From the ¶ Unitat de Biologia Cel.lular i Molecular, Institut
Municipal d'Investigació Mèdica, Universitat Pompeu Fabra,
c/Dr. Aiguader 80, 08003 Barcelona, Spain and Unitat de
Biofísica, Departament de Bioquímica i Biologia
Molecular, Facultat de Medicina, Universitat Autònoma de
Barcelona, 08193 Bellaterra, Spain
Received for publication, January 9, 2001, and in revised form, March 8, 2001
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ABSTRACT |
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In addition to its structural role in cellular junctions, The domains of As mentioned above, phosphorylation of Expression of Recombinant Proteins--
Expression and
purification of full-length Transient Transfections and Analysis of
Transfectants--
Assays were performed in SW-480 cell line, which
although it contains high levels of Analysis of Protease Sensitivity of -Catenin plays a dual role as a key effector
in the regulation of adherens junctions and as a transcriptional
coactivator. Phosphorylation of Tyr-654, a residue placed in the last
armadillo repeat of
-catenin, decreases its binding to E-cadherin.
We show here that phosphorylation of Tyr-654 also stimulates the
association of
-catenin to the basal transcription factor
TATA-binding protein. The structural bases of these different
affinities were investigated. Our results indicate that the
-catenin
C-terminal tail interacts with the armadillo repeat domain, hindering
the association of the armadillo region to the TATA-binding protein or
to E-cadherin. Phosphorylation of
-catenin Tyr-654 decreases
armadillo-C-terminal tail association, uncovering the last armadillo
repeats. In a C-terminal-depleted
-catenin, the presence of a
negative charge at Tyr-654 does not affect the interaction of the
TATA-binding protein to the armadillo domain. However, in the case of
E-cadherin, the establishment of ion pairs dominates its association
with
-catenin, and its binding is greatly dependent on the absence of a negative charge at Tyr-654. Thus, phosphorylation of Tyr-654 blocks the Ecadherin-
-catenin interaction, even though the
steric hindrance of the C-tail is no longer present. These results
explain how phosphorylation of
-catenin in Tyr-654 modifies the
tertiary structure of this protein and the interaction with its
different partners.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Catenin was initially described as a protein involved in the
regulation of E-cadherin function, since it binds to the cytoplasmic domain of this protein and is necessary for linkage of E-cadherin to
the actin cytoskeleton (1). Sequences involved in E-cadherin and
-catenin binding have been identified in
-catenin; association of
E-cadherin requires armadillo repeats 4-12 situated in the central
part of
-catenin (2). On the other hand,
-catenin binding is
limited to a short 31-amino acid sequence in the first armadillo repeat
of
-catenin (3). It has been proposed that the interactions of
-catenin with these two proteins are regulated by tyrosine
phosphorylation (4, 5). In the case of E-cadherin, we have recently
demonstrated that phosphorylation of tyrosine residue 654 diminishes
the association of
-catenin to this protein by a factor of 10 (6).
This residue is modified in vivo by effectors that
concomitantly decrease
-catenin-E-cadherin binding (6). On the other
hand, there is no direct evidence so far that modification of any Tyr
residue on
-catenin inhibits its interaction with
-catenin.
-catenin
is a critical component of the wnt-signaling pathway that governs cell fate in early embryogenesis (7, 8). Activation of this
pathway induces the stabilization of free
-catenin, its translocation to the nucleus, and its binding to members of the LEF-1/TCF family of transcription factors (7, 8). Interaction of
-catenin with these factors converts them to transcriptional activators (9) and stimulates the expression of several genes containing Tcf-4-responsive sequences in their promoter (10-14). In
the absence of wnt stimulus, cytosolic
-catenin is
degraded through a mechanism requiring its binding to the tumor
suppressor gene product adenomatous polyposis coli (8). The exact role of adenomatous polyposis coli in the regulation of
-catenin levels has not been perfectly explained, although it is thought to facilitate the formation of a complex between
-catenin and axin/axil, glycogen synthase 3-B, and
-TCRP/slimb (8).
-catenin involved in transcriptional activation have
been localized in the N- and C-terminal parts of this molecule (15,
16). The C-terminal tail of
-catenin, when fused to LEF-1, has been
shown to be sufficient to promote transactivation (15). Although the
mechanism underlying this activation is not totally known, the N- and
C-terminal transactivation domains of
-catenin interact with a
growing list of nuclear factors that include the TATA-binding protein
(TBP)1 (16), Pontin (17),
Teashirt (18), Sox17 and 13 (19), histone deacetylase (20), SMAD4 (21),
the retinoic acid receptor (22), and the CREB binding protein and
related proteins (23-26). One of the essential roles of
-catenin-Tcf-4 complex consists in recruiting the basal
transcriptional machinery to the promoters of wnt-sensitive
genes. A key component of this transcriptional complex is TBP, which
interacts with two different domains of
-catenin necessary for
transactivation (16).
-catenin Tyr-654 severs
-catenin-E-cadherin binding (6). Since this residue is located in a
domain involved in TBP binding (16), we have investigated the possible
role of this phosphorylation in the interaction between
-catenin and
TBP.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin, fragments 1-106 and 696-end,
and
-catenin point mutants Tyr-86
Glu, Tyr-86
Phe, Tyr-654
Glu and Tyr-654
Phe have been previously described (6). A DNA
fragment corresponding to the complete 12 armadillo repeats (amino
acids 138-683) was amplified from entire
-catenin cDNA by
polymerase chain reaction using oligonucleotides corresponding to
nucleotide sequences 358-372 and 2035-2047. The 1.7-kilobase
amplification fragment was inserted in the
BamHI-SmaI sites of a pGEX-6P-1 plasmid and
expressed in Escherichia coli as a glutathione
S-transferase (GST) fusion protein. Armadillo fragments
comprising repeats 7-12 (amino acids 422-683) and 10-12 (amino acids
575-696) were generated cutting the entire armadillo domain cDNA
with EcoRI-EcoRV or
EcoICRI-EcoRV and inserting in pGEX
6P-2 digested with EcoRI-SmaI or pGEX 6P-3
digested with SmaI. The
-catenin deletion mutants used in
this study are presented in Fig. 1, indicating which part of the
molecule they comprise. The 1-80-amino acid fragment of Tcf-4 was
generated from pcDNA3-hTcf-4 cutting with BamHI and
SmaI and inserting in pGEX-6P-1 plasmid. Phosphorylation of
-catenin mutant forms by recombinant pp60c-src
protein kinase (from Upstate Biotechnology, Inc.) was performed as
described (6). To avoid a possible interference of this kinase in the
binding assay, once phosphorylated the GST-
-catenin protein was
purified by chromatography on glutathione-Sepharose 4B as indicated below.
-Catenin Binding Assays--
The indicated amounts of
-catenin proteins or the 12 armadillo repeats were incubated with
different concentrations of N- and C-terminal-GST-
-catenin tails (or
GST as a control) at ratios from 1:1 to 5:1 (GST protein
versus
-catenin) for 30 min at 20 °C. Incubations were
performed in binding buffer: 50 mM Tris-HCl, pH 7.3, 150 mM NaCl, 3 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and 0.1% (w/v)
Triton X-100 in a final volume of 200 µl. In some experiments binding
to the cytosolic domain of E-cadherin (cytoE-cadh) or to the Tcf-4
-catenin binding domain (Tcf-4-(1-80)) was performed in these same
conditions. Protein complexes were isolated by incubation with 40 µl
of a 50% (w/v) suspension of glutathione-Sepharose 4B for 30 min at
20 °C. Beads were collected by spinning in a microcentrifuge and
washed three times with binding buffer. Samples were separated by
SDS-polyacrylamide gel electrophoresis, and the presence of bound
proteins in the complex was analyzed by Western blot with specific
monoclonal antibodies (mAbs) against
-catenin C terminus
(Transduction Laboratories, Lexington, KY),
-catenin armadillo core
(Alexis Biochemicals, San Diego, CA), E-cadherin cytosolic domain
(Transduction Labs), or Tcf-4 N terminus (Santa Cruz Biotechnology).
Lysate pull-down assays were performed incubating 12 pmol of GST or
GST-
-catenin with 50 µg of SW-480 total cell extract in the
conditions mentioned above. Samples were purified by
glutathione-Sepharose chromatography and the presence of TBP or Tcf-4
in the complex was determined by Western blot with specific mAbs from
Transduction Laboratories and Santa Cruz Biotechnology, respectively.
Immunoblots were developed with peroxidase-conjugated secondary
antibody followed by enhanced chemiluminiscence detection system (ECL,
Pierce). The autoradiograms were scanned, and the values obtained were
either compared with known amounts of recombinant proteins included as
reference (
-catenin binding assays) or with the value obtained for
wild-type full-length
-catenin (pull-down assays).
-catenin (like most intestinal
epithelial cells), it is deficient in E-cadherin (27). Absence of
E-cadherin precludes that the observed differences in TBP binding by
the different
-catenin mutants could be attributed to impaired
transport to the nucleus due to a distinct association to E-cadherin.
Cells were grown in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% fetal calf serum (Life
Technologies). When 80% confluent, cells were transfected with the
indicated plasmids using LipofectAMINE (Life Technologies) according to the instructions of the manufacturer. After transfection, cells were
incubated for 48 h in Dulbecco's modified Eagle's medium plus
10% fetal calf serum. Cell extracts were prepared in radioimmune precipitation buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% sodium deoxycholate, 1% Nonidet P-40, 0.1%
SDS, 1 mM EDTA) supplemented with 10 µg/ml aprotinin, 20 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and
0.25 mM Na3VaO4. Lysates were
centrifuged at 13,000 rpm in a microcentrifuge for 5 min at 4 °C.
250 µg of extract were incubated in a final volume of 0.3 ml with 20 µl of a 50% (w/v) suspension of nickel nitrilotriacetic acid-agarose
(Qiagen, Hilden, Germany) for 30 min at 4 °C. Beads were washed with
radioimmune precipitation buffer, and bound proteins were eluted with
electrophoresis sample buffer. Samples were separated by 10%
SDS-polyacrylamide gel electrophoresis and analyzed by Western blot. To
reprove the membranes, blots were stripped as described (28). The
absence of signal after stripping was always checked by incubating with the correspondent secondary antibody and ECL reagent.
-Catenin-mediated Transcriptional
Activity--
-Catenin-mediated transcription was performed
transfecting NIH-3T3 fibroblasts, SW-480 cells, or E-cadherin-deficient
MiaPaca-2 pancreas cells with a plasmid containing three copies of the
Tcf-4 binding site upstream a firefly luciferase reporter gene (plasmid TOP-FLASH) as described (29). The activity of the product of the
Renilla luciferase gene under the control of a constitutive thymidine kinase promoter (Promega) was used as control. Assays were
always performed in triplicate; the average of the results of 3-4
independent transfections ± S.D. is given.
-Catenin--
1 µg of the different
forms of
-catenin, phosphorylated or not by
pp60c-src, were incubated in the presence of
trypsin (60 ng) at 24 °C in a final volume of 100 µl in a buffer
containing 90 mM Tris-HCl, pH 8.5, 2 mM
CaCl2, and 4 mM dithiothreitol. Reactions were
stopped at different digestion times from 1 to 90 min with
electrophoresis loading buffer and boiled for 4 min. The extent of the
digestion was determined analyzing the samples by SDS-polyacrylamide
gel electrophoresis and Western blot with a mAb anti-
-catenin C
terminus, which recognizes an epitope situated between amino acids 696 and 781 of this protein. A quantitation of the reaction was performed scanning the autoradiograms and representing the amount of full-length
-catenin at the different times of incubation relative to the initial time.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Catenin is a good substrate of pp60c-src
tyrosine kinase in vitro; this kinase modifies specifically
Tyr-86 and Tyr-654, located in the N-terminal domain and in the last
armadillo repeat of
-catenin, respectively (6) (see Fig.
1). Although Tyr-86 is phosphorylated with a higher stoichiometry, only modification of Tyr-654 alters the
interaction of
-catenin with E-cadherin. Since Tyr-654 is located in
the domain of interaction with TBP, we examined whether tyrosine
phosphorylation of
-catenin influences the association with this
factor. As shown in Fig. 2,
phosphorylation of
-catenin by pp60c-src greatly
increased its interaction with TBP in pull-down assays (by 6-fold). To
analyze the specific influence of Tyr-654 phosphorylation,
-catenin
mutants were used in which Tyr-86 and Tyr-654 were replaced by Phe. The
same amounts of pulled down TBP were obtained when phosphorylated
wild-type
-catenin or phosphorylated Tyr-86
Phe mutant were used
as bait (Fig. 2). In this case the amount of Tyr(P) incorporated to the
-catenin form was greatly reduced, since only Tyr-654 was
phosphorylated (Fig. 2). On the other hand, binding of TBP to the
Tyr-654
Phe mutant was not increase after phosphorylation,
demonstrating that phosphorylation of this residue is involved in the
augmented interaction of TBP and
-catenin (Fig. 2).
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Fig. 1.
Diagram of
-catenin. The three different domains that
form this protein are shown. The 12 armadillo repeats of
-catenin
are represented with numbered boxes, and the two tyrosine
residues phosphorylated by pp60c-src are also
indicated. The deletion mutants used in this article are depicted,
indicating which parts of the molecule they comprise. wt,
wild type.
View larger version (48K):
[in a new window]
Fig. 2.
Phosphorylation of Tyr-654 enhances binding
of -catenin to TBP but not to Tcf-4. 11 pmol of GST or GST-
-catenin fusion proteins were phosphorylated by
pp60c-src in the conditions indicated under
"Experimental Procedures." Pull-down assays were performed
incubating the GST proteins with 50 µg of total cell extracts
prepared from SW-480 cells. Protein complexes were pelleted down by
affinity on glutathione-Sepharose beads, and proteins bound to the
complex were analyzed by SDS- polyacrylamide gel electrophoresis and
Western blot with anti-TBP mAb. Membranes were stripped and re-analyzed
with mAb against Tyr(P),
-catenin, or Tcf-4. wt,
wild-type
-catenin; Y86F, Y654F, Y86E, and Y654E correspond to
-catenin mutants Tyr-86
Phe, Tyr-654
Phe, Tyr-86
Glu,
and Tyr-654
Glu, respectively. The estimated molecular weights of
the bands detected with each antibody are shown. The autoradiograms
were scanned in a densitometer, and the results obtained
(numbers below the lanes) are presented relative
to the value obtained for wild-type
-catenin (or phosphorylated
wild-type
-catenin in the case of the analysis with Tyr(P) mAb).
Only the upper band in the analysis of
-catenin was employed for
this analysis; the lower band corresponds to a degradation product of
this protein occasionally observed in our preparations that does not
interfere in the assay.
To confirm these results, binding of TBP to -catenin mutants Tyr-86
Glu and Tyr-654
Glu was determined. These forms were generated
to mimic the effect of phosphorylation in these two residues.
-Catenin Tyr-654
Glu interacted much better with TBP than the
wild-type form; the amount of pulled down TBP was eight times greater
(Fig. 2). Therefore, it could be demonstrated that the introduction of
a negative charge in Tyr-654 enhances
-catenin binding to TBP.
We also noticed that phosphorylation of Tyr-86 exerted an opposite
effect on -catenin association to TBP.
-Catenin Tyr-86
Glu
consistently pulled down a lower amount of TBP than wild-type
-catenin (approximately a 30% less); phosphorylation of Tyr-86 in
-catenin Tyr-654
Phe exerted a similar action (Fig. 2). Although
consistently detected, this negative effect of Tyr-86 phosphorylation
on TBP binding was clearly less important than the positive effect
observed after Tyr-654 phosphorylation. Probably for this reason, no
significant differences were observed in the interaction of TBP to the
Tyr-654
Glu mutant or to the double mutant Tyr-86
Glu/Tyr-654
Glu (data not shown).
The effects of -catenin phosphorylation on its association to a well
known co-factor, Tcf-4, were determined. No differences in the amount
of this protein pulled down by GST-
-catenin were observed after
phosphorylation of this molecule or when
-catenin Tyr-86
Glu and
Tyr-654
Glu mutants were analyzed (Fig. 2). The same results were
obtained when in vitro binding of recombinant
-catenin
and Tcf-4 was determined (not shown).
The in vivo association between -catenin and TBP was also
investigated. SW-480 cells were chosen for these assays because they
contain very little E-cadherin, and most of the
-catenin is not
retained in the membrane by this molecule. Cells were transfected with
wild type or Tyr-654
Glu
-catenin labeled with polyhistidine and
the X-Press® tag to facilitate their purification and identification. Transfected forms were purified by Ni2+-agarose, and the
amount of associated TBP was determined. As shown in Fig.
3A, TBP associated in
vivo better with
-catenin mutant Tyr-654
Glu than with the
wild-type form (2.5-fold better). This higher association correlated
with a greater stimulation of
-catenin-Tcf-4-mediated transcription.
Overexpression of wild-type
-catenin in SW-480 cells induced a
significant increase (60% stimulation) in the activity of a reporter
gene placed under the control of a
-catenin- and Tcf-4-sensitive
promoter (TOP plasmid) (30). Expression of Tyr-654
Glu
-catenin
mutant raised the activity of this promoter to a higher extent (194%
stimulation) (Fig. 3B). Similar stimulations of TOP activity
were obtained in other cell lines (Fig. 3B).
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Our results indicate that phosphorylation of -catenin Tyr-654
regulates not only the interaction with E-cadherin but with TBP as
well. The structural basis of these differences was investigated. Three
different regions can be distinguished in
-catenin with distinct
charge distributions: the N- and C-terminal tails, with pIs close to
4.5, and the armadillo repeat domain, which presents a basic pI of 8.3 (31). It has been proposed that
-catenin C-terminal region directly
interacts with the armadillo domain (32-34). The association between
the complete armadillo domain (amino acids 138-683) and the N- and
C-terminal regions of
-catenin was studied using binding assays with
recombinant proteins. Both N-tail (amino acids 1-106) and C-tail
(amino acids 696-end) interacted with the armadillo
domain. Binding of the C terminus to the
armadillo domain requires sequences upstream of the last six armadillo
repeats, since a recombinant protein comprising only repeats 7-12
(amino acids 422-683) associated to the C-terminal tail much worse
than the complete armadillo domain (Fig. 4B). A possible
effect of phosphorylation of
-catenin Tyr-654, located in the last
armadillo repeat, on armadillo-C-tail association was studied.
Phosphorylation of this residue decreased armadillo-C-tail interaction
(Fig. 4A) but did not modify the binding of the armadillo
domain with the N-tail (Fig. 4C). Consequently,
-catenin
C-terminal tail also interacted better with the wild-type armadillo
domain than with an armadillo form containing the Tyr-654
Glu
mutation (Fig. 4A).
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These results suggest that, in its native conformation, -catenin is
folded with its C-tail interacting with the armadillo repeats.
Phosphorylation of Tyr-654 disrupts this interaction and releases the
C-terminal tail. To prove this model, experiments of limited trypsin
proteolysis of
-catenin were performed, and the extent of unfolding
of the C-tail was followed by measuring the rate of disappearance of
-catenin reactivity using an antibody that recognizes only the
intact C-tail. As shown in Fig. 5, the
-catenin mutant Tyr-654
Glu presented a higher susceptibility to
proteolysis than the wild-type form. A faster degradation of the
wild-type protein was also observed when it was phosphorylated by
pp60c-src. In this case, differences in sensitivity
to trypsin proteolysis were less evident, probably due to the
incomplete phosphorylation of Tyr-654 in our conditions (6).
Phosphorylation of either wild type or Tyr-86
Phe mutant produced
the same patterns of trypsin digestion (not shown), discarding possible
effects due to phosphorylation of Tyr-86 in our assay.
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We have also analyzed whether binding of the armadillo repeat domain to
the C-tail affected the interaction of -catenin with E-cadherin or
Tcf-4. Interaction of the armadillo repeats with the cytosolic domain
of E-cadherin (cytoE-cadh) was disrupted by the addition of the C-tail
(696-end), indicating that both protein domains interact within the
same region of the armadillo domain (Fig.
6A). On the contrary, the
addition of
-catenin N-tail (1) did not modify
armadillo-cytoE-cadh association (Fig. 6A). It is remarkable
that the armadillo domain bound cytoE-cadh significantly better than
full-length
-catenin (Fig. 6A), supporting the conclusion that removal of the C-tail facilitates the interaction with cytoE-cadh. On the other hand, binding of the armadillo domain to a Tcf-4 fragment containing the
-catenin-binding site was not modified by
the addition of both
-catenin terminal tails (Fig.
6B).
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The binding site for TBP to the -catenin C-terminal domain has been
ascribed to amino acids 630-729, with residues 630-675 contributing
critically to this association (16). In our hands, TBP binds uniquely
to the armadillo domain (amino acids 138 to 683) and not to the C-tail
(amino acids 696-end) (Fig.
7A). As in the case of
cytoE-cadh,
-catenin armadillo domain also bound TBP significantly
better than full-length
-catenin (Fig. 7A), indicating
that the C-tail restricted the interaction with TBP. The association of
TBP to the 12 armadillo repeats was also competed by preincubation with
-catenin C-tail (Fig. 7A) but not with Tcf-4 (data not
shown).
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At this point, we also considered the possibility that phosphorylation
of Tyr-654 might be inducing alterations in TBP binding independent of
the presence of the C-tail. As shown in Fig. 7A, this is not
the case; either the wild-type armadillo domain as well as the
phosphorylated form of this protein or the Tyr-654 Glu mutant
pulled down similar amounts of TBP. Thus, these results indicate
that changes in TBP binding upon phosphorylation of Tyr-654 are
basically due to the release of
-catenin C-terminal tail from the
armadillo domain, allowing a better interaction of the last armadillo
repeats with TBP.
Our results on the binding of the armadillo domain to TBP and to
E-cadherin differ in their sensitivity to tyrosine phosphorylation. As
shown in Fig. 6 and 7A, whereas phosphorylation of Tyr-654 decreases binding of E-cadherin, it does not modify the interaction of
TBP to the armadillo domain. This result suggests that both proteins
interact with this domain in a different way. One possibility is that
TBP is not binding through an interaction based in the establishment of
ion pairs, as it has been proposed for E-cadherin. Another possibility
is that both proteins interact with different surfaces of the armadillo
domain. To explore these possibilities, binding of TBP to full-length
-catenin or to the armadillo domain was performed in the presence of
an excess of cytoE-cadh. As shown in Fig. 7B, the addition
of a 10-fold molar excess of cytoE-cadh did not modify the amount of
TBP bound to the armadillo domain, whereas it increased the amount of
TBP pulled down by full-length
-catenin. This result suggests that,
although TBP and E-cadherin interact with overlapping armadillo
repeats, both proteins bind to different faces of
-catenin.
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DISCUSSION |
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-Catenin has been shown to act both as a regulator of
E-cadherin-dependent cell-to-cell adhesion and as an
essential mediator in the wnt-signaling pathway (8, 35).
Experimental data indicate that the presence of
-catenin in the
cellular junctions is controlled by tyrosine phosphorylation (5,
36-40). We have previously demonstrated that phosphorylation of
Tyr-654, a residue located in the 12th and last armadillo repeat of
-catenin, modifies the association of this protein to E-cadherin
(6). The armadillo repeat domain has been shown to be essential for the
binding of
-catenin to its many binding partners, as E-cadherin and
the transcription factor Tcf-4. However, binding of both proteins does
not show the same requirements; whereas Tcf-4 associates mainly to
repeats 3-8 (41), E-cadherin requires the last 8 repeats (2, 9, and 42). Therefore, it makes sense that, as we show in this article (Fig.
2), modification of a residue placed at the 12th armadillo repeat does
not affect Tcf-4 binding.
Armadillo repeat 12 has also been characterized as part of the C
terminus-transactivating element required for activation of gene
expression (16). Our results indicate that phosphorylation of
-catenin Tyr-654 increases binding of this protein to TBP both
in vitro and in vivo, and this greater
association correlates with a higher stimulation of Tcf-4-
-catenin
transcriptional activity. This higher stimulation of Tcf-4
transcriptional activity observed in vivo by
-catenin
Tyr-654
Glu mutant is not a consequence of its impaired association
to E-cadherin, since it is observed in cells that present very low
levels of E-cadherin. In any case, our data suggest that
phosphorylation of Tyr-654 is relevant not only for disruption of
-catenin-E-cadherin binding but for stimulation of the interaction
of
-catenin to the basal transcriptional machinery as well. These
results are consistent with the fact that the nonjunctional pool of
-catenin is preferentially phosphorylated on tyrosine (37).
According to our results, phosphorylation of Tyr-654 affects binding of
-catenin to TBP by releasing the restriction created by the C-tail.
This restriction is evidenced by the fact that the armadillo domain
interacts better with TBP than the complete
-catenin and also by the
inhibitory effect of the C-tail on the binding of TBP to the armadillo
domain. These data have suggested a working model, presented in Fig.
8, which proposes that, when not
phosphorylated and not bound to any ligand,
-catenin would adopt a
folded conformation in which the C-terminal tail and the N-tail
interact with the armadillo repeat domain. This conformation would
prevent the binding to armadillo repeats of low affinity ligands
and would select those (such as E-cadherin) presenting high association
constants. Phosphorylation of tyrosine residue 654 would remove the
C-tail and allow a better access of TBP to the last armadillo repeats.
As indicated, association of Tcf-4, which takes place mainly through
armadillo repeats 3-8, would not be affected by the C-tail.
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Thus, the presence or absence of a negative charge at Tyr-654 would act
as a key for opening or closing -catenin and would affect the
association of this protein to factors binding to the last armadillo
repeats and, possibly, to the C-tail as well. In some cases, as for TBP
(Fig. 7), after removal of the C-tail the presence or not of a
phosphate in Tyr-654 does not modify the interaction of proteins with
the armadillo domain. However, in other cases the introduction of a
negative charge at this position might hamper the binding of
-catenin with factors like E-cadherin that interact mainly by
charge complementarity (31) (see Fig. 6). Thus, for these proteins the
negative effect caused on the establishment of ion pairs would
predominate over the removing of the steric interference caused by the
C-tail.
We have also demonstrated that, although E-cadherin and TBP associate
to the same armadillo repeats, they do not appear to compete directly
for binding to the armadillo domain. As mentioned before, these results
suggest that E-cadherin and TBP are interacting with different
surfaces of this domain, as depicted in Fig. 8. The interference of the
C-tail on the binding of these two proteins indicates that the C-tail
can interact with, and possibly hide, both binding surfaces.
Accordingly, interaction of E-cadherin with -catenin displaces the
C-tail from its binding to the armadillo domain and facilitates the
interaction of factors as TBP that associates to the other side of this
domain (see Fig. 8). Although a simultaneous interaction of E-cadherin
and TBP with
-catenin is evidently not physiological (E-cadherin and
TBP are localized in different cellular compartments), it is possible
that a similar role to E-cadherin might be played by other factors
interacting with the same binding surface.
Nevertheless, a definitive validation of our model of -catenin
regulation by tyrosine phosphorylation would require the
determination of the complete structure of this molecule and the
characterization of the effect of the two terminal tails in the
interaction of
-catenin with its numerous protein partners (35).
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ACKNOWLEDGEMENTS |
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We thank Santiago Roura, Josep Baulida, and Esteve Padrós for their help at different stages of the study and Drs. E. Batlle, R. Kemler, and H. Clevers for reagents.
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FOOTNOTES |
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* This work was supported by La Marató de TV3 Grant 983110 (to A. G. H.), Ministerio de Ciencia y Tecnología Grant PM99-0064 (to M. D.), FEDER-Fondo Nacional I+D Fund Grants 2FD97-1491-C02-01 and 2FD97-1491-C02-02 (to A. G. H. and M. D., respectively), and Direcció General de Recerca Grants 1999SGR00245 and 1999SGR00102.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipients of predoctoral fellowships awarded by Ministerio de Educación y Ciencia and CIRIT (Generalitat de Catalunya), respectively.
To whom correspondence should be addressed. Tel.:
34-93-581-1870; Fax: 34-93-581-1907; E-mail: mireia.dunach@uab.es (for
M. Duñach) or Tel.: 34-93-221-1009; Fax: 34-93-221-3237; E-mail: agarcia@imim.es (for A. Garcia de Herreros)
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M100194200
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ABBREVIATIONS |
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The abbreviations used are: TBP, TATA-binding protein; GST, glutathione S-transferase; cytoE-cadh, cytosolic domain of E-cadherin; mAb, monoclonal antibody; Tyr(P), phosphotyrosine.
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