 10% FBS (serum-treated) or 0% FBS serum (serum-starved). Cell monolayers were detached by incubating in HBSS containing 0.02% crystallized trypsin (Worthington) and 10 mM CaCl2 for 5 min at 37 °C. After trypsinization, single cell suspensions were made by trituration with a Pasteur pipette. Cell viability as assessed by trypan blue dye exclusion was greater than 95%. Cells were washed twice in HBSS (Life Technologies, Inc.) and incubated at 3 × 105 cells per well in 500 µl of HBSS containing 1% BSA and 100 µg/ml DNase (Worthington) with or without 1 mM CaCl2 and in the presence or absence of 100 µg/ml anti E-cadherin or control antibodies as indicated.
For EGF treatment, single cell suspensions prepared as described above were incubated with 200 ng/ml EGF in the aggregation wells for the times indicated. A sample of each treatment was further controlled for EGFR levels by Western blotting. For treatment with Geldanamycin (50), PD 130305 (51), or mAb 225 (52), cells were grown at subconfluent density for 48 h and were incubated with 1 µM Geldanamycin, 1 µM PD 130305, or with 20 mM mAb 225 for 18 h prior to trypsinization. Aggregation assays were performed at 37 °C at 100 rpm for the indicated times in triplicate wells, in 24-well non-tissue culture treated plates (No. 1147; Becton Dickinson, Franklin Lakes, NJ) that had been blocked with phosphate-buffered saline, 2% BSA for 30 min at 37 °C. Assays were stopped at time 0 and 30 min (unless otherwise indicated) by fixing the cells in 1% glutaraldehyde. The extent of cell-cell binding was monitored by measuring the disappearance of single cells using a Coulter counter. Standard deviations of the mean values are included. ![]() |
RESULTS |
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Serum Withdrawal Increases the Levels of E-cadherin Complexes with
the Actin Cytoskeleton--
In the absence of serum, MDA-MB-468 cells
display E-cadherin-dependent aggregation which correlated
with a 3-fold increase in E-cadherin protein levels. This increase in
E-cadherin was accompanied by the association of vinculin with the
E-cadherin·-catenin complex (46). This result prompted the
examination of additional cytoskeletal proteins that may be recruited
to the E-cadherin adhesion complex to support cell-cell adhesion in
these cells as a consequence of serum withdrawal. High levels of actin
(Fig. 1A, lane 2) and
-actinin (Fig. 1A, lane 5), in addition to vinculin (Fig.
1A, lane 8), were observed to be associated with E-cadherin immunoprecipitates. In contrast, actin,
-actinin, or vinculin were
not detected in immunoprecipitates of E-cadherin from serum-treated cells (Fig. 1A, lanes 1, 4 and 7, respectively).
To control for specificity of the antibodies, total cell lysates were
reacted with antibodies to each of these proteins (Fig. 1A, lanes
3, 6, and 9). Background immunoreactivity was
consistently observed when E-cadherin immunoprecipitates were probed
with the monoclonal antibody to
-actinin (Fig. 1A, lanes
4 and 5). When 3 times the amount of lysate was used
for immunoprecipitation from serum-treated cells to account for the
reduced amount of E-cadherin (Fig. 1B, lanes 1 and
2), similar levels of actin (Fig. 1B, lanes 3 and 4), vinculin (Fig. 1B, lanes 5 and 6),
and
-actinin (data not shown) were found in the immune complexes,
which suggests that the association of these molecules with E-cadherin
in serum-treated MDA-MB-468 cells is restricted by the low levels of
E-cadherin expression.
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Detergent-soluble Complexes of Actin with E-cadherin Are Not
Observed in -Catenin Positive Cell Lines--
Actin had not been
previously shown to co-precipitate with E-cadherin from
detergent-soluble lysates, presumably because this complex is believed
to be detergent-insoluble in most cell lines (45, 55, 56). To explore
whether complexes of E-cadherin with actin are detectable in other
epithelial cell lines, analysis for the co-precipitation of these
molecules was carried out in MCF-10A, MDA-MB-361, and A431 cells, all
of which express normal levels of
-catenin and display aggregation
properties that are not affected by serum starvation (Ref. 46 and data
not shown). In contrast to MDA-MB-468 cells, actin was not found in
immunoprecipitates of E-cadherin from lysates of the epithelial cancer
cell lines MCF-10A, MDA-MB-361, or A431 cells (Fig.
