* Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606-8502, Japan; and Department of Cell Biology,
Vanderbilt University, School of Medicine, Nashville, Tennessee 37232-2175
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Abstract |
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p120ctn binds to the cytoplasmic domain of cadherins but its role is poorly understood. Colo 205 cells grow as dispersed cells despite their normal expression of E-cadherin and catenins. However, in these cells we can induce typical E-cadherin-dependent aggregation by treatment with staurosporine or trypsin. These treatments concomitantly induce an electrophoretic mobility shift of p120ctn to a faster position. To investigate whether p120ctn plays a role in this cadherin reactivation process, we transfected Colo 205 cells with a series of p120ctn deletion constructs. Notably, expression of NH2-terminally deleted p120ctn induced aggregation. Similar effects were observed when these constructs were introduced into HT-29 cells. When a mutant N-cadherin lacking the p120ctn-binding site was introduced into Colo 205 cells, this molecule also induced cell aggregation, indicating that cadherins can function normally if they do not bind to p120ctn. These findings suggest that in Colo 205 cells, a signaling mechanism exists to modify a biochemical state of p120ctn and the modified p120ctn blocks the cadherin system. The NH2 terminus-deleted p120ctn appears to compete with the endogenous p120ctn to abolish the adhesion-blocking action.
Key words: E-cadherin; catenin; colon carcinoma; Colo 205; p120ctn ![]() |
Introduction |
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CELL-CELL adhesion is a dynamic process, especially
during morphogenetic cell rearrangement such as
intercalation and delamination of cells, and in
many pathological phenomena including tumor invasion. To clarify the mechanisms of how cell-cell adhesion is regulated is an important issue for understanding such biological phenomena. Cadherin-mediated adhesion is thought
to be controlled by catenins which are associated with the
cytoplasmic domain of cadherin. A region in the COOH
half of the cadherin cytoplasmic domain directly binds
-catenin, which in turn associates with
-catenin (Barth et
al., 1997
).
-Catenin interacts with the actin-based cytoskeleton directly or indirectly, involving other cytoskeletal
proteins such as
-actinin and vinculin (Knudsen et al.,
1995
; Rimm et al., 1995
; Watabe-Uchida et al., 1998
).
These molecular interactions are essential for this system
to exert its full adhesion activity (Hirano et al., 1992
;
Watabe et al., 1994
; Ozawa and Kemler, 1998a
; Watabe-Uchida et al., 1998
), although homophilic interactions between the cadherin extracellular domains per se can be
achieved without catenins (Brieher et al., 1996
; Yap et al.,
1997
; Chitaev and Troyanovsky, 1998
). It is thought that
cadherin function may be regulated by biochemical modifications of the catenins. Indeed, phosphorylation of
catenins, in particular
-catenin, has been implicated in
the instability of cell-cell adhesion (e.g., Matsuyoshi et al.,
1992
; Hamaguchi et al., 1993
; Serres et al., 1997
; Takahashi et al., 1997
; Balsamo et al., 1998
).
One such catenin is p120ctn, a member of the Armadillo/
-catenin gene family (Peifer et al., 1994
). The p120ctn protein was originally identified as a target for p60v-src kinase
(Reynolds et al., 1989
, 1992
) and later found to be a protein associated with the cytoplasmic domain of cadherins
(Reynolds et al., 1994
; Daniel and Reynolds, 1995
; Shibamoto et al., 1995
; Staddon et al., 1995
). Other proteins related to p120ctn, constituting a subfamily of Armadillo/
-catenin, have also been identified (Heid et al., 1994
;
Hatzfeld and Nachtsheim, 1996
; Mertens et al., 1996
;
Paffenholz and Franke, 1997
; Sirotkin et al., 1997
). Recent
studies show that p120ctn binds to the juxtamembrane portion of the cadherin cytoplasmic domain, which is different
from the region to which
-catenin binds (Finnemann et al.,
1997
; Lampugnani et al., 1997
; Yap et al., 1998
). In contrast to the well-known function of
-catenin, that of
p120ctn remains largely unknown. However, some biological effects of its ectopic expression have been reported,
e.g., overexpression of p120ctn induces extensive dendrite-like processes in fibroblasts (Reynolds et al., 1996
) and
perturbs gastrulation in Xenopus laevis embryos (Geis et al.,
1998
; Paulson et al., 1999
). In addition, a recent report
shows that overexpression of
-catenin in MDCK cells, a protein related to p120ctn, alters their morphology and motility (Lu et al., 1999
).
In the present work, we studied a unique aggregation
property of colon carcinoma Colo 205 cells (Semple et al.,
1978). They grow as dispersed cells not forming compact
aggregates, despite the expression of all general components of the E-cadherin-catenin complex. We found that
typical E-cadherin-dependent aggregation could be induced by treatment with staurosporine, a kinase inhibitor, or with low concentrations of trypsin. Correlating with this
adhesive change, the electrophoretic mobility of p120ctn
was altered. Furthermore, when NH2 terminus-deleted
p120ctn molecules were introduced into Colo 205 cells,
these constructs induced an E-cadherin-dependent compact aggregate formation, similar to effects induced by
staurosporine and trypsin. Together with other findings,
our results suggest that p120ctn can function as an inhibitory regulator in the cadherin adhesion system.
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Materials and Methods |
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Antibodies and Other Reagents
Mouse mAbs HECD-1 (Shimoyama et al., 1989) and SHE78-7 to human
E-cadherin (Takara Shuzo Co., Ltd.), rat mAb NCD-2 to chicken N-cadherin (Nakagawa and Takeichi, 1998
), mouse mAb to p120ctn (Transduction Laboratories), mouse mAb M2 to FLAG (F-3165; Sigma Chemical
Co.), rabbit polyclonal antiserum to FLAG (SC-807; Santa Cruz Biotechnology, Inc.), mouse mAb to
E-catenin (Transduction Laboratories), rat
mAb
18 to
E-catenin (Watabe et al., 1994
), mouse mAb 5H10 to
-catenin (Johnson et al., 1993
), and rabbit polyclonal antiserum to
-catenin (Shibamoto et al., 1995
) were used. Anti-MUC1 antibody
MY.1E12 (Yamamoto et al., 1996
) was a gift from T. Irimura (University
of Tokyo, Tokyo, Japan).
Antibodies used for detection of primary antibodies were as follows: goat Cy-3-labeled species-specific antibody to mouse IgG (AP-124C; Chemicon International, Inc.), donkey biotinylated species-specific antibody to rabbit IgG (RPN1004; Nycomed-Amersham), FITC-labeled streptavidin (RPN1233; Nycomed-Amersham), sheep HRP-linked species-specific antibody to mouse IgG (NA9310; Nycomed-Amersham) and rat IgG (NA932; Nycomed-Amersham), goat HRP-linked antibody to rabbit IgG (NA934; Nycomed-Amersham), and Sepharose 4B-linked goat antibody to mouse IgG (62-6541; Zymed Labs, Inc.).
The following protein kinase inhibitors were used: staurosporine (#19-123; Upstate Biotechnology), calphostin C (C-159; Research Biochemicals
Inc.), tyrphostin (EI-215; Biomol), genestein (G-103; Research Biochemicals International), and herbimycin A (OP-12, Kyowa Medex Co., Ltd.).
UCN-01 (Takahashi et al., 1990) was a kind gift of T. Tamaoki (Kyowa
Medex Co., Ltd.). O-sialoglycoprotein endopeptidase was purchased from
Cedarlane Labs., Ltd.
Cells and cDNA Transfection
Human colon carcinoma cell lines Colo 205 (Semple et al., 1978) and HT-29 (Fogh and Trempe, 1975
), and MDCK cells (Gaush et al., 1966
) were
used. These cells were cultured in a 1:1 mixture of DME and Ham's F12
supplemented with 10% FCS (DH10). For transfer of Colo 205 cells, an
aliquot of the suspended cell culture was moved to new dishes with fresh
DH10 medium. When necessary, they were trypsinized as described below. For HT-29 and MDCK cells, they were rinsed with 1 mM EDTA in
Ca2+- and Mg2+-free saline, then treated with 0.05% crude trypsin (#0152-13; Difco Laboratories) and 1 mM EDTA in the same solution for 5 min at 37°C, and finally suspended in DH10.
Colo 205 cells were transfected by electroporation or by use of adenoviral expression vectors. For electroporation, trypsinized cells (4 × 105) were suspended in 200 ml of Hepes-buffered (pH 7.4) saline with 1 mM Ca2+ and 1 mM Mg2+ (HBS). 20 µg of an expression vector was added to the suspension, which was electrified at 1,160 µF at 250 V. For adenovirus-mediated transfection, 106 cells were suspended in 500 µl of DH10 with 5 × 107 plaque-forming units of adenovirus, and incubated for 4 h. Cells were washed twice with DH10, and after 48 h samples were collected. MDCK cells were transfected by electroporation under the same conditions as for Colo 205 cells, except that 1,060 µF at 200 V was used. HT-29 cells were transfected with adenoviral vectors only.
Cell Aggregation and Trypsin Treatment
Colo 205 cells were washed twice with Ca2+- and Mg2+-free Hepes-buffered saline supplemented with 1 mM EDTA, and completely dissociated into single cells by pipetting. Then, 2 × 105 cells were resuspended in 1 ml of DH10 with or without 1 µg/ml of SHE78-7, a blocking antibody to human E-cadherin, and placed in a Nunc 6-well plate (#152795). Cells were incubated for 3 h at 37°C on a gyratory shaker (KS6320; Marysol) at 80 rpm.
For trypsin treatment to induce cell adhesion, cells were rinsed twice
with DH (DH10 without FCS), and incubated in DH with crystalline
trypsin (T-8253; Sigma Chemical Co.) of various concentrations at 37°C.
For biochemical analysis, the incubation was generally stopped at 30 min,
and the cells were rinsed twice with HBS containing 0.1% trypsin inhibitor (T-6522; Sigma Chemical Co.) and subjected to further analysis. 0.01%
trypsin in the presence (TC) or absence (TE) of 1 mM Ca2+ treatments
were performed as described previously (Takeichi, 1977).
cDNA Construction
Using mouse p120ctn cDNA (GenBank accession number Z17804) as a
template, we generated FLAG-tagged p120ctn (FLf)1 and other constructs
by PCR. The following primers were used: primer N, 5'-GAATTCATGGACGACTCAGAGGTG-3'; primer A, 5'-GAATTCATGTTAGCAAGCTTGGATAGTTTG-3'; primer CR, 5'-GAATTCGATATCCTACTTGTCATCGTCGTCCTTGTAGTCAATCTTCTGCATCAAGG-GTGC-3'; primer AR, 5'-CTGCAGGAAATCCACTGTATCATT-3'
Primer CR and
AR were antisense primers. The primer CR contained
an EcoRV restriction site, and complementary sequences for stop codon
and FLAG epitope DYKDDDDK (Hopp et al., 1988
) at the 5' portion, as
underlined. Primers N and CR were used for amplifying FLf, and primers
A and CR for
N346f, a mutant molecule in which the NH2-terminal 346 amino acids (aa) of p120ctn were deleted. For construction of other NH2-terminal deletion mutants
N101f,
N157f,
N244f, and
N323f, the fragment FLf was internally digested with BglII/EcoRV, NcoI/EcoRV, SmaI/
EcoRV, and Bsu36I/EcoRV, respectively. We assume that translation of the constructs will start at the next ATG downstream of the deletion. For
FL
Rf and
N346
Rf, in which aa 641-819 were deleted, oligonucleotides corresponding to aa 346-640 were synthesized by PCR by use of primer A and
AR, digested by PstI, and inserted into the internal PstI
sites of FLf or
N346f. The obtained fragments were subcloned into the
pCA-pA expression vector (Niwa et al., 1991
) and pAdV-CA-pA adenoviral construction vector (Nakagawa and Takeichi, 1998
).
For construction of cN/JM(), encoding a chicken N-cadherin mutant
in which the juxtamembrane portion of the cytoplasmic region is deleted,
the catenin binding region with COOH-terminal 70 aa and the extracellular-transmembrane region were amplified by PCR, respectively, using
pCMV-cN/FLAG-pA (Nakagawa and Takeichi, 1998
) as a template. For
amplifying the catenin binding region, primers 5'-CGCCGTACGACCATGAGATCTAATGAGGGACTTAAAGCAGCC-3' and 5'-GCGGCCGCTTACTTGTCATCGTCGTCCTTGTAGTC-3' were used. For
the extracellular-transmembrane region, primers 5'-GCGCGTACGACCATGTGCCGGATAGCGGGAACGCCG-3' and 5'-CGCGAGATCTCTGACGCTCCTTATCCGGCG-3' were used. Obtained products
were linked through the BglII sites underlined above, and subcloned into
pCA-pA or pAdV-CA-pA. All PCR-generated fragments of DNA obtained above were completely sequenced, and no sequence error was detected.
Construction of Recombinant Adenovirus
Recombinant adenovirus AdV-CA-lacZ expressing -galactosidase with
the CAG promoter was a gift from K. Moriyoshi (Kyoto University, Kyoto, Japan). Construction of recombinant viruses was performed according to methods described previously (Moriyoshi et al., 1996
). In brief,
HEK 293 cells (ATCC CRL 1573) cultured with DH10 in 6-well plates
(Iwaki Co.) were cotransfected with viral genome fragments (0.2 µg) and
linearized adenoviral shuttle vector plasmids (1 µg) by use of LipofectamineTM (#18324-012; Life Technologies, Inc.). The next day, the cells
were divided into collagen-coated 24-well plates (Iwaki Co.). 10 d later,
wells became full of dead cells, caused by viral propagation, and the debris
was screened for proper protein expression by immunostaining and immunoblotting with the anti-FLAG antibody M2. We obtained AdV-CA-FLf, AdV-CA-
N101f, AdV-CA-
N157f, AdV-CA-
N244f, AdV-CA-
N346f, which expressed different forms of p120ctn proteins under the
control of the CAG promoter (Niwa et al., 1991
), and AdV-cN/JM. The
full-length N-cadherin expression vector AdV-Ncad was described previously (Nakagawa and Takeichi, 1998
). The recombinant adenoviruses
were amplified, and purified by CsCl2 step gradient centrifugation (Kanegae et al., 1994
). FCS was added to the purified adenovirus solutions at a
final concentration of 10%. Aliquots of the virus solution were stored at
80°C until used.
Cell Extraction and Immunoprecipitation
Colo 205 cells were removed from dishes by pipetting, rinsed twice in
HBS, and lysed in TBS-Ca (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM
CaCl2) supplemented with 1% Triton X-100, 1% NP-40, 1 mM PMSF, 1 mM
NaVO3, and 50 mM NaF (TBS-Triton) for 30 min at 4°C on a rocking platform. The soluble fraction was collected by centrifugation and preincubated with 50 µl of secondary antibody-conjugated beads and 5% BSA on
ice for 20 min. After preincubation, incubations with primary and secondary antibodies were sequentially performed on ice for 1 h each, followed
by washing three times with TBS-Triton. Finally, samples were boiled in
150 µl of SDS sample buffer with 7.5 µl of -mercaptoethanol for 5 min.
Gel Electrophoresis, Immunoblotting, and Immunohistochemistry
Proteins were separated in SDS-PAGE and electrophoretically transferred to membranes. The transferred proteins were detected by the enhanced chemiluminescence system (RPN 2106; Nycomed-Amersham). Immunohistochemical detection of cadherin and catenins was performed as described in Watabe-Uchida et al. (1998).
Phosphatase Treatment
Immunoprecipitated materials were washed twice with phosphatase reaction buffer (50 mM Tris-HCl, pH 8.5, 50 mM MgCl2, 1 mM PMSF, 1 mM DTT, 0.1% Triton X-100, 0.1% NP-40). 27 µl of the reaction buffer and 3 µl of alkaline phosphatase (#567-744; Boehringer Mannheim GmbH) were added to the precipitate, and the mixture was then incubated at 30°C for 1 h. After incubation, 1 µl of 0.5 M EDTA and 30 µl of 2× SDS sample buffer were added, and the mixture was boiled for 5 min.
Phosphorous-32 Metabolic Labeling and Phosphoamino Acid Analysis
For 32P-metabolic labeling, cells were cultured in phosphate-free DME
(#21097-035; Life Technologies Inc.) with 0.5 mCi of 32P (NEX-053, New
England Nuclear Life Science Products, Inc.) for 24 h. Then, cells were
harvested and trypsinized when necessary. From their detergent extracts
prepared as above, p120ctn was immunoprecipitated and separated by
SDS-PAGE. From the gels, the labeled p120ctn-protein band was excised
after comparing with their autoradiograms, and the collected gel pieces
were homogenized in 500 µl of freshly prepared 50 mM NH4HCO3. After addition of 25 µl -mercaptoethanol and 5 µl 10% SDS, the samples
were boiled for 5 min and agitated at room temperature for 2 h. Supernatants were collected after centrifugation at 15,000 rpm for 5 min, mixed
with 20 µg RNase A and 250 µl ice-cold TCA (100% wt/wt), and incubated on ice for 1 h. After centrifugation, the pellets were air-dried and
proteins were digested by incubating with 50 µl 6 N HCl at 110°C for 90 min. Digested products were air-dried again and resuspended in buffer
(2.2% formic acid, 7.8% glacial acetic acid), pH 1.9, with unlabeled phosphoserine, phosphothreonine, and phosphotyrosine. Samples were spotted on TLC plate and separated by two-dimensional electrophoresis at 1.5 kV for 40 min with the pH 1.9 buffer, and at 1.0 kV for 30 min with buffer
(5% glacial acetic acid, 0.5% pyridine), pH 3.5. Finally, labeled phosphoamino acids were visualized by the BAS-1000 image analyzing system
(FUJIX Inc.).
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Results |
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Induction of Compact Aggregate Formation in Colo 205 Cells
Colo 205 cells grow as dispersed cells occasionally forming
loose, small clusters (Fig. 1 A) as seen in catenin-deficient cells (Hirano et al., 1992; Oyama et al., 1994
; Shimoyama
et al., 1992
), and they only lightly attach to the culture
dish. Despite this behavior, they express an apparently
normal set of E-cadherin,
E-catenin,
-catenin, and
p120ctn proteins (Fig. 1 E). Moreover, the expression levels
of these proteins are similar to those in HT-29 cells, which
can organize epithelial sheets (see Fig. 7 A). To determine
whether the cadherin-catenin system in Colo 205 cells is
entirely inactive, we rotated the cultures to facilitate cell
aggregation. Under these culture conditions, Colo 205 cells clumped into larger aggregates (Fig. 1 C) and this clumping was inhibited by addition of the E-cadherin
blocking antibody SHE78-7 (Fig. 1 D), indicating that
E-cadherin is functional. However, their aggregates were
still loose and never formed tightly associated compact aggregates as generally produced by the cadherin adhesion
system (Takeichi, 1988
). These observations suggest that Colo 205 cells expose functional E-cadherin molecules on
their surface, but their cadherin system has some deficit in
exerting its full activity to organize compact cell aggregates. Interestingly, when the cultures of Colo 205 cells
were starved by not refreshing the culture medium for a
few days, they occasionally formed compact aggregates
(Fig. 1 B), implying that their cadherin system is reversibly
impaired and can be reactivated under certain physiological conditions.
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As an initial attempt to investigate how the cadherin system is impaired in Colo 205 cells, we examined the effect of various biochemical reagents, including kinase inhibitors and activators, on their aggregation. Among the reagents tested, staurosporine showed a marked effect. It induced compact cell clustering in Colo 205 cultures within a few hours after administration (Fig. 2, A-D), during which the initial sign of adhesion induction was observed within 2 h. This effect was saturated by 6 h. The compaction of cell clusters was E-cadherin-dependent, as it was inhibited by SHE78-7 (Fig. 2 E). Staurosporine also slightly promoted spreading of cells, but this was not inhibited by SHE78-7 (Fig. 2 E, arrows). Since staurosporine is known to inhibit protein kinase C (PKC), we tested other PKC inhibitors, but none were effective (Table I). Tyrosine kinase inhibitors were also negative, except herbimycin A showed a weak compaction-inducing activity, but only when cells were cultured overnight with this inhibitor (data not shown). PKC activators and phosphatase inhibitors, such as phorbol esters and okadaic acid, also had no effects (data not shown). Thus, among the reagents tested, staurosporine exhibited an exceptionally strong effect on Colo 205 aggregation. This effect of staurosporine was reversible as its removal caused redispersion of Colo 205 cells (data not shown).
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Besides metabolic inhibitors, we found that trypsin is a
strong inducer for compact Colo 205 aggregation. When
low concentrations of trypsin (0.01-0.001%) were added
to serum-free cultures, Colo 205 cells became tightly associated with each other, deforming their morphology
(Fig. 2, F-H). This adhesion induction was quick, beginning within 15 min after the addition of trypsin, and the effect was almost saturated at 30 min. The trypsin-mediated induction of aggregation was completely blocked by
SHE78-7 (Fig. 2 I), indicating that it was an E-cadherin-
dependent process. Under these conditions, which included a physiological concentration of Ca2+, cadherins
are not digested with trypsin (Takeichi, 1977). Trypsin concentration of 0.001% was sufficient for the above adhesion induction, whereas 0.0001% was not effective. Thus,
we found two different classes of reagents to induce compact Colo 205 aggregation, trypsin as a quicker inducer
and staurosporine as a slower inducer.
Localization of MUC1, E-Cadherin, and Catenins in Colo 205 Cells
Previous studies indicated that mucins, such as MUC1
(episialin; Wesseling et al., 1996; Kondo et al., 1998
) and
epiglycanin (Kemperman et al., 1994
), reduce cell-cell adhesion presumably by steric hindrance. As Colo 205 cells
express MUC1 (Baeckstrom et al., 1991
), trypsin may remove such antiadhesive mucins. To check this possibility,
we immunofluorescently stained for MUC1 before and after trypsin treatment, and found that this proteoglycan was
equally present on the surface of both trypsinized and untrypsinized cells (Fig. 2, J and K), even being localized at
intercellular contact sites in the adhesion-induced aggregates. Treatment of cells with O-sialoglycoprotein endopeptidase, which inactivates epiglycanin and enhances
adhesion of mammary carcinoma cells (Kemperman et al.,
1994
), showed no effects on Colo 205 aggregation (data not shown). From these observations, we assumed that the
effect of trypsin was not to remove steric hindrance molecules, but to digest some signaling proteins on the cell surface, such as receptors, affecting intracellular physiological
states. This idea is supported by results of other experiments described below.
Immunostaining for E-cadherin in Colo 205 cells show a diffuse distribution of this molecule on their surface (Fig. 3, A and A'). When compaction was induced in their aggregates by staurosporine or trypsin treatment, E-cadherin became highly concentrated into cell-cell contact sites (Fig. 3, C, C', E, and E'). Catenins displayed a similar distribution (see p120ctn in Fig. 3, B, B', D, D', F, and F'). These observations suggest that the E-cadherin-catenin complex can be redistributed to cell-cell contact sites under the above compaction-inducing conditions.
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Changes in Electrophoretic Mobility of p120ctn
To study if any changes were induced in the cadherin- catenin system following the above treatments, we immunoprecipitated E-cadherin from Colo 205 cells untreated or treated with staurosporine or trypsin. The amount of catenins coprecipitating with E-cadherin was not changed after these treatments (Fig. 4 A). However, we noted a significant change in the pattern of the p120ctn band. In untreated Colo 205 cells, p120ctn was detected as a broad, diffuse band that appeared to comprise multiple components (Fig. 4 A, lanes p120 c), although the pattern varied slightly from experiment to experiment. After compaction-inducing treatments, the band pattern was altered. Its most intense portion was shifted to the position corresponding to the bottom (electrophoretic front) of the original band (Fig. 4 A, lanes p120, t and s). Similar electrophoretic patterns of these proteins were observed in Western blots of the whole cell lysates (Fig. 4 B). The changes in the p120ctn band pattern coincided with the onset of adhesion induction by each reagent, starting within 15 min with trypsin (Fig. 4 C) and within 2 h with staurosporine (Fig. 4 D). A certain level of p120ctn-band shift was already detected at 5 min of incubation with trypsin. When trypsin was removed from the culture, the original electrophoretic profile of p120ctn was eventually restored within overnight incubation (Fig. 4 C), together with a recovery of the dispersed cell morphology (data not shown). Removal of staurosporine gave similar results.
|
Next, we asked whether the p120ctn change occurred before, or as a result of, adhesion induction. We treated Colo
205 cells with 0.01% trypsin in the presence (TC) or absence (TE) of 1 mM Ca2+. TC treatment leaves E-cadherin-mediated adhesion intact, whereas TE destroys
E-cadherin-dependent adhesion (Takeichi, 1977). We found that both treatments resulted in a similar p120ctn-band shift (Fig. 4 E). This finding indicates that the p120ctn
band change was not brought about as a result of adhesion
induction but directly through trypsin treatment signals.
We also found that the trypsin-induced p120ctn-band shift
took place even when E-cadherin was blocked with antibodies (Fig. 4 C). This was also the case in the staurosporine treatment (data not shown). When other kinase inhibitors listed in Table I were tested, only herbimycin A
induced a similar p120ctn-band shift at a 24-h incubation
period (data not shown).
We sought to understand the molecular nature of the p120ctn-band shift. When p120ctn immunoprecipitates collected from untreated Colo 205 were incubated with alkaline phosphatase, the diffuse p120ctn band was transformed into a sharp band positioned at the front of the original (Fig. 4 F), which comigrated exactly with the p120ctn from staurosporine or trypsin treated cells. This suggests that the band shift observed may have been brought about by p120ctn dephosphorylation also. We then analyzed phosphorylated residues in p120ctn by 32P-metabolic labeling. The results showed that the major phosphorylated residues were serine (Fig. 4 G). However, their labeling intensity was however, not significantly reduced after trypsin treatment of cells. Antiphosphotyrosine antibodies detected only weak signals from the p120ctn bands in these cells (data not shown). It is possible that the p120ctn-band shift is caused by dephosphorylation of a subset of the phosphorylated residues. Alternatively, the quantitative differences in phosphorylation suggested by the phosphatase treatments may be underrepresented by amino acid analysis methodology.
Truncated p120 Induces Compaction in Cell Aggregates
The above findings suggest that the process of p120ctn-band shift could be associated with the induction of compact Colo 205 aggregation. To pursue this possibility, we
attempted to modify the activity of p120ctn by expressing a
series of its deletion constructs attached to a FLAG-tag at
the COOH terminus in Colo 205 cells (Fig. 5 A). Transient
expression of these molecules was achieved by electroporation of cDNAs or infection by adenoviral expression
vectors. Generally, the adenoviral infection yielded much
higher cDNA transfection efficiencies. Successfully transfected cells were detected by immunofluorescence staining
with antibodies to the FLAG-tag. We first tested the full-length p120ctn as a control and found no particular effect
on the aggregation of Colo 205 cells (Fig. 6 A). Then, we
transfected them with N346f, leaving the Armadillo repeat domain intact. Notably, cell groups expressing this
construct were transformed into tightly associated aggregates in which the introduced molecules were sharply concentrated at cell-cell contact sites (Fig. 6 B). Then we
tested other deletion constructs. Concerning shorter NH2-terminal deletions,
N323f displayed the same adhesion-inducing effect as
N346f (Fig. 6 C), and
N244f also
showed a positive effect, but their aggregation appeared
looser than that observed with the former (Fig. 6 D). On
the other hand,
N157f and
N101f had no effect on cell
adhesion (Fig. 6, E and F). These observations indicate
that the critical sites are located between 245 and 323. We
tested two other constructs, FL
Rf and
N346
Rf, in
which the Armadillo repeat domain was partially deleted,
and found that they had no effect on cell aggregation (Fig.
6, G and H). The last two molecules did not bind to E-cadherin, as previously found (Daniel and Reynolds, 1995
;
data not shown), whereas all the others tested did. Consistently, FL
Rf and
N346
Rf were distributed only in
the cytoplasm (Fig. 6, G' and H') whereas the others
were located along the cell membrane, as well as the cytoplasm (Fig. 6, A'-F'). Cell membrane distributions were
categorized into two groups: the adhesion-inducing mutant molecules were concentrated into cell-cell contact
sites (e.g., Fig. 6, B'and C', arrows) whereas the noneffective proteins were located randomly along the cell membrane.
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In the preceding experiments, the p120ctn-band shift was
correlated with adhesion induction. Therefore, we also analyzed the band profile of the NH2 terminus deletion constructs. In Western blotting of the lysates of cells transfected with these constructs, all NH2-terminally deleted
molecules showed broad electrophoretic bands (Fig. 5 B),
although the proportion of the bottom to the upper components in the band tended to increase in the adhesion-inducing construct N346f. When FLf and
N346f were
collected by immunoprecipitation and treated with phosphatase, only the bottom component remained undigested
in both samples (Fig. 5 C), as found in endogenous p120ctn,
indicating that the bottom bands of different p120ctn constructs were equivalent to each other in terms of phosphatase resistance.
We examined whether or not the binding of p120ctn to
E-cadherin was altered by NH2-terminal deletions. We
transfected Colo 205 cells with FLf or mutant N346f, and
immunoprecipitated E-cadherin from them (Fig. 5 D).
Anti-FLAG antibody D8 detected only the ectopic proteins, and the antibodies recognizing the COOH-terminal region of p120ctn detected both the transfected and endogenous molecules (Fig. 5 D, middle and right panels). Comparison of the band profiles in these samples with those in
Western blotting of whole cell lysates (Fig. 5 E) indicates
that the proportion of the E-cadherin-bound p120ctn to its
entire pool was not different between FLf and
N346f. The small difference seen in electrophoretic mobility between the endogenous and full-length ectopic molecules is
probably due to their difference in species origin, the
former from the mouse and the latter from the human.
These results suggest that the binding affinity of p120ctn for
E-cadherin was not altered by the NH2-terminal deletion.
p120ctn in Other Cell Lines
We tested two other epithelial lines, HT-29 and MDCK
cells, to ask whether they also respond to the NH2 terminus-deleted p120ctn constructs, as well as the compaction-inducing reagents. Human colon carcinoma HT-29 cells,
which express normal levels of E-cadherin and catenins
(Fig. 7 A), organize into epithelial sheets in confluent cultures (Fig. 7 B, c; Fogh and Trempe, 1975; Chantret et al., 1988
). When these cells are dispersed with trypsin in the
absence of Ca2+ and seeded into new plates they require a
lag period to reestablish epithelial sheets, e.g., at 18-24 h
after cell transfer, the cultures still contain many round,
dispersed cells (Fig. 7 B, b), although they eventually established epithelial sheets at 48 h. We analyzed p120ctn in
these HT-29 cells, and found that its band pattern dynamically changed with the cycle of cell transfer. p120ctn derived from freshly trypsinized HT-29 cells showed a single band (Fig. 7 F). 24 h after the transfer p120ctn was shifted
to an upper position (Fig. 7 F). At 48 h, when cells formed
confluent epithelial sheets, the p120ctn band returned to
the lowest position (Fig. 7 F). We tested if the p120ctn-band
shift observed after overnight culture could be canceled by
retreatment with trypsin (0.001%; 30 min) or staurosporine (7 nM; 6 h) by adding them to HT-29 cells precultured
for 18 h. The results showed that both treatments abolished the mobility shift of the p120ctn band (Fig. 7 G). The
range of the p120ctn-band shifting was similar to that found
in Colo 205 cells. In correlation with this p120ctn band
change, tight cell-cell association was induced in HT-29 cells preincubated for 18 h and then incubated with staurosporine (7 nM) for 6 h or with trypsin (0.001%) for 30 min
(Fig. 7 B, e and f), as seen in the case of Colo 205 cells.
On the other hand, MDCK, which is widely used as a
model of polarized epithelial cells, reorganized epithelial
sheets within 18 h after transfer. Addition of staurosporine
or trypsin to such MDCK cultures had no effects on cell-
cell association (Fig. 7 C), although we observed subtle
morphological alterations in the treated cultures. Analysis
of p120ctn in MDCK cells detected two major bands as reported by Mo and Reynolds (1996), the upper band likely
corresponds to the p120ctn band in human colon carcinoma
lines (Fig. 7 F). The p120ctn pattern in MDCK was not
changed by trypsin-mediated cell transfer. When MDCK
cells were treated with staurosporine, the electrophoretic mobility of p120ctn was slightly enhanced (Fig. 7 G), as described by Ratcliffe et al. (1997)
. However, this position
shift of the p120ctn band was subtle compared with that observed in the above carcinoma lines. Trypsin treatment of
MDCK cells did not affect the mobility of p120ctn (Fig. 7 G).
Next, we transfected the above cell lines with the NH2
terminus-truncated N346f, as well as with the control
FLf immediately after cell transfer, and examined the effects after 24 h.
N346f, but not FLf, enhanced HT-29
cell-cell association (Fig. 7 D).
N346f-expressing cells
formed compact aggregates, whereas FLf had no effects.
Rather, we observed that high-level overexpressions of
FLf tended to disperse epithelial sheets (data not shown). In MDCK cells, neither construct affected the cell-cell
contact morphology, they simply accumulated at cell-cell
contact sites (Fig. 7 E).
Induction of Colo 205 Aggregation by N-Cadherin Lacking the p120ctn-binding Site
These findings suggest that in particular cells such as Colo
205, p120ctn inhibits cadherin-mediated adhesion under
certain physiological conditions. If this is the case, cadherin activity might be restored under circumstances
where it cannot interact with p120ctn. To test this possibility, we transfected Colo 205 cells with two different constructs of N-cadherin cDNA, one encoding its entire portion and the other encoding a mutant molecule in which the juxtamembrane region of the cytoplasmic domain,
which is known to contain the p120ctn-binding site (Yap et al.,
1998), has been deleted (Fig. 8 A). Immunoprecipitation
with anti-N-cadherin antibodies from the transfected cells
confirmed that the mutant N-cadherin was unable to associate with p120ctn (Fig. 8 B), but coprecipitated normally
with
-catenin. Morphological and immunostaining examinations of these transfectants showed that the expression
of full-length N-cadherin had no effect on cell-cell adhesion, suggesting that cadherins were generally blocked in
the Colo 205 environment, irrespective of their type. In
contrast, the mutant N-cadherin induced a strong aggregation of Colo 205 cells (Fig. 8 C). These findings suggest
that cadherins can function normally in these cells unless
they interact with p120ctn, supporting the idea that p120ctn
can act as an inhibitor of cadherin function.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This work was initiated to understand why Colo 205 cells
cannot undergo typical E-cadherin-dependent association despite their normal expression of E-cadherin and associated proteins. We found that the treatment of these
cells with two independent reagents, trypsin and staurosporine, could reactivate the E-cadherin adhesion system.
Although the initial mechanisms for their actions remain unknown, we assume that trypsin removes or truncates
some cell surface proteins involved in intracellular signaling, leading to an activation or suppression of their downstream cascade and via this pathway, modulates the cadherin system. The response of Colo 205 cells to the trypsin
treatment was rapid, suggesting that the proposed signaling system probably does not require transcription of new
genes. As another possible mechanism of the trypsin action, we considered that this enzyme might have facilitated
cell adhesion by digesting such surface components as mucins, known to physically prevent cell-cell contacts. However, this possibility is less likely, because we could induce
compact cell aggregation by intracellular manipulation of
Colo 205 cells without enzymatic treatment. Moreover, no
correlation was found between the distribution of MUC1,
a major Colo 205 mucin, and adhesion induction. Concerning staurosporine, its action could be connected with
the proposed signaling cascade directly or indirectly. Of
the many kinase inhibitors tested, only staurosporine effectively induced Colo 205 cell adhesion within a short incubation period. This suggests that staurosporine has a
specific target in its action on Colo 205 adhesion, although we cannot specify it because this antibiotic can inhibit multiple classes of enzymes. It was reported that retinoic acid
treatment results in a similar adhesion induction (Nakagawa et al., 1998), suggesting that this reagent is another
effector to activate the cadherin system.
Since the initial or intermediate steps of the proposed
signaling system could be complex, we focused our analysis on its putative terminal step, asking if the cadherin-
catenin complex per se was modified during the adhesion
induction, and we identified an alteration in the electrophoretic mobility of p120ctn. This change could be triggered in the absence of E-cadherin-mediated adhesion,
implying that it resulted directly from the trypsin or staurosporine-triggered signaling cascade. A trypsin/staurosporine-sensitive mobility shift of p120ctn was also observed with another carcinoma line HT-29. Although transient in these cells, the shift nonetheless correlated
perfectly with adhesion induction. In contrast, MDCK
cells neither showed such modulation of p120ctn, nor responded to the adhesion-inducing reagents. Although
staurosporine treatment slightly enhanced the electrophoretic mobility of p120ctn, even in MDCK cells (Ratcliffe et al., 1997), the magnitude of the band shifting in
this case was much smaller as compared with that for Colo
205 and HT-29 cells.
These findings prompted us to further examine the role for p120ctn by structure-function analysis, and we found that NH2-terminally truncated p120ctn constructs by themselves could reactivate the E-cadherin system. This observation implies that p120ctn inhibits cadherin-mediated adhesion in Colo 205 cells. Thus, we postulate that the NH2-terminally deleted p120ctn lacks this activity, and competes with endogenous molecules, ultimately resulting in the reactivation of the cadherin system. This hypothesis is strongly supported by the finding that ectopic N-cadherin induced aggregation of Colo 205 cells only when it was unable to bind to p120ctn. This finding also suggests that different types of cadherin, in general, cannot function in the intracellular environment of Colo 205 if they bind to p120ctn.
The correlation between the band shifting of p120ctn and
adhesion induction suggests that the putative inhibitory
activity of this molecule might be elicited by certain biochemical modifications. In Colo 205 cells, the original
p120ctn band was broad. Although multiple splicing products of p120ctn are expressed in many carcinoma lines (Mo
and Reynolds, 1996), the broad band observed in Colo 205 cells appears to result from phosphorylation at various levels because phosphatase treatment of the Colo 205 p120ctn
resulted in the generation of a sharp single band. This
phosphatase-treated p120ctn was similar in electrophoretic
mobility to that found in the adhesion-induced Colo 205 cells. These findings suggest the possibility that p120ctn acquires the inhibitory activity when hyperphosphorylated,
and the staurosporine/trypsin-induced signals reduce the
level of phosphorylation. We found that the major phosphorylated residue in p120ctn of Colo 205 cells was serine,
consistent with a previous observation by Ratcliffe et al.
(1997)
. Our results do not exclude the possibility that
other residues are also phosphorylated at low levels, as it
has been reported that tyrosine is phosphorylated in
p120ctn of other cell lines (Kinch et al., 1995
; Calautti et al.,
1998
; Hazan and Norton, 1998
; Rosato et al., 1998
). The
net amount of serine phosphorylation in p120ctn did not
appear reduced after its electrophoretic mobility shift, although phosphoamino acid analysis may not adequately
detect small changes in phosphorylation status. If dephosphorylation is indeed involved in this process, one possibility is that only a specific subset of phosphorylation
sites are required for the p120ctn change in motility. The
NH2-terminally deleted p120ctn, which induced cell adhesion, still contained slower-migrating, phosphatase-sensitive components, although their proportion tended to be
reduced. Possibly, the key phosphorylation sites directly affecting the electrophoretic mobility of p120ctn are located outside the NH2-terminal region. These observations also suggest that hyperphosphorylation may be insufficient to confer the adhesion inhibitory activity on p120ctn
if its NH2 terminus is absent. A model for the signaling
cascade to regulate p120ctn phosphorylation has been proposed by Ratcliffe et al. (1997)
.
How does p120ctn inhibit cadherin function? It is interesting to note that the juxtamembrane domain of the cadherin cytoplasmic region where p120ctn binds can inhibit
cell adhesion when overexpressed (Kintner, 1992), and
also is required for clustering of cadherin molecules (Yap
et al., 1998
) which is thought to be essential for cadherin-mediated cell adhesion. Another report shows that deletion of a similar region in E-cadherin resulted in activation
of this adhesion molecule, enhancing its lateral interactions in the cell membrane of a leukemia line (Ozawa and
Kemler, 1998b
), suggesting that this domain can inhibit
lateral clustering of cadherins. p120ctn is the only molecule
known to bind to the juxtamembrane domain of cadherins
and could be the major effector of these activities. For example, p120ctn may directly regulate lateral clustering of
cadherins and this activity may be blocked by hyperphosphorylation or other mechanisms controlled by the NH2
terminus. In support of this hypothesis, our immunostaining observations suggest that clustering of E-cadherin into
cell-cell contact sites is enhanced by the adhesion-induction treatments. Alternatively, p120ctn may have no function in the default state and might serve as an inhibitor of
cadherin function only when posttranscriptionally modified. The results of our N-cadherin experiments, as well as the above leukemia study (Ozawa and Kemler, 1998b
), indicate that cadherins can be active without p120ctn.
We have demonstrated a p120ctn-dependent inhibition
of the cadherin system in carcinoma cells. It is known that
cadherin-mediated adhesion is perturbed in tumors in a
variety of ways, including mutation of cadherin or catenin
genes, and these events have been implicated in cancer invasion and metastasis (reviewed by Shiozaki et al., 1996).
Here, we have identified a novel physiological mechanism
of inhibiting cadherin function. Many metastasizing tumors maintain strong cadherin and catenin expression.
Such tumor cells must have a mechanism to destabilize
cell-cell adhesion, at least transiently, for detaching from
the original tumors. Biochemical modification of p120ctn
could be used for such processes. Colo 205 cells may be an
extreme case in which the p120ctn-dependent inhibitory
mechanism is constitutively turned on, whereas in HT-29 it
operates only transiently, as these cells can eventually form normal looking epithelial sheets. The mechanism for
the transient modification of p120ctn in HT-29 remains unknown.
Finally, the question arises as to how such an activity of
p120ctn is involved in normal cellular events. Cell-cell adhesion is dynamic and requires regulation during many
types of morphogenetic processes. p120ctn could be implicated in such regulation as suggested by previous observations. The juxtamembrane domain of the cadherin cytoplasmic domain to which p120ctn binds was implicated in
cell motility (Chen et al., 1997) and in axon outgrowth
(Riehl et al., 1996
). Overexpression of p120ctn in Xenopus
embryos perturbs gastrulation (Geis et al., 1998
; Paulsen
et al., 1999). p120ctn, whose action can be modulated posttranscriptionally, could play a role in the control of such
adhesion-related cellular behavior. As we suggested in
light of the effect of trypsin, there might be a signaling cascade originating at the cell surface to regulate the p120ctn
activity. Identification of components of such cascades is
an important future issue for unraveling the morphogenetic regulatory mechanisms of cell-cell adhesion.
![]() |
Footnotes |
---|
Address correspondence to Masatoshi Takeichi, Department of Biophysics, Faculty of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. Tel.: 81-75-753-4196. Fax: 81-75-753-4197. E-mail: takeichi{at}take.biophys.kyoto-u.ac.jp
Received for publication 5 February 1999 and in revised form 23 March 1999.
Shinichi Nakagawa's current address is Department of Anatomy, Downing Street, University of Cambridge, Cambridge CB2 3DY, United Kingdom.
We thank T. Moriguchi, Y. Gotoh, and E. Nishida (Kyoto University) for advice and technical support on phosphoamino acid analysis. We also thank M.J. Wheelock (University of Toledo) for 5H10 antibody, T. Irimura (University of Tokyo) for anti-MUC1 antibody, K. Moriyoshi (Kyoto University) for recombinant adenoviruses, and T. Tamaoki (Kyowa Medex) for UCN-01.
This work was supported by the program Grants-in-Aid for Creative Fundamental Research, and for Specially Promoted Research of the Ministry of Education, Science, Sports, and Culture of Japan.
![]() |
Abbreviations used in this paper |
---|
aa, amino acid; FLf, full-length FLAG-tagged p120ctn.
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