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INTRODUCTION |
The cadherins represent a family of transmembrane receptors that
mediate homophilic, Ca2+-dependent cell-cell
adhesion. In epithelial cells, the members of this family, such as the
classical E-, N-, and P-cadherins, are primarily found at the adherens
junctions of adjacent cells (1).
-Catenin as well as plakoglobin
(
-catenin) associate directly with the highly conserved cytoplasmic
domain of classical cadherins in a mutually exclusive manner (2, 3).
The cadherin-catenin complex is linked via
-catenin either directly
(4) or indirectly to the actin filament network via the actin-binding
proteins
-actinin or vinculin (5, 6). The association of the
cadherin-catenin complex with the cytoskeleton is essential for tight
cell-cell interaction.
Nevertheless, cadherin/catenin-mediated cell-cell contacts have to be
highly dynamic because, particularly during embryonic development or
wound healing, adherens junctions have to be rapidly disassembled and
reassembled (7). Down-regulation of cadherins results in the separation
of neighboring cells, a phenomenon that is observed during embryonic
development at the epithelial-mesenchymal transition
(EMT)1 of forming mesoderm
(8) as well as in tumor cells, allowing their invasion and
dissemination throughout the body (9). During epithelial-mesenchymal
transition, cells transiently lose their epithelial features and
acquire a fibroblastoid morphology (10). The critical importance of an
intact cadherin-catenin complex is underscored by the observation that
down-regulation of any of its components resulting in the loss of the
tumor-suppressive actions of adherens junctions correlates with tumor
invasion and metastasis (11). Moreover, the integrity of adherens
junctions appears to be dynamically regulated by tyrosine
phosphorylation. Transfection of a v-src oncogene (12, 13) or treatment
with growth factors (14, 15) causes unstable cell-cell adhesion and
migration of cells, and inhibition of PTPs enhances this destabilizing effect (16). The model in which reversible tyrosine phosphorylation serves to regulate cadherin-mediated cell-cell adhesion is further supported by the demonstration of cadherin-catenin complex association with the receptor tyrosine kinases EGF receptor and human EGF receptor
2/Neu (17, 18) as well as with the transmembrane PTPs µ,
, and
(19-21).
-Catenin and plakoglobin are mammalian homologues of the
Drosophila protein Armadillo, whose function is critical for
normal segmental pattern formation during development (22). The
presence of a repeating 42-amino acid sequence motif defines members of the "armadillo family" (23). Data obtained in the
Drosophila and Xenopus systems suggest an
additional function for
-catenin independent of cadherin-mediated
cell adhesion. This involves translocation of
-catenin to the
nucleus that is preceded by its accumulation in the cytoplasm. Thus,
free
-catenin is involved in transcriptional regulation of specific
genes that are essential for embryonic development (24). The signals
resulting in a free pool of
-catenin include the binding of
Wingless/Wnt to its transmembrane receptor Frizzled and the inhibition
of the serine/threonine kinase Zeste-White 3 (Shaggy) or its vertebrate
homologue glycogen synthase kinase 3 (25). Further functions for
-catenin and plakoglobin are indicated by their association with the
adenomatous polyposis coli (APC) tumor suppressor protein, which is
thought to serve as a cytoplasmic effector of
-catenin, negatively
regulating the accumulation of cytosolic
-catenin in concert with
glycogen synthase kinase 3 (26) and axin/conductin (27-29) by inducing ubiquitin-dependent degradation of
-catenin
(30).
In this report we show that during epithelial migration,
-catenin
accumulates in the cytosol in a free, uncomplexed and
tyrosine-phosphorylated form. However, in confluent cells,
-catenin
maintains epithelial cell integrity as an essential part of the
cadherin-catenin tumor suppressor system. Our findings suggest that
tyrosine phosphorylation regulates the function of
-catenin as a
signaling molecule during epithelial cell migration. We further
demonstrate PTP LAR to be a modulator of epithelial cell migration,
which strongly supports a function of this protein tyrosine phosphatase
in the regulation of cell-cell contacts and epithelial cell integrity.
Moreover, we show that ectopic expression of PTP LAR inhibits tumor
formation in nude mice. A dysfunction of PTP LAR may therefore lead to
tumor invasion and metastasis.
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MATERIALS AND METHODS |
Cloning of
-Catenin,
-Catenin, and Plakoglobin
(
-Catenin)--
Human
-catenin (accession number D14705),
-catenin (Z19054), and Plakoglobin (M23410) were amplified from
cDNA generated from MCF7 cells by PCR. PCR products were cloned in a eukaryotic expression vector under the control of the cytomegalovirus promoter and confirmed by sequence analysis.
Cell Lines and Cell Culture--
All cell lines were obtained
from the American Tissue Culture Collection. NBT II cells (CRL-1655)
were grown in minimal essential medium supplemented with 1%
nonessential amino acids, Earle's balanced salt, 2 mM
glutamine, 1 mM sodium pyruvate, and 10% fetal calf serum
(Sigma). MCF7 cells (HTB22) were grown in RPMI medium supplemented with
2 mM glutamine and 10% fetal calf serum, human embryonic
293 kidney cells (CRL 1573) in Dulbecco's modified Eagle's medium
supplemented with 1 mg/ml glucose, 2 mM glutamine, and 10%
fetal calf serum. Fetal calf serum was routinely heat-inactivated. All
media and supplements were obtained from Life Technologies, Inc.