2, lanes 2, 5 and
8, respectively), even though the levels of
immunoprecipitated E-cadherin were similar to those found in
serum-starved MDA-MB-468 cells (Fig. 2, lanes 1, 4, 7 and
10, respectively). Actin was present in the
detergent-soluble fraction of these cells (Fig. 2, lanes 3, 6 and 9), indicating that the lack of actin in
E-cadherin immunoprecipitates from these cells was not due to the
insolubility of actin in these lysates. These results show that the
absence of detectable actin in E-cadherin immunoprecipitates in these
cell lines correlates with their expression of
-catenin.
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EGF Treatment Attenuates Cell-Cell Adhesion and Dissociates
Cytoskeletal Components from the E-cadherin Complex--
EGF treatment
has been shown to promote cell scattering and reduce cell-cell adhesion
by an unknown mechanism that correlated with increased tyrosine
phosphorylation of -catenin (34, 35, 36). The effect of EGFR
activation on cell-cell aggregation was therefore compared with its
effects on the association of cytoskeletal proteins with E-cadherin in
MDA-MB-468 cells. EGF caused an abrupt decline in
E-cadherin-dependent aggregation after 10 min of EGF
treatment of serum-starved cells (Fig.
3A). The effect of EGF on
cell-cell aggregation was abolished after 30 min of treatment,
consistent with decreased EGFR levels due to down-regulation of the
protein (Ref. 9, data not shown). The total levels of E-cadherin or
catenins (
-catenin, plakoglobin/
-catenin, and p120cas)
were not reduced by treatment with EGF (Fig. 3B, lanes 1-8) and therefore do not account for the reduced cellular aggregation caused by EGFR stimulation.
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Inactivation of EGFR Function Stimulates Cell-Cell Adhesion and Association of E-cadherin with the Actin Cytoskeleton in Serum-treated MDA-MB-468 Cells-- To verify whether EGFR activity was directly associated with the non-adhesive state of serum-treated MDA-MB-468 cells, cells were incubated with EGFR function-blocking reagents, and both cell-cell aggregation and the co-precipitation of actin and vinculin with E-cadherin were examined. Treatment of serum-treated MDA-MB-468 cells for 18 h with either the EGFR function-blocking antibody (mAb 225) (52) or Geldanamycin (50) down-regulated EGFR levels (Fig. 6A, lanes 1-4) and caused a 16- and 14-fold increase in E-cadherin-dependent cell adhesion, respectively (Fig. 6C). Similarly, treatment of MDA-MB-468 cells with 1 µM PD 130305, a specific EGFR tyrosine kinase inhibitor (51), which inhibited EGFR autophosphorylation at 0.1 µM (Fig. 6B, lanes 1-3), caused a 9-fold increase in E-cadherin-dependent cell-cell aggregation (Fig. 6C). In comparison, the increase in cell aggregation induced by serum starvation was of greater magnitude (32-fold) (see Fig. 3A) and may be caused by the more complete inhibition of EGFR autophosphorylation (Fig. 5, lane 1).
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DISCUSSION |
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The present study examines the mechanism of regulation of
E-cadherin-dependent cell adhesion by tyrosine
phosphorylation. The effect of EGFR activation on cell adhesion was
compared with the association of E-cadherin complexes with actin and
the actin bundling proteins, vinculin and -actinin. The MDA-MB-468
cell line was used as a model because it has been shown to express high
levels of the EGF receptor (49) and contains complexes of cadherins
with the cytoskeleton which are detergent-extractable, thus permitting
the biochemical analysis of these molecular interactions (46). A recent
study showed that high levels of vinculin were associated with the
E-cadherin/
-catenin complex in these
-catenin-deficient cells,
and vinculin was shown to bind
-catenin, suggesting that it could
mediate the interaction of E-cadherin with the actin-based cytoskeleton
(46). In the present study, the loss of cell adhesion caused by EGF
treatment was associated with the decoupling of actin,
-actinin, and
vinculin from E-cadherin complexes. The withdrawal of these
cytoskeletal components from the E-cadherin adhesion complex was
accompanied by a dramatic phosphorylation of
-catenin,
plakoglobin/
-catenin, and p120cas on tyrosine residues.
These results suggest a model whereby tyrosine phosphorylation of
-catenin or plakoglobin/
-catenin causes vinculin to dissociate
from the E-cadherin-catenin complex, thus causing the loss of cell-cell
adhesion in these cells.
Previous studies have indirectly linked tyrosine phosphorylation of
-catenin to reduced cell-cell adhesion and increased cell migration
(29-31, 34, 36), but no mechanism was elaborated in these studies. In
the present study, while tyrosine phosphorylation did not affect the
association of catenins with E-cadherin, it was associated with the
loss of actin,
-actinin, and vinculin from E-cadherin
immunoprecipitates. In accord with some studies (37), vinculin tyrosine
phosphorylation was undetected in these cells, although it has been
found at low levels in src transformed cells (57, 58). The
results from the present study therefore support a model whereby
tyrosine phosphorylation modulates the interaction of the
E-cadherin-catenin complex with the actin cytoskeleton. In this model,
vinculin dissociation from phosphorylated
-catenin or
plakoglobin/
-catenin also removes
-actinin and actin from the
adhesion complex; these two molecules have both been shown to be
associated with vinculin (59, 60). However, EGF appears to affect more
dramatically the association of E-cadherin with actin and
-actinin
than with vinculin, which suggests that other molecules may be involved
in the linkage of the E-cadherin complex with the actin
cytoskeleton.
The high detergent extractability of cadherin complexes with the actin
cytoskeleton appears to correlate with the absence of -catenin
expression in MDA-MB-468 cells. Cadherin complexes with actin were not
detectable in
-catenin positive cell lines, and except for studies
showing binding of E-cadherin-catenin complexes with the actin-binding
protein DNase I (61) or the co-sedimentation of filamentous actin
(F-actin) with
-catenin in vitro (28), no previous
evidence exists for the interaction of E-cadherin with F-actin in
epithelial cells in vivo. The adhesion proteins, E-cadherin,
-catenin, and plakoglobin/
-catenin in MDA-MB-468 cells, were
predominantly found in the Triton-soluble pool, and a comparable amount
of these proteins was observed in both soluble and insoluble-Triton
fractions of
-catenin positive cell lines (data not shown). These
results are consistent with the notion that coupling of the
E-cadherin-catenin complex to the actin cytoskeleton via
-catenin
renders these complexes insoluble in Triton X-100, which results in
their removal from the soluble pool (45, 55, 56). In contrast, both
actin and
-actinin have been found in Triton-soluble
immunoprecipitates of N-cadherin, in chicken retinal cells, and in
fibroblasts (62, 63). The greater detergent solubility of complexes of
actin with N-cadherin may be due to a more labile association of
N-cadherin with the cytoskeleton. These results are consistent with the
proposed dynamic nature of N-cadherin-mediated adhesion (64, 65).
The conditions used in this study appear to permit the extraction of
the filamentous form of actin in the Triton-soluble phase since the
association of actin with E-cadherin in the Triton-soluble fraction was
inhibited by the actin depolymerizing agent, cytochalasin D. Triton
X-100 extraction is known to preserve the integrity of the actin
cytoskeleton (53, 54), and thus the interaction of E-cadherin with the
actin cytoskeleton has been observed in Triton X-100-insoluble
fractions (45). However, F-actin is generally assumed to be associated
with the Triton-insoluble pool (53, 54), and thus its association with
E-cadherin complexes may not be detected in soluble Triton X-100
extracts. In the present study, shorter fragments of F-actin that may
be either arrested in an earlier stage of polymerization or that result
from mechanical shearing during cellular extraction (53, 54) may allow
the detection of E-cadherin/actin interactions in the soluble phase. It
remains also an intriguing possibility that vinculin may couple the
E-cadherin complex to a pool of F-actin that is distinguishable in its
Triton solubility from that which is coupled through -catenin.
A model emerges from the collective data in which -catenin and
vinculin may differentially support cell adhesion. The increased solubility of cadherin-vinculin-actin complexes implies that vinculin may be promoting a more dynamic interaction of cadherins with the
cytoskeleton than does
-catenin. The reversible effects of serum
starvation and EGF on both cell-cell aggregation and the association of
E-cadherin with the cytoskeleton in MDA-MB-468 cells suggest that the
adhesion promoted by vinculin may also be more sensitive to changes in
growth factors levels. In support of this hypothesis, in the
-catenin positive A431 or BT549 cell lines, which also express both
EGFR and vinculin (32, 46), EGF had no effect on both cell-cell
adhesion or on the association of
-catenin with cadherin complexes,
even though it induced
-catenin tyrosine
phosphorylation.2 These
results raise the possibility that vinculin may bind
-catenin with a
lower affinity than does
-catenin or to a site on
-catenin distinct from the
-catenin binding site that is sensitive to tyrosine phosphorylation.
Other possible mechanisms that modulate the connection of E-cadherin
with the cytoskeleton may be downstream of EGFR tyrosine phosphorylation. One such mechanism may be the activation of the Rho
family of small GTP-binding proteins that is dependent on tyrosine
kinase activity (66) and results in rapid reorganization of the actin
cytoskeleton (reviewed in Refs. 67 and 68) and decreased cadherin
activity (69). In addition, vinculin function may be regulated by
depletion of phosphatidylinositol pools in response to EGF
(for review, see Ref. 70). Phosphatidylinositol has been shown to
regulate vinculin activity by inducing conformational changes that
unfold the tail from the head domain of vinculin (59, 60), thus
exposing both the -actinin and actin binding sites (71, 72). Thus,
reduced levels of phosphatidylinositol as a result of breakdown
mediated by EGF stimulation may convert vinculin from an open/active to
a close/inactive conformation that is unable to bind actin,
-actinin, and possibly
-catenin. Another likely possibility is
that EGF-induced tyrosine phosphorylation of paxillin, which may
increase its affinity for vinculin (73), could result in recruitment of
vinculin from adherens junctions to focal contacts, thus resulting in
weaker cell-cell adhesion.
The present study has shown that EGFR directly modulates E-cadherin-dependent adhesion through the connection of E-cadherin to the cytoskeleton and that vinculin may play a significant role in this modulation. Elucidation of the precise roles of the various intracellular proteins associated with cadherins will provide a further understanding of cadherin-based cellular adhesion.
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ACKNOWLEDGEMENTS |
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We thank Dr. Stuart Aaronson for support; Lan Kang for technical support; Drs. Zhen Fan and John Mendelsohn for providing the antibody mAb 225; and Drs. Neal Rosen and David Rimm for stimulating discussions.
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FOOTNOTES |
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* This work was supported by the Department of Surgery at the Memorial Sloan-Kettering Cancer Center and SPORE in breast cancer grant 1P50CA68425 (to L. N.) from the National Cancer Institute.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 correspondence should be addressed: The Derald H. Ruttenberg Cancer Center, Mount Sinai Medical Center, One Gustave Levy Place, Box 1130, New York, NY 10029. Tel.: 212-824-8168; Fax: 212-987-2240; E-mail: rhazan{at}smtplink.mssm.edu.
1 The abbreviations used are: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; FBS, fetal bovine serum; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; HBSS, Hanks' balanced saline solution; mAb, monoclonal antibody; DMSO, dimethylsulfoxide.
2 R. B Hazan, unpublished results.
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REFERENCES |
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