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INTRODUCTION |
Wnt-1 was originally identified as a proto-oncogene in mice (1)
and was found to be a vertebrate homolog of the Drosophila segment polarity gene wingless. To date at least 15 additional vertebrate homologs have been identified (for review see
Ref. 2). Members of the Wnt family of extracellular glycoproteins are
able to activate at least two different signaling pathways in
vertebrates (reviewed in Refs. 3 and 4). The canonical Wnt pathway
is involved in different developmental processes such as cell fate
specification and cell migration. The major cytoplasmic effector of
this pathway,
-catenin, accumulates in the cytoplasm in response to
a Wnt signal. Subsequently,
-catenin can enter the nucleus where it
binds to the N terminus of
TCF/LEF1 transcription
factors to function as a transcriptional coactivator. Deregulation of
the Wnt/
-catenin pathway leads to carcinogenesis. Mutations
increasing the stability and thus the cytoplasmic/nuclear pool of
-catenin were found in colon carcinomas and malignant melanomas (4). The identification of c-myc as a target gene of TCF-4/
-catenin links Wnt signaling with cell proliferation and
thus with carcinogenesis (5). Another important target gene of TCF/LEF
in this context leading to cell cycle deregulation was identified as
cyclin D1 (6).
Members of the TCF/LEF family were originally identified as T- and
B-lymphocyte-specific transcription factors. Sequence-specific DNA
binding is mediated by the HMG box, and the target motif of these
factors is given by the sequence (C/G)TTTG(A/T)(A/T) (7). Members of
this family are unable to activate transcription of reporter gene
constructs that carry multiple copies of their minimal binding motifs
(8) and thus lack activation properties on their own. This raises the
question how these proteins work as transcriptional regulators. One
well characterized target gene regulated by LEF-1 is the T-cell
receptor
gene, TCR
. Activation of the TCR
promoter by LEF-1
is strictly context-dependent. Under these circumstances LEF-1 functions as an architectural transcription factor by bending DNA, thus facilitating stable interactions of other known transcription factors like CREB/ATF, PEBP2
, and Ets-1 (9).
In addition, LEF-1 interacts with proteins thought to function as
transcriptional activators like ALY (10) and SMADs (11, 12). More
recently, a functional interaction of LEF-1 as well as XTCF-3 with
-catenin has been demonstrated (13-15). As a major player in
canonical Wnt signaling,
-catenin has been shown to function as a
transcriptional coactivator directly binding TBP (16), pontin52 (17,
18), and p300/CBP (19, 20). p300/CBP synergizes with
-catenin in
activation of target genes. A synergistic effect specific for
-catenin-mediated activation of Xtwin was also reported for SMAD4;
however, a direct binding between both
-catenin and SMAD4 remained
elusive (11).
Despite binding to the same target site, members of the TCF/LEF family
differ in their function. When overexpressed on the ventral side of
Xenopus embryos, mLEF-1 induces the formation of a secondary
body axis via activation of the Wnt target gene siamois (13,
14), whereas XTCF-3 fails in this assay (15). In epithelial A6 cells
LEF-1 activates the Wnt target gene fibronectin, whereas
XTCF-3 does not (21). Two hybrid studies demonstrated that unlike
LEF-1, XTCF-3 binds transcriptional corepressors of the groucho family
(22) as well as CtBP (23). This led to the model that XTCF-3 functions
as a transcriptional repressor in the absence of
-catenin by
recruiting corepressors, whereas LEF-1 is unable to function as a
repressor due to lack of interaction with grouchos or CtBP (23).
However, Levanon et al. (24) showed physical and functional
interaction of the human groucho homolog TLE-1 with hLEF-1. The
contradictory results may be explained by different splice variants of
LEF-1 that were used by Roose et al. (22) and Levanon
et al. (24), respectively.
Analysis of the genomic structure of the human TCF-1
gene and analysis of the detected mRNA transcripts revealed
extensive alternative splicing events. These result in a maximum of 96 theoretical splice variants, at least 5 of which have been shown to be
expressed in vivo (25). Alternative splice variants also
have been reported for TCF-3, TCF-4, and LEF-1. These
observations further support the idea that the binding of corepressors
or coactivators depends on the expressed splice variant.