Migration Assays--
For in vitro wound assays, NBT
II cells were plated at a density of 7 × 104
cells/cm2. After 24 h, the confluent monolayer was
scratched with a pipette tip to create a cell-free area, growth factors
were added, and wound closure was documented by photography. For
scatter assays, cells were plated at a cell density of 1 × 104 cells/cm2. Growth factors were added after
24 h, and the morphology of cells and the dispersion of small
colonies were documented by photography. Quantification of migration
was performed by counting single cells with fibroblastoid migration
morphology compared with cells in groups with epithelial morphology of
different randomly chosen microscopic fields. 1000 cells/dish were
counted. All migration assays were performed in triplicates. Tyrosine
kinases (TK), mitogen-activated protein kinase, or phosphatidylinositol
3-kinase were inhibited by genistein (250 µM; Sigma),
PD98059 (50 µM; Biolabs), or LY295002 (20 µM; Biomol), respectively, added 1 h before the
addition of the growth factors. As a control, the solvent
Me2SO was used. DNA synthesis was inhibited by the specific
inhibitor of DNA polymerase
aphidicolin (1, 5 µM;
Serva) 1 h before adding the growth factors.
Tumor Formation in Nude Mice--
NBT II cells were resuspended
at a cell density of 2 × 106/50 µl in minimal
essential medium and injected subcutaneously into the flank region of
Swiss nude mice (IGR Villejuif, Paris, France). Tumor formation was
monitored by measuring the width (W) and length (L) of the tumors with W < L.
The tumor volume was calculated according to the formula
(W2 × L ×
/6). Assays were
performed at least in triplicates.
Generation of Recombinant Retroviruses and Retrovirus-mediated
Gene Transfer--
Full-length PTP LAR (H. Saito, Harvard Medical
School, Boston, MA) was subcloned into pLXSN vector (31). Stable NBT II
cell lines were generated by retroviral gene transfer as described (32). Polyclonal and clonal cell lines were selected in medium containing 0.5 mg/ml G418 (Life Technologies, Inc.). Ectopic expression was confirmed by immunoprecipitation and Western blot analysis.
Transient Expression, Cell Lysis, and
Immunoprecipitation--
Transient transfection of human 293 embryonic
kidney cells was performed as described (33). For biochemical analysis,
NBT II cells were plated at a density of 1 × 104
cells/cm2 at the day before lysis. When indicated, cells
were pretreated before lysis with sodium orthovanadate (1 mM) for the indicated period of time. After washing with
ice-cold phosphate-buffered saline (PBS), cells were lysed in ice-cold
lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl,
1 mM EDTA, 10% glycerol, 20 mM pyrophosphate, 1% Triton X-100, 100 mM NaFl, 2 mM
phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 20 µg/ml
leupeptin, 0.7 µg/ml pepstatin, 0.2 mM ammonium
molybdate, 2 mM sodium orthovanadate) and precleared by
centrifugation at 12,500 × g for 10 min at 4 °C.
The protein concentrations of the supernatants were adjusted to be
equal. HNTG buffer (20 mM HEPES, pH 7.5, 150 mM
NaCl, 10 mM pyrophosphate, 10 mM NaFl, 0.2 mM ammonium molybdate, 10% glycerol, 0.1% Triton X-100, 2 mM sodium orthovanadate) was added in a 1:1 ratio, and immunoprecipitations were performed for 2 h at 4 °C. Protein A- or protein G-Sepharose was added for an additional 2 h.
Precipitates were washed three times with HNTG buffer, and beads were
resuspended in SDS sample buffer. For subsequent Western blot analysis,
proteins separated by SDS-PAGE were transferred to nitrocellulose
(Schleicher and Schuell) and incubated with the respective antibody.
Proteins were visualized by the ECL system (Amersham Pharmacia
Biotech). Before reprobing, blots were stripped by incubation for
1 h in 68 mM Tris-HCl, pH 6.8, 2% SDS, and 0.1%
-mercaptoethanol at 50 °C.
In Vitro Binding Assays--
The plasmid coding for the GST-hPTP
LARi fusion protein was constructed by PCR amplification of
the cDNA sequence between amino acids 1259 and 1881 of human PTP
LAR and cloned into the appropriate pGEX vector (Amersham Pharmacia
Biotech). In vitro mutagenesis (34) of the original vector
pSP65-LAR yielded PTP LAR with either inactivated PTP domain 1 PTP LAR
D1 C1522S or inactivated PTP domain 2 PTP LAR
D2 C1813S, which were also amplified by PCR between amino
acids 1259 and 1881 of human PTP LAR and subcloned into pGEX vector.
The integrity of the subcloned PCR products was confirmed by sequence
analysis. GST fusion proteins were expressed in Escherichia
coli and purified as described (35). 3 µg of GST-hPTP
LARi fusion protein or a 3-fold molar excess of GST were
incubated at 4 °C with equal amounts of cell lysates, immobilized on
glutathione-Sepharose (Sigma), and washed three times with HNTG. Bound
proteins were separated by SDS-PAGE for Western blotting.
Affinity Precipitation--
The plasmid coding for the
GST-E-cadherin cytoplasmic fusion protein was constructed by
amplification of the cDNA sequence between amino acids 734 and 884 of murine E-cadherin (36) by PCR and cloned into the appropriate pGEX
vector. For affinity precipitation, equal concentrations of precleared
NBT II cell lysates were incubated with 5 µg of purified
GST-E-cadherin cytoplasmic fusion protein or a 3-fold molar excess of
GST and immobilized on glutathione-Sepharose. The resulting complexes
were washed three times with HNTG, and bound proteins were separated by
SDS-PAGE for Western blotting.
Antisera--
Monoclonal antibody against phosphotyrosine (4G10)
was obtained from UBI, and
-catenin,
-catenin, plakoglobin, and
E-cadherin antibodies were obtained from Transduction Laboratories. A
second antibody against E-cadherin was raised against a GST fusion
protein containing amino acids 834-913 of human E-cadherin. Monoclonal antibody 11.1A (M. Streuli, Dana Farber Cancer Institute, Boston, MA)
recognizes the extracellular domain of human PTP LAR (37). Rabbit
antiserum 320 (Y. Schlessinger, New York University Medical Center, New
York) is directed against a peptide corresponding to the C terminus of
PTP LAR (amino acids 1868-1881).
In Vitro Dephosphorylation Assay--
-Catenin was
transiently overexpressed in human 293 embryonic kidney cells. Cells
were serum-starved, treated with pervanadate (0.3 µM
H2O2, 0.1 mM sodium orthovanadate)
for 10 min, and lysed. Immunoprecipitations were performed as described
above, except that HNTG without pyrophosphate, ammonium-molybdate, and
sodium orthovanadate was used. PTP activity toward
tyrosine-phosphorylated
-catenin was assayed in 200-µl reactions
at 25 °C containing 25 mM HEPES, pH 7.5, 5 mM EDTA, 10 mM dithiothreitol, and 1 mg/ml bovine serum albumin with 200 ng of GST-hPTP LAR cytoplasmic, GST-hPTP
LAR D1 C1522S, or GST-hPTP LAR D2 C1813S added.
Reactions were stopped by washing with HNTG and subsequently separated
by SDS-PAGE. The tyrosine phosphorylation status was analyzed by Western blotting using anti-phosphotyrosine antibody.
Immunofluorescence--
NBT II cells were plated at a density of
1 × 104 cells/cm2 or 7 × 104 cells/cm2. 24 h later, cells were
fixed with 2% paraformaldehyde in PBS (pH 7.4, 125 mM
sucrose). Autofluorescence was quenched with PBS glycine (100 mM glycine, 0.1% borohydrate in PBS), and the cells were
permeabilized with 0.5% saponin in PBS (5 min). Nonspecific antibody
binding was blocked for 1 h with phosphate-buffered gelatin (PBS,
0.5% bovine serum albumin, 0.045% cold water fish gelatin, 5% donkey
serum). Primary antibody incubation was performed at room temperature
for 2 h after dilution in phosphate-buffered gelatin. After three
washes in phosphate-buffered gelatin, primary antibody binding was
detected with isotype specific secondary antibody, either fluorescein
isothiocyanate(DTAF)- or Cy3-conjugated (The Jackson Laboratories). For
double-labeling experiments, antibody decoration was performed
consecutively. Coverslips were mounted under Permafluor mounting medium
(Immunotech, France) and viewed either with a conventional fluorescence
microscope (Leica, FRG) or with a CLSM laser confocal microscope
(Leica, FRG). Controls were recorded at identical settings.
 |
RESULTS |
Relocalization of the Cadherin-Catenin Complex upon Induction of
Epithelial Cell Migration--
Migration of epithelial cells in tissue
culture represents in many aspects a model system of the
epithelial-mesenchymal transition (38). We chose for our studies the
rat bladder carcinoma cell line NBT II (39) because certain growth
factors and components of the extracellular matrix induce migration of
these cells (40-42). Results of an in vitro wound assay are
shown in Fig. 1A for EGF and
the commercial serum substitute Ultroser G. To rule out proliferation as the cause of wound closure, we used aphidicolin to inhibit DNA
synthesis, which did not interfere with migration (data not shown). In
contrast, inhibition of phosphatidylinositol 3-kinase by LY295002, TK
by genistein, or mitogen-activated protein kinases by PD98059
completely abolished cell migration (data not shown). The protein
levels of the components of the cadherin-catenin complex remained
unchanged during migration (data not shown), and even overexpression of
E-cadherin could not prevent disruption of intercellular contacts (43).
However, the localization of members of the cadherin-catenin complex
during EMT was altered. E-cadherin (green fluorescence) and
-catenin (red fluorescence) were located along the entire cell-cell contact region of adjacent, subconfluently plated cells (Fig.
1B, upper panel). Fluorescence on contact-free
membrane portions was only weak or absent. Some weak
-catenin
staining was detected in the nucleus of control cells, most probably
because of the fact that these subconfluent cells did not establish
their adherens junctions completely. However, upon induction of
migration by EGF, E-cadherin and
-catenin redistributed over the
entire cell surface and into the cytoplasm. Interestingly, increased nuclear localization of
-catenin was also detectable during
epithelial cell migration.

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Fig. 1.
Migration of epithelial NBT II cells.
A, in vitro wound assay. 24 h after plating,
the confluent cell monolayer was scratched as described in "Material
and Methods." EGF (100 ng/ml) or Ultroser G (2%)
were added. Wound closure was documented by photography. The
calibration bar indicates 80 µm. B,
immunofluorescence studies. 24 h after plating, EGF (100 ng/ml)
was added to the subconfluent cells. 6 h later the cells were
fixed for subsequent immunolabeling, and images were recorded by
conventional fluorescence microscopy. The upper panel shows
untreated controls, whereas the lower panel presents cells
induced to migration by EGF. The green fluorescence
indicates the presence of E-cadherin, whereas the red
fluorescence represents -catenin. Controls incubated without
primary antibody remained negative for a fluorescence signal (data not
shown). The calibration bar indicates 20 µm.
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Tyrosine Phosphorylation of the Cadherin-Catenin
Complex--
Because tyrosine phosphorylation seems to be involved in
the regulation of adherens junctions (12, 13), we investigated the
specificity of tyrosine kinases on the cadherin-catenin complex. Transient cotransfection of TKs with members of the cadherin-catenin complex demonstrated that only TKs that play a role in epithelial cell
migration, such as EGF receptor, c-Src, or fibroblast growth factor
receptor 2, are capable of phosphorylating
-catenin and plakoglobin.
However,
-catenin and E-cadherin were not substrates of these TKs.
These observations could be confirmed in nontransfected NBT II cells
stimulated to migrate. After cell lysis, all members of the
cadherin-catenin complex were immunoprecipitated with specific antibodies. This technique allows the precipitation of the members of
the cadherin-catenin complex regardless of their localization and
binding partners and, therefore, also allows precipition of E-cadherin-bound
-catenin. Tyrosine phosphorylation levels were analyzed by Western blotting with an anti-phosphotyrosine antibody (Fig. 2, top) followed by the
detection of E-cadherin (Fig. 2, middle) or the catenins
(Fig. 2, bottom) by specific antibodies. Tyrosine
phosphorylation of
-catenin and plakoglobin, but not of
-catenin
and E-cadherin, was detectable already 30 min after induction of
migration. The phosphorylation was transient, lasted for about 9 h, and was no longer detectable after 24 h (data not shown). For
immunoprecipitation, pretreatment of the cells with 1 mM
sodium orthovanadate, a specific inhibitor of PTPs, was necessary to
detect tyrosine phosphorylation. This indicated that the
phosphorylation state of
-catenin and plakoglobin was tightly
regulated by TKs and PTPs. The correlation of epithelial cell colony
dispersion with tyrosine phosphorylation of
-catenin and plakoglobin
could also be demonstrated in HT29 and HaCaT cells (data not shown), suggesting a common regulatory mechanism during the induction of cell
migration.

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Fig. 2.
Tyrosine phosphorylation of
-catenin and plakoglobin during epithelial cell
migration. NBT II cells were plated at a density of 1 × 104 cells/cm2. 24 h later, growth factors
were added to induce migration (EGF, 100 ng/ml; acidic fibroblast
growth factor (aFGF), 30 ng/ml including 50 µg/ml heparin;
Ultroser G: 2%). 90 min before lysis sodium orthovanadate (1 mM) was added (right) or not (left). As a positive control,
cells were treated for 10 min with pervanadate. After lysis the
cadherin/catenin complex was immunoprecipitated and precipitates were
separated by SDS-PAGE. Tyrosine phosphorylation levels were analyzed by
Western blotting with an anti-phosphotyrosine antibody
(top). The membranes were reprobed with specific antibodies
(Ab) to E-cadherin (middle) or the catenins
(bottom) to assure equal amounts of precipitated proteins.
Reproducibly it was observed that highly tyrosine-phosphorylated
-catenin (as here after pervanadate treatment) was not efficiently
immunodecorated in the reblot. Arrows indicate the proteins
of interest, and molecular mass standards are shown in kilodaltons on
the left. IP, immunoprecipitation.
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|
The Increased Free Pool of
-Catenin during Epithelial Cell
Migration--
We next examined the consequences of the
-catenin
phosphorylation. To this end,
-catenin from EGF-treated cells was
affinity-precipitated with a GST fusion protein comprising the entire
cytoplasmic domain of E-cadherin, and the levels of free, uncomplexed
-catenin (Fig. 3, top) as
well as its phosphotyrosine content (Fig. 3, bottom) were
analyzed by immunoblotting. As previously demonstrated, this strategy
allows, in contrast to immunoprecipitation, specifically and
selectively the precipitation of the free, non-E-cadherin-bound pool of
-catenin (44). The induction of migration by EGF correlated with an
increase of the free pool of
-catenin, which had a significantly higher phosphotyrosine content than
-catenin obtained by
immunoprecipitation (compare Fig. 2 with Fig. 3). The free pool of
-catenin in nonstimulated control cells was unaffected. Although we
could detect increases in free, tyrosine-phosphorylated
-catenin
even without adding sodium orthovanadate (see Fig. 8), we added it in
this set of experiments to improve detection. These findings indicate
that phosphorylation by TKs leads to an increase of the free,
uncomplexed pool of
-catenin during epithelial cell migration.

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Fig. 3.
During epithelial cell migration, the free,
uncomplexed pool of -catenin is increased and
correlates with enhanced tyrosine phosphorylation. NBT II cells
were plated at a density of 1 × 104
cells/cm2. 24 h later, EGF (100 ng/ml) was added for
the indicated time intervals. 30 min before lysis, sodium orthovanadate
(1 mM) was added. Subsequently, affinity precipitations
were performed with 5 µg of a GST/E-cadherin cytoplasmic protein or
GST in a 3-fold molar excess. Proteins were separated by SDS-PAGE, and
the levels of free, uncomplexed -catenin were analyzed by Western
blotting with an anti- -catenin-antibody (top). Western
blotting with an anti-phosphotyrosine antibody shows the increase in
the level of tyrosine phosphorylation in free, uncomplexed -catenin
(bottom). Note that in comparison to Fig. 2, cells were
pretreated only for 30 min with sodium orthovanadate. Arrows
indicate the proteins of interest, and molecular mass standards are
shown in kilodaltons on the left.
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Colocalization of the Cadherin-Catenin Complex with PTP
LAR--
Pretreatment with sodium orthovanadate led to an increased
tyrosine phosphorylation of
-catenin and plakoglobin in migrating NBT II cells. PTPs may therefore act as steady state equilibrium antagonists of TKs for regulatory events at adherens junctions. Because
the transmembrane PTPs µ and
were shown to associate with the
cadherin-catenin complex (19, 20), we extended the search for other
PTPs at adherens junctions. As shown in Fig. 4, PTP LAR (red fluorescence)
colocalized with the cadherin-catenin complex (green
fluorescence) as indicated by the yellow fluorescence signal at adherens junctions of epithelial cells. PTP LAR was shown to
be localized at focal adhesions of epithelial MCF7 cells (45). In NBT
II cells, we detected PTP LAR predominantly at adherens junctions but
also at focal adhesions; however, with a lower signal intensity.
Because motile cells have to rapidly disassemble and reassemble
adherens junctions and focal adhesions, the localization of PTP LAR
supports its potential regulatory function during epithelial cell
migration.

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Fig. 4.
Members of the cadherin-catenin complex
colocalize with PTP LAR in NBT II cells. 24 h after plating,
confluent monolayers of NBT II cells were fixed and processed for
indirect immunofluorescence. Cells were labeled with antibodies against
either -catenin, plakoglobin, or E-cadherin (green
fluorescence, upper panel) and simultaneously with an
antibody against PTP LAR (red fluorescence, middle
panel) as indicated. Laser confocal fluorescence images show the
colocalization (yellow superimposition, lower
panel) of PTP LAR with the cadherin-catenin complex at
adherens junctions. Controls without primary antibodies remained
negative for a fluorescence signal. The calibration bar
indicates 20 µm.
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Association of
-Catenin and Plakoglobin with PTP LAR--
To
investigate the potential association of PTP LAR with the
cadherin-catenin complex in intact cells, subconfluent human MCF7 cells
were stimulated with EGF, lysed, and immunoprecipitated with antibodies
either against PTP LAR or the cadherin-catenin complex. Western
blotting analysis with specific antibodies to
-catenin and
E-cadherin (Fig. 5A) or plakoglobin and
E-cadherin (Fig. 5B) detected these proteins in anti-human PTP LAR
immunoprecipitates with the monoclonal antibody 11.1A against the
extracellular domain of human PTP LAR and vice versa.
Interestingly, the association was constitutive and independent of EGF
stimulation. No specific signal of members of the cadherin-catenin
complex was obtained with the rabbit polyclonal antiserum 320 to PTP
LAR, suggesting that the antigenic epitope was inaccessible in the
complex. To investigate which component of the cadherin-catenin complex
mediated the interaction with PTP LAR, we used a GST fusion protein
comprising the entire cytoplasmic domain of PTP LAR (GST-PTP
LARi) to affinity purify the individual components of the
cadherin-catenin complex. The members of the cadherin-catenin complex
were individually and transiently overexpressed in human 293 embryonic
kidney fibroblasts that had been treated with the tyrosine phosphatase
inhibitor pervanadate before lysis.
-Catenin (Fig. 5C,
left) and plakoglobin (Fig. 5C, right)
were found to associate specifically with the cytoplasmic domain of PTP
LAR under these conditions. E-cadherin and
-catenin did not interact
with PTP LAR directly under the same experimental conditions (data not
shown). The phosphorylation state of
-catenin or plakoglobin, which
were both tyrosine-phosphorylated after treatment with pervanadate did
not affect their association with the GST-PTP LAR cytoplasmic fusion
protein. This interaction required the complete cytoplasmic domain of
PTP LAR, because deletion mutants of PTP LAR did not bind
-catenin
or plakoglobin efficiently (data not shown).

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Fig. 5.
Members of the cadherin-catenin complex
associate with PTP LAR. A and B, association
of the cadherin-catenin complex with PTP LAR in intact cells. Human
epithelial MCF7 cells were plated at 1 × 104
cells/cm2 and stimulated 24 h later with EGF (100 ng/ml) as indicated. Cells were lysed, and immunoprecipitations with
antibodies against PTP LAR (monoclonal antibody (mAb) 11.1A,
rabbit polyclonal antiserum #320 (polycl. Ab))
or members of the cadherin-catenin complex were performed as indicated.
NIS indicates the nonimmune-serum control. Proteins were
separated by SDS-PAGE and analyzed by Western blotting with antibodies
against E-cadherin (A, top), -catenin
(A, middle), plakoglobin (B,
top), or PTP LAR (A and B,
bottom; A, long exposures were shown on the very
bottom). Arrows indicate the proteins of interest, and
molecular mass standards are shown in kilodaltons on the left.
C, in vitro association of -catenin and
plakoglobin with PTP LAR. -Catenin and plakoglobin were transiently
overexpressed in human embryonic kidney 293 fibroblasts. After 24 h of serum starvation, the cells were stimulated with pervanadate for
10 min before lysis. Equal amounts of lysates were incubated with the
GST-PTP LAR cytoplasmic fusion protein (GST-hPTP LARi) or a
3-fold molar excess of GST. Lysates of control vector-transfected 293 cells were incubated with the GST-PTP LAR cytoplasmic fusion protein
(GST-hPTP LARi). Complexes were
immobilized on glutathione-Sepharose, and precipitates were separated
by SDS-PAGE. Bound proteins were analyzed by Western blotting with an
anti- -catenin antibody (left) or an antibody against
plakoglobin (right). Arrows indicate the proteins
of interest, and molecular mass standards are shown in kilodaltons on
the left.
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|
-Catenin Is a Substrate of PTP LAR in Vitro--
The
association of
-catenin and plakoglobin with PTP LAR prompted us to
investigate whether these proteins could represent actual substrates
for PTP LAR. GST fusion proteins of PTP LAR comprising either the
entire cytoplasmic domain (Fig. 6, GST-hPTP LAR cytopl.) or
mutants of PTP LAR with catalytically inactivated PTP domain 1 (GST-hPTP LAR PTP D1 C1522S) or inactivated PTP
domain 2 (GST-hPTP LAR PTP D2 C1813S) were
incubated with tyrosine-phosphorylated
-catenin from transiently
overexpressing, pervanadate-treated human 293 cells. After different
time intervals, the reactions were terminated and analyzed with an
anti-phosphotyrosine-specific antibody. A significant reduction in the
tyrosine phosphorylation signal within the first 5 min after incubation
of
-catenin with GST-hPTP LAR cytopl. or GST-hPTP
LAR PTP D2 C1813S (Fig.
6, top, left, and
right) was revealed. No change in phosphorylation levels was
observed after incubation with GST-hPTP LAR PTP D1
C1522S (Fig. 6, top and middle), confirming
results of enzymatic activity measurements of the GST fusion proteins
using p-nitrophenyl phosphate as a substrate (data not
shown). These data are consistent with previous findings that the first
PTP domain of PTP LAR is essential for catalytic activity, whereas the
second is catalytically inactive (46). Blots were reprobed with an
anti-
-catenin-specific antibody to confirm that equal amounts of
protein were used in the assay (Fig. 6, bottom). We
therefore conclude that
-catenin is a substrate for PTP LAR.

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Fig. 6.
-Catenin is a substrate of PTP
LAR in vitro. -Catenin was transiently
overexpressed in human embryonic kidney 293 fibroblasts. After 24 h of serum starvation, the cells were stimulated with pervanadate
before lysis. -Catenin was subsequently immunoprecipitated with an
anti- -catenin antibody (IP-Ab) , and precipitates were
incubated for the indicated time intervals with GST-hPTP LAR
cytoplasmic (cytopl.) fusion protein (left),
GST-hPTP LAR PTP D1 C1522S mutant
(middle), or GST-hPTP LAR PTP D2 C1813S-mutant
(right). Reactions were terminated after the indicated
periods of time, and proteins were separated by SDS-PAGE. Tyrosine
phosphorylation levels of -catenin were analyzed by Western blotting
with an anti-phosphotyrosine antibody (top). Western
blotting with an anti- -catenin antibody revealed that equal amounts
of -catenin were immunoprecipitated (bottom).
Arrows indicate the proteins of interest.
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PTP LAR Inhibits Epithelial Cell Migration and Tumor Formation in
Nude Mice--
We next examined whether PTP LAR has a direct
regulatory function during epithelial cell migration. We therefore
ectopically expressed human PTP LAR in NBT II cells at levels
comparable with the endogenous protein, thereby yielding polyclonal as
well as clonal cell lines with about twice the PTP LAR expression
relative to the parental cells (data not shown). With these cell lines we performed scatter assays to quantify migration after EGF treatment. This modest ectopic expression of hPTP LAR in NBT II cells
significantly inhibited EMT and motility (Fig.
7A) to about 40% that of the vector control-infected cell lines (NBT II pLXSN, Fig.
7B) without affecting the kinetics of the onset of
migration. No differences in activation and autophosphorylation of the
EGF receptor and its association with the adapter protein Shc were
detected (data not shown), suggesting that ectopic hPTP LAR does not
function by inactivating the EGF receptor. Furthermore, downstream
events of EGF signaling like DNA synthesis and proliferation rate of these NBT II-hPTP LAR cell lines were not affected by ectopic expression of hPTP LAR (data not shown).

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Fig. 7.
Ectopic overexpression of human PTP LAR
inhibits epithelial cell migration and tumor formation of rat NBT II
cells in nude mice. A and B, scatter assay
of NBT II pLXSN and NBT II hPTP LAR cells. A, NBT II cells
infected with the empty vector pLXSN (left) or human PTP LAR
(right) were plated at 1 × 104
cells/cm2 and stimulated 24 h later with EGF (100 ng/ml) as indicated. After 7 h, the migration morphology was
documented by photography. B, quantification of the scatter
assay (±S.D.). To this end, 1000 cells/dish from randomly chosen
microscopic fields were counted; assays were performed in triplicates.
A cell was judged as a migrating cell, when it had changed from a
cobblestone-like, epithelial morphology to a migrating, fibroblastoid
phenotype. C, tumor formation in nude mice by NBT II pLXSN
and NBT II hPTP LAR cells. NBT II cells infected with the empty vector
pLXSN (filled triangle) or human PTP LAR (filled
square) were injected with 2 × 106 cells/50 µl
subcutaneously into the flank region of Swiss nude mice. Tumor
formation was monitored. The graph shows the average tumor
volume of three tumors per polyclonal and clonal cell line,
respectively.
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The epithelial cell migration in vitro resembles a
simplified model system of tumor formation and metastasis in
vivo. Therefore we tested whether ectopic expression of hPTP LAR
correlated also with an decreased capability of these cells to form
tumors in vivo in nude mice. To this end, we injected NBT II
hPTP LAR cells subcutaneously into the flank region of Swiss nude mice.
Interestingly, these cells displayed significantly reduced tumor growth
in comparison to NBT II pLXSN cells (Fig. 7C). The
capability to form tumors of polyclonal and clonal cell lines did not
differ. The parental NBT II cells form tumors to the same extent as the
vector control-infected cell lines, which rules out a clonal artifact
(data not shown). These data strongly suggest that PTP LAR serves as a
negative regulator of epithelial cell migration and tumor formation.
PTP LAR Inhibits Tyrosine Phosphorylation and the Increase of the
Free Pool of
-Catenin--
These data prompted us to investigate
biochemical parameters, which correlated with the inhibition of
EGF-induced migration in hPTP LAR-expressing NBT II cells. We therefore
affinity-precipitated
-catenin in control and hPTP LAR-expressing
NBT II cells using a GST-E-cadherin cytoplasmic fusion protein. Because
we were interested in the function of PTP LAR, we did not treat the
cells with the inhibitor of PTPs, sodium orthovanadate. Nevertheless,
even under these conditions we were able to detect an increase in the
free, uncomplexed and tyrosine-phosphorylated pool of
-catenin in
vector-infected control cells, demonstrating that an increase in free,
tyrosine-phosphorylated
-catenin occurred also in cells without
sodium orthovanadate pretreatment (Fig.
8, left). However, in NBT
II-hPTP LAR-expressing cells, neither tyrosine phosphorylation nor an
increase in the free pool of
-catenin were detectable (Fig. 8,
right). Because
-catenin is a substrate of PTP LAR
in vitro and ectopic expression of human PTP LAR in NBT II
cells does not interfere with EGF receptor-mediated signaling, we
suggest that PTP LAR represents a specific negative regulator of
-catenin tyrosine phosphorylation, which prevents an increase in
free
-catenin, thereby inhibiting epithelial cell migration.

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Fig. 8.
Ectopic expression of human PTP LAR in NBT II
cells inhibits the phosphorylation in tyrosine residues of
-catenin as well as the increase of the free pool
of -catenin. NBT II cells infected with
the empty vector pLXSN (left) or human PTP LAR
(right) were plated at 1 × 104
cells/cm2 and stimulated after 24 h with EGF (100 ng/ml) for the indicated time intervals. Sodium orthovanadate
pretreatment was omitted to avoid irreversible inhibition of
protein-tyrosine phosphatase activity. Cells were lysed, and in
vitro associations were performed with 5 µg of the
GST-E-cadherin cytoplasmic (cytopl.) protein or GST in a
3-fold molar excess. Proteins were separated by SDS-PAGE, and the
levels of free, uncomplexed -catenin were analyzed by Western
blotting with an anti- -catenin antibody (top). Western
blotting with an anti-phosphotyrosine antibody shows the increased
tyrosine phosphorylation in free, uncomplexed -catenin only in the
control-infected NBT II cell line (bottom).
Arrows indicate the proteins of interest, and molecular mass
standards are shown in kilodaltons on the left.
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DISCUSSION |
Cadherin-catenin complex-mediated cell-cell adhesion as
well as adhesion-independent functions of catenins have been implicated in the modulation of multicellular differentiation, proliferation, and
malignant transformation of epithelial cells (7, 8, 11). Cell migration
is an essential process during embryonic development and in epithelial
regeneration of adult organisms, for example in wound healing, and
requires precise control, which is altered or lost when tumor cells
become invasive and metastatic. In this study we have defined at the
cellular and biochemical level distinct mechanisms regulating
epithelial cell migration. Furthermore, we have characterized in motile
cells the impact of tyrosine phosphorylation of
-catenin and its
regulation by tyrosine kinases and the protein-tyrosine phosphatase
LAR.
Growth factors such as EGF, acidic fibroblast growth factor, or
hepatocyte growth factor/scatter factor have been shown to induce
migration of epithelial cells (38). A correlation has been suggested
between migration and tyrosine phosphorylation of
-catenin (12, 14),
but its significance remained unclear. Inhibition of tyrosine kinase
signaling inhibits epithelial cell migration of NBT II cells. Receptor
tyrosine kinases like the EGF receptor (17) or cytoplasmic tyrosine
kinases like Src are able to mediate tyrosine phosphorylation of
-catenin (13). In NBT II cells, a dominant-negative c-Src mutant was
able to inhibit migration (47). We demonstrate here that after
induction of migration, the pool of free, uncomplexed
-catenin is
increased and that this increase correlates with enhanced
-catenin
tyrosine phosphorylation. Therefore, tyrosine phosphorylation may
result in a reduced interaction of
-catenin with both E-cadherin and the actin-cytoskeleton. Because the integrity of the cadherin-catenin complex is essential for strong cell-cell adhesion, this reduced interaction may lead to an overall decrease in intercellular contacts. Interestingly, a fusion protein of E-cadherin and
-catenin was reported to be able of mediating the interaction of E-cadherin with the
cytoskeleton independent of
-catenin. However, these cells were no
longer capable of migration (48). In light of these data, tyrosine
phosphorylation of
-catenin may lead to disruption of the contact
between E-cadherin and the cytoskeleton and to an increased pool of
free
-catenin. This suggests a function for
-catenin independent
of cadherin-mediated cell adhesion.
Besides their role at adherens junctions, a signaling function of
-catenin or its Drosophila homologue Armadillo was shown to be essential for normal embryonic development in
Drosophila and Xenopus. Interference with the
signaling function of free, uncomplexed
-catenin abolished proper
vertebrate development (24). Subsequent studies in
Drosophila and Xenopus led to the discovery of a
signaling cascade that regulates the cytoplasmic pool of
-catenin.
Without a signal,
-catenin is localized mainly at adherens
junctions, and any free
-catenin is down-regulated in a
ubiquitin-dependent manner (30). An extracellular signal such as Wnt or Wingless (49, 50) leads via the receptor Frizzled (51)
to an inhibition of glycogen synthase kinase 3/Zeste White 3 activity,
thus stabilizing
-catenin/Armadillo in its free form (44, 52) by
inhibiting APC- and ubiquitin-dependent degradation (30,
53). We could show an increased pool of free
-catenin in the
cytoplasm and the nucleus in migrating cells after EGF treatment,
suggesting another way of regulating the free pool of
-catenin
during epithelial cell migration beside Wnt/Wingless signaling.
Tyrosine phosphorylation of
-catenin may also be able to stabilize
free
-catenin, because it was shown for the homologous plakoglobin
that the tyrosine-phosphorylated form did no longer associate with APC
(54), thereby possibly preventing APC-mediated degradation. Indeed, we
were able to demonstrate that the free pool of
-catenin has an
increased phosphotyrosine content during EGF-dependent
epithelial cell migration. Additionally, EGF is also able to inhibit
glycogen synthase kinase 3 activity (55), and tyrosine phosphorylation
of
-catenin was shown to correlate with carcinoma formation and
tumor invasion (56). Only free
-catenin or plakoglobin is able to
associate with members of the high mobility group (57) family of
transcription factors, namely LEF1, T-cell factor 3, and T-cell factor
4 (58-62). However, recently it was demonstrated that
-catenin and
plakoglobin differ in their nuclear translocation and complexing with
LEF1 and that LEF1-dependent transactivation is
preferentially driven by
-catenin (63). The transcription factors
LEF1 and T-cell factor are able to induce dorsal mesoderm when
expressed together with
-catenin in Xenopus embryos
(58-60). Data from LEF1-deficient and LEF1-transgenic mice demonstrate
an important role of this transcription factor during EMT, because it
is normally up-regulated during EMT and inductive processes between
mesenchyme and epithelium (64-66). It is tempting to speculate that a
common regulatory system exists that coordinates the expression of
genes for mesenchymal and epithelial phenotypes during embryonic
development as well as in epithelial cell migration. During EMT or cell
migration this common regulator would switch off expression of
epithelial genes while switching on genes for the mesenchymal
phenotype. The
-catenin·LEF1 complex may represent this common
regulator, and molecules that control the free pool of
-catenin may
be essential for proper development or wound healing.
PTPs were proposed to play an important role in the regulation of
cell-cell contacts because treatment with sodium orthovanadate, a
potent inhibitor of phosphatase activity, diminished normal cell
contact inhibition in epithelial cells and led to increased tyrosine
phosphorylation at adherens junctions (16). PTPs may therefore act as
steady state equilibrium antagonists of TKs for regulatory events at
adherens junctions. Consistent with these findings we show that
tyrosine phosphorylation of
-catenin and plakoglobin in migrating
epithelial cells was significantly increased after pretreatment with
sodium orthovanadate. Little is known about interacting proteins or
in vivo substrates of transmembrane PTPs. PTP LAR was
recently shown to interact and colocalize at focal adhesions with LAR
interacting protein 1 (LIP-1) and the multidomain protein Trio.
However, neither of these proteins appears to be a substrate for PTP
LAR (45, 67). Moreover, a PTP LAR-like PTP was reported to interact
with the cadherin-catenin complex at adherens junctions of
neurosecretory PC12 cells (68). We demonstrate here the colocalization
and interaction of PTP LAR with the cadherin-catenin complex in
epithelial cells. Furthermore, we show that only
-catenin and
plakoglobin are able to associate directly with PTP LAR, and we present
evidence that
-catenin is a substrate of PTP LAR. PTP LAR is of
special interest because it is localized at adherens junctions and at
focal adhesions (this report and Refs. 45, 67, 68); thus PTP LAR could
represent an essential regulator of the disassembly and reassembly of
cell-cell as well as cell-extracellular matrix adhesions during
epithelial cell migration. We demonstrate that modest ectopic
overexpression of PTP LAR significantly inhibited epithelial cell
migration. A similar situation is found in Drosophila, where
PTP LAR, contrary to vertebrates, is almost exclusively expressed in
developing neurons. In flies lacking PTP LAR, motor axons bypass their
normal target region and instead continue to extend without stopping (69). PTP LAR, PTP µ, PTP
as well as PTP DEP-1 were found to be
expressed at elevated protein levels with increased cell confluence
(20, 70-72), thereby contributing to the observed increased tyrosine
phosphatase activity in confluent cells (73). The increase in
phosphatase activity in confluent cells suggests a role of PTPs in the
regulation and stabilization of cell-cell contacts and epithelial cell
integrity. In contrast, during wound healing, cells at the wound edge
are in a subconfluent situation, where PTP action is reduced. This
could favor tyrosine kinase signaling through stimulation by growth
factors such as EGF, transforming growth factor
, and keratinocyte
growth factor, which results in an increase in cell migration and
proliferation and, thus, to accelerated wound healing, because the
expression of these growth factors was increased during a wound
situation (74, 75, 76).
The importance of free and tyrosine-phosphorylated
-catenin during
epithelial cell migration is underscored by our observations that
interfering with this parameter by overexpressing hPTP LAR inhibits
epithelial cell motility. Furthermore, expression of hPTP LAR in NBT II
cells inhibited tumor formation in nude mice, although the growth
characteristics of these cells in vitro were not altered.
Ectopic expression of hPTP LAR to about twice the endogenous level was
sufficient to result in the biological effects observed. Such modest
ectopic expression of hPTP LAR appears to be critical, because high
overexpression results in completely altered growth characteristics and
apoptosis of the cells (77). The data presented in this report strongly
support an important role for PTP LAR in the regulation of cell-cell
adhesion and epithelial cell migration as well as tumorigenesis by
controlling the free pool of signaling
-catenin. Because mutations
or deletions in either APC or
-catenin leading to a stabilized pool
of free
-catenin have been correlated with tumor formation (61, 62,
78), loss of PTP LAR function may also contribute to tumor formation and metastasis. As a potential tumor suppressor gene product, PTP LAR
could therefore serve as a prognostic marker for human cancer.