In this study we report on the isolation of novel XTCF-4 variants.
Furthermore, we analyzed the transactivation potential, DNA binding
affinities, protein-protein interactions, and posttranslational modifications of these variants in comparison to mLEF-1/XLEF-1 and
XTCF-3. Our studies revealed that the complex formation of XTCF-4 and
-catenin and the ability of this complex to activate target genes
depends on the absence of two short amino acid motifs, LVPQ and SFLSS.
Unexpectedly, these motifs neither influenced groucho nor SMAD4 binding
to the transcription factors. Instead, we found that
XTCF-4-
-catenin-DNA complex formation was accompanied by a
dephosphorylation event observed in XTCF-4 isoforms lacking these motifs.
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EXPERIMENTAL PROCEDURES |
DNA Constructs Used in This Study--
Additional TCF-4 variants
were isolated exactly as described (26). In short, TCF-4 clones were
isolated from an A6
-ZAP cDNA library. Complete cDNA clones
were obtained by 5'-rapid amplification of cDNA ends and combining
overlapping fragments. The XTCF-3 clone was isolated from a gastrula
stage cDNA library and completed by 5'-rapid amplification of
cDNA ends. The Xenopus LEF-1 and the XTCF-3
grg
constructs were kind gifts of O. Destreé (22, 27). Independently,
an identical XLEF-1 clone has been isolated in our laboratory by means
of reverse transcriptase-PCR. The C-terminal truncated form of XTCF-3
has been constructed by PCR methods using proofreading Pwo
polymerase (Roche Molecular Biochemicals). Primer sequences used for
this construct were 5'-GCGCCTCGAGATGCCTCAGCTCAACAGCGGC-3' and
5'-GGGCCCCTCGAGTCTCTCTCAACTAGTCACTGGATCTGGTCAC-3'. The correct sequence
of this construct was verified by DNA sequencing. All of these
constructs were subcloned into pCS2+ containing a cytomegalovirus promoter as well as an sp6 promoter.
For the isolation of a full-length Xenopus ESG-1 clone, a
ZAP Xenopus gastrula cDNA library was screened with
a 600-base pair fragment of ESG-1 (28) obtained by PCR amplification of a Xenopus gastrula library. Only partial cDNA clones
were isolated, the longest encoding amino acids 34-756. The N-terminal
region of ESG-1 (756 base pairs) was amplified by PCR of a
Xenopus oocyte cDNA library and subcloned into the
BamHI site of ESG-1. The obtained full-length sequence was
95% homologous to the previously isolated ESG-1 and submitted to
GenBankTM (accession number AF289027). For GST pull-down
assays with GST-SMAD4, human SMAD4 cloned into pGex4T3 (Amersham
Pharmacia Biotech). The truncated version of XTCF-3 lacking the HMG
domain and C terminus (amino acids 1-322) which was used as a negative control was prepared by PCR and subcloned into the
EcoRI-XhoI sites of pCS2+. The primers used were
5'- CGGGAATTCATGCCTCAACTAAACAGCGGC-3' and
5'CGGCTCGAGACTGGGCTTCTTCTCTTCTTCC-3'.
Cell Culture and Reporter Gene Assays--
Transfection of
epithelial A6 cells with different expression constructs and luciferase
reporter gene assays were performed as described (21), except for the
use of Effectene transfection reagent (Qiagen) instead of Lipofectin
(Life Technologies, Inc.). Cell extracts used in bandshift studies were
prepared in 50 mM Tris/HCl, pH 7.5, 150 mM
NaCl, 0.5% Nonidet P-40, 1 mM DTT, and 1 mM
phenylmethylsulfonyl fluoride. The used phosphatase inhibitors were
sodium vanadate (10 mM), sodium fluoride (10 mM), and sodium molybdate (10 mM).
Bacterial Expression of HMG Box Transcription Factors and in
Vitro Translation--
For expression of His-tagged fusion proteins,
cDNAs were subcloned into pRSETA (Invitrogen) with PCR techniques
using proofreading Pwo polymerase (Roche Molecular
Biochemicals) according to the manufacturer's instructions.
Transformed BL21(DE3) bacteria were induced by 1 mM
isopropyl-1-thio-
-D-galactopyranoside for 2 h at
room temperature. After centrifugation, bacterial pellets were lysed by
sonification in lysis buffer (50 mM
NaH2PO4, 300-1000 mM NaCl, 10 mM imidazole, pH 8.0) including protease inhibitors (complete, Roche Molecular Biochemicals), and the cleared supernatant was loaded onto nickel-nitrilotriacetic acid resins for 1 h at 4 °C in a batch procedure. After washing with 50 mM
NaH2PO4, 300-1000 mM NaCl, 10-30
mM imidazole, pH 8.0, the protein was eluted 10× with 1 ml
of 50 mM NaH2PO4, 300-1000
mM NaCl, 100-300 mM imidazole, pH 8.0. Each
fraction was analyzed by SDS-PAGE followed by Coomassie staining using
standard procedures. For Western blot analyses of His-tagged fusion
proteins, a commercially available antibody was used (RGS-His by
Qiagen). For the electrophoretic mobility shift assays proteins were
dialyzed against 1000-fold access of binding buffer (20 mM
Tris/HCl, pH 8.0, 50 mM NaCl, 5% glycerol, 0.1 mg bovine
serum albumin per ml, 1 mM dithiothreitol, 1 mM MgCl2), and protein concentrations were determined by
Bradford assay. In vitro translation was done using a
coupled transcription/translation kit (Promega).
Far Western Analyses--
Bacterially expressed fusion proteins
were separated by SDS-PAGE and transferred to a nitrocellulose
membrane. Blocking was done in 3% bovine serum albumin in TBST
overnight at 4 °C. The ESG protein was translated in
vitro in the presence of [35S]methionine and added
to the blocked nitrocellulose filter in TBST for 8 h at 4 °C.
After 3 washes in TBST, 0.25% bovine serum albumin the filter was
exposed to a PhosphorImager screen and further analyzed. The negative
control was a His-tagged fusion protein comprising the cytoplasmic
domain of Xenopus cadherin-11 (29).
GST Pull-down Assay--
Interaction assays were performed by
addition of bacterially expressed GST or GST-SMAD4 proteins, 2-4 µl
of 35S-labeled TCF/LEF proteins, and 300 µl of
binding buffer (20 mM Tris/HCl, pH 8.0, 50 mM
KCl, 2.5 mM MgCl2, 1 mM DTT, 10%
glycerol, and 0.2% Nonidet P-40) to 20 µl of glutathione-Sepharose
beads. Binding reactions were rolled for 4 h at 4 °C and washed
four times with wash buffer (20 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, and 0.2% Nonidet P-40).
The beads were boiled in sample buffer and subjected to SDS-PAGE. After
staining with Coomassie to verify the integrity of GST and GST-SMAD4,
the gel was dried and subjected to PhosphorImager analysis to visualize
bound TCF/LEF proteins.
Quantitative Electrophoretic Mobility Shift Assay
Studies--
The oligonucleotide sequences used were as shown in Fig.
3B and as published (21). Before labeling, oligonucleotides
were purified by denaturing PAGE. Both DNA strands were labeled
separately and annealed to form the duplex DNA. Competition studies
were performed with a 100-fold excess of either unlabeled probe or an
unrelated oligonucleotide as previously published (21),
5'-CAATAAAAAAGGGATCTCGCCTGTTAATGA-3'. For quantitative gel shift
analysis, the total concentration of the dimer in the binding reaction
was 13 nM. Binding conditions using different
concentrations of fusion proteins were done exactly as described (21).
The protein concentrations used are given in the corresponding figures.
Quantification of the band shift studies was performed using a
PhosphorImager (Fuji). The results shown in Fig. 4, A and
B, are the mean of 3-5 independent experiments. Error
bars give standard error means. Data analysis was done using CricketGraph III software (Computer Associates International, Inc.
Islandia, NY). For supershift analysis 1 µl of either XTCF-4 antibody
(Biomol) or
-catenin antibody (Sigma) was added.
Dephosphorylation Procedure--
Phosphatase treatment lysates
were prepared as described above in 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM DTT, and
1 mM phenylmethylsulfonyl fluoride. Phosphatase treatment
was done by adding 1 µl of potato acidic phosphatase (Roche Molecular
Biochemicals) and 0.5 µl of calf intestine alkaline phosphatase
(Roche Molecular Biochemicals) to 1 ml of lysis buffer. Controls were
in the absence of phosphatase in presence of 10 mM NaF and
10 mM sodium molybdate. Optimal results were obtained after
10 min of treatment at 30 °C as longer treatment (30 min) resulted
in a nearly complete loss of DNA binding.
Phosphorylation of Fusion Proteins by Casein Kinase
II--
Phosphorylation of bacterially expressed fusion proteins by
casein kinase II (CKII) was performed with commercially available CKII
(New England Biolabs) according to the manufacturer's instructions within 10 min.
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RESULTS |
Screening for XTCF-4 Reveals Different Splice Variants--
In our
attempt to isolate Xenopus members of the TCF/LEF family, we
recently identified a Xenopus homolog of TCF-4 (26). In
addition to the published XTCF-4 sequence, which we now refer to as
XTCF-4B (GenBankTM accession number AF207708), we isolated
in this screen two highly identical variants that differ in two short
amino acid motifs (Fig. 1A).
Whereas XTCF-4A (GenBankTM accession number AF287150)
contains both of these stretches, XTCF-B lacks the sequence SFLSS at
position 288-292. XTCF-4C (GenBankTM accession number
AF287151) additionally lacks 4 amino acids (LVPQ) at position 256-259.
In comparison to other known members of the TCF/LEF family, XTCF-4A
resembles XTCF-3, whereas XTCF-4C is similar to murine LEF-1 (see Fig.
1A). As identical gaps are present in the mLEF-1 sequence,
we exclude the possibility that these sequence variations might be an
artifact. Based on the fact that the multiple TCF-1 isoforms are due to
alternative splicing (25), we assume that the isolated XTCF-4 variants
were also generated by alternative splicing.

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Fig. 1.
Isolation of different XTCF-4 variants that
show distinguishable transactivation behavior. A, the
sequences of the variable region XTCF-4A, -B, and -C. Homologous
sequences of mLEF-1, XLEF-1, and XTCF-3 are given. B,
schematic representation of the constructs used in this study (not to
scale). The box in dark shading represents the
HMG box responsible for DNA binding, whereas the box in
light gray shading is the -catenin-binding site. In
XTCF-3 grg the putative groucho binding site has been eliminated.
C, activation of the 499/+20 fibronectin reporter gene
construct in A6 cells. Luciferase activity was normalized to the
promoter activity in the absence of transfected TCF/LEF factors.
D, activation of the promoter depends on the presence of a
functional TCF/LEF site as mutations in the TCF/LEF target site in the
499/+20 mutant construct lead to a loss of activation in A6
cells. Each bar in C and D represents
the mean of 7-18 independent experiments; error bars
indicate S.E. The dashed line in C and
D indicates the level of 2-fold activation of the 499/20
luciferase construct in A6 cells in comparison to basal activity.
E, in XTC fibroblasts the FN promoter construct is repressed
by XTCF-3, XTCF-3 C, XTCF-4A, and XTCF-4B. F, repression
of the promoter depends on the presence of a functional TCF/LEF site as
judged by use of the 499/+20 mutant construct. Each
bar in E and F represents the mean of
3-6 independent experiments; error bars indicate S.E. The
dashed line in E and F indicates the
basal activity of the 499/20 luciferase construct in XTC cells in the
absence of transfected TCF/LEF factors.
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We also isolated Xenopus homologs of TCF-3
(GenBankTM accession number AF287149) and mLEF-1
(GenBankTM accession numbers AF287147 and AF287148) that
are almost identical to the published sequences (15, 27). The isolated Xenopus homologs of mLEF-1 not only lack the two described
short amino acid stretches but also the region in between (Fig.
1A). Comparison with the recently characterized genomic
structure of the human TCF-1 gene (25) reveals that
this region is encoded by the alternatively used exon IVA of hTCF-1
(underlined in Fig. 1A). So far we have not been
able to detect a Xenopus homolog of XLEF-1 or TCF-1
encompassing this exon. For functional studies, these splice variants
were subcloned into pCS2+ containing a cytomegalovirus promoter
allowing expression of the constructs in culture cell lines.
Differential Ability of TCF/LEF Transcription Factors to
Activate Target Gene Expression--
To compare the transactivating
activities of these HMG box transcription factors, we used the
Xenopus fibronectin gene as a direct target for
the classical Wnt/
-catenin pathway in Xenopus. As shown
previously, in epithelial A6 cells mLEF-1 activates a
499/+20
fibronectin (FN) reporter gene construct significantly stronger than
XTCF-3 (Ref. 21 and Fig. 1C). We first tested whether in
this assay XLEF-1 behaves in a similar fashion to its mouse counterpart
even though it lacks the sequences corresponding to the TCF-1 exon IVA.
Indeed, under comparable conditions XLEF-1 was able to activate the FN
promoter to the same extent as the mouse homolog (Fig. 1C).
We conclude that the sequence differences between mLEF-1 and XLEF-1 are
nonrelevant with respect to activation of the fibronectin promoter.
Given the similarity with respect to the LVPQ and SFLSS motifs between
XTFC-4A and XTCF-3 on the one hand and XTCF-4C and mLEF-1 on the other,
we next investigated the ability of XTCF-4A and-C to activate target
gene expression. Whereas XTCF-4A was only weakly active (1.2-fold
activation, n = 14, not significant), XTC-4C gave a
strong response of the reporter gene construct (2.6-fold activation,
n = 17 with p < 0.001 in Student's
t test) (Fig. 1C). Thus, in their ability to
activate the FN promoter, XTCF-4A behaves like XTCF-3 (1.3-fold
activation, n = 15, not significant), whereas XTCF-4C
shows properties similar to mLEF-1 (2.0-fold activation, n = 7, with p < 0.01 in Student's
t test). Unlike XTCF-4C and mLEF-1, XTCF-4B was not able to
activate strongly the reporter gene (1.5-fold activation,
n = 18) (Fig. 1C).
Fibronectin is highly expressed in XTC fibroblasts, and this is due to
an activated Wnt signaling pathway (21). We therefore analyzed the
different TCF/LEF factors also in this cell line. As the Wnt pathway is
already active in these cells, transfection of XLEF-1 did not lead to a
further activation of the FN promoter. However, transfection of XTCF-3,
XTCF-4A, or XTCF-4B led to an inhibition of FN expression (Fig.
1E). In contrast, XTCF-4C behaved as XLEF-1. Thus, those
TCF/LEF factors that failed to activate the FN promoter in epithelial
A6 cells are working as transcriptional repressors in XTC fibroblast,
whereas those TCF/LEF factors that activate the reporter in A6 cells do
not show repressing activity in XTC cells.
In all cases, activation of the reporter gene construct was dependent
on the presence of a functional TCF/LEF target site that we further
refer to as the Wnt-responsive element, WntRE. Introduction of
point mutations into the WntRE of the
499/+20 fragment (21), thereby
interfering with binding of the transcription factors to their target
site, completely eliminated the responsiveness of the promoter upon
transfection of TCF/LEF members both in epithelial A6 cells or XTC
fibroblasts (Fig. 1, D and F).
In summary, despite the high percentage of amino acid sequence identity
between different variants of XTCF-4 that differ in the LVPQ and SFLSS
motifs, these factors show distinguishable transactivation behavior in
two different cell lines.
Differential Ability of TCF/LEF Factors to Activate Target Genes Is
Not Due to Complex Formation with groucho, CtBP, or SMAD4--
In
previous reports it had been speculated that LEF-1 functions as a
transcriptional activator, whereas XTCF-3 acts as a repressor of
transcription (22, 23). This assumption was based on the following
observations: (i) mLEF-1 activates expression of the homeobox
transcription factor siamois and induces axis duplication in early
Xenopus embryos, whereas XTCF-3 does not, and (ii) XTCF-3 binds to transcriptional repressors of the groucho family, whereas for
LEF-1 contradictory results have been obtained (22, 24). In agreement
with this groucho-repressor model was the observation that deletion of
the groucho-binding site in XTCF-3 converts the repressor into an
activator (22), and we were able to confirm this observation using
fibronectin as a target for Wnt signaling (21).
We therefore asked whether the differential potential of TCF/LEF
members to activate the fibronectin promoter might be due to
differences in their ability to interact with transcriptional repressors of the groucho family. For this purpose, a far Western was
performed (9) with purified His-tagged fusion proteins of mLEF-1,
XLEF-1, XTCF-3, XTFC-4A, and XTCF-4C using SDS-PAGE. After
electrophoresis, proteins were transferred onto a nitrocellulose membrane and overlaid with radioactively labeled Xenopus
ESG-1, a member of the groucho family of transcriptional repressors. In
contrast to Roose et al. (22), but supporting Levanon
et al. (24), we found that all members of the TCF/LEF family
can interact with ESG-1 in vitro (Fig.
2). The cytoplasmic domain of
Xenopus cell-cell adhesion molecule cadherin-11 was used as negative control demonstrating the specificity of interaction between
ESG-1 and TCF/LEF factors. Based on these results, we conclude that all
members of the TCF/LEF family have the potential to interact with
members of the groucho family and that there is no simple correlation
between the ability of a TCF/LEF factor to bind groucho members and
their role as transcriptional activators or repressors,
respectively.

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Fig. 2.
Interaction of TCF/LEF transcription factors
with ESG-1 groucho. A, fusion proteins were purified on
nickel-agarose and separated on polyacrylamide gels. XLEF-1 and mLEF-1
were stained by Coomassie (left), whereas the other proteins
(right) were detected by an anti-His-tag antibody (Qiagen).
The asterisk labels the band for XLEF-1. Molecular weights
are indicated. Xcad-11 is a His-tagged fusion protein of the
intracellular domain of Xenopus cadherin-11 that served as a
negative control (29). B, overlay blot with
35S-labeled Xenopus ESG-1, a member of the
groucho family. Lanes were as in A. Note that all proteins
are interacting with ESG-1 the exception of Xenopus
cadherin-11. C, SMAD4 binds directly to TCF/LEF
transcription factors. In vitro translated
35S-labeled TCF/LEF proteins (lanes 1-6)
were precipitated with GST (lanes 7-12) or SMAD4-GST
(lanes 13-18). All tested factors bound GST-SMAD4 except
TCF-3 lacking the HMG domain and C terminus.
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Another transcriptional corepressor that had been shown to interact
with the C-terminal region of XTCF-3 is CtBP (23). Binding of CtBP to
XTCF-3 depends on two PLSL(T/V) motifs in the C terminus of XTCF-3 that
are also present in hTCF-4 but not XTCF-4. Since this region is missing
in mLEF-1, this opens the possibility that CtBP might be involved in
repressing fibronectin expression by XTCF-3. We therefore constructed a
C-terminal deletion construct of XTCF-3 (Fig. 1B) that has
been shown to be unable to bind CtBP (23), and we tested the ability of
this construct to activate the FN-promoter in A6 cells. In several
independent experiments XTCF-3
C was unable to activate transcription
from the
499/+20 construct (Fig. 1C). This failure in
activation is not due to a nonfunctional protein given the following
two observations. First, XTCF-3
C is able to bind the Wnt-RE (see
Fig. 5A below) and thus is functional with respect to DNA
binding. Second, in XTC cells this construct was still able to repress
FN expression (Fig. 1E) showing that this construct is still
functional with respect to target gene regulation. This experiment
together with the ability of XTCF-3
grg to activate fibronectin
expression (21) demonstrate that CtBP is not the major corepressor
component of the XTCF-3-mediated transcription factor complex on the FN promoter.
A complex of SMAD4, LEF-1, and
-catenin has recently been shown to
regulate the transcriptional activation of the Xenopus homeobox transcription factor twin (11, 12). Subsequent
in vitro precipitation experiments demonstrated that SMAD4
binds directly to the HMG box of LEF-1 (11). In the context of this study we therefore aimed to analyze this interaction with respect to
other members of the TCF/LEF family. To determine whether SMAD4 preferentially binds to members of this family, GST pull-down experiments were performed. In vitro translated
35S-labeled TCF/LEF proteins were precipitated with
GST-SMAD4 or with GST alone. As shown in Fig. 2C, a strong
interaction with SMAD4 was observed for all TCF/LEF factors tested,
none of them interacting with GST alone. As a negative control, XTCF-3
lacking the HMG domain and the C terminus was used (XTCF3 (amino acids 1-322)). We conclude from these experiments that all
Xenopus members of the TCF/LEF family can interact with
SMAD4 thereby excluding the possibility that differential complex
formation with SMAD4 accounts for the observed differences in
transactivation of HMG box transcription factors.
Different Members of the TCF/LEF Family Bind to Their Target
Sequence with Different Affinity--
We next reasoned that the
differences in transactivation activity of TCF/LEF members might be due
to differences in DNA binding affinity. Different TCF/LEF fusion
proteins were purified as judged by Coomassie staining (Fig.
3A), and binding affinities
toward two well defined WntRE in Xenopus were determined.
The DNA duplexes were derived from the TCF/LEF-binding sites found in
the Xenopus fibronectin and siamois
promoters (21, 30) (Fig. 3B). We recently reported that
mLEF-1 specifically binds to the WntRE of the fibronectin promoter
(21). Here we demonstrate that XTCF-3, XTCF-4A, and-4C are also able to
bind specifically to the FN-WntRE (Fig. 3C). Whereas
addition in excess of unlabeled oligonucleotides containing the
FN-WntRE prevented binding of the fusion proteins to the labeled fragment, addition of an unspecific competitor did not (Fig.
3C). Identical results were obtained with the sia-WntRE
indicating the specific binding of TCF/LEF factors (not shown).

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Fig. 3.
Sequence-specific binding of XTCF-3, XTCF-4A,
and XTCF-C. A, different TCF/LEF fusion proteins were
purified and analyzed by Coomassie staining. B, target
oligonucleotides containing a TCF/LEF target site (WntRE) were derived
from the Xenopus siamois and
fibronectin promoters. C, all TCF/LEF factors
specifically interact with the fibronectin-derived oligonucleotide. The
specific competitor in this experiment was an excess of the unlabeled
oligonucleotide representing the FN-WntRE. The unspecific competitor
used was as indicated under "Experimental Procedures."
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To determine the apparent dissociation constant to the used
oligonucleotides derived from the siamois or the FN promoter, respectively, we performed quantitative bandshift studies with variable
amounts of TCF/LEF fusion proteins (Fig.
4, A and B). The
percentage of bound oligonucleotide was plotted as a function of the
fusion protein concentration, and the protein concentration that
resulted in a binding of 50% of the oligonucleotide was estimated as
the apparent KD value. The KD
values observed in our experiments were within a range of 0.3 and 4.6 µM and thus within the same order of magnitude as
recently determined for the murine LEF-1 HMG box only (31). In
addition, to verify these constants by a different plotting procedure
we performed classical Scatchard plot analysis. For this purpose, the
ratio of bound protein to free protein concentration (bound/free) is
plotted versus the concentration of bound protein. By using
this procedure, the KD value can be estimated by
using the slope of the obtained line. Additionally, the total number of
binding sites represented by the used oligonucleotide can also be
estimated by the abscissa intercept (for a more detailed description of Scatchard plots see Ref. 32).

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Fig. 4.
Determination of binding constants of TCF/LEF
factors. A, quantitative bandshift studies were done
with the proteins as indicated on the siamois-derived oligonucleotide,
and the resulting shift in mobility was quantified by a PhosphorImager
analysis. The results are represented in two different ways. First, the
percentage of bound oligonucleotide is given as a function of the
concentration of protein added (left side).
Kd is estimated by the protein concentration with
50% occupancy of binding sites. Second, results are shown as Scatchard
plots (right side), confirming the Kd
values as well as the theoretical concentration of binding sites (13 nM). The linear slope to determine Kd
was calculated using Cricket Graph III software (Computer Associates
International, Inc., Islandia, NY). The regression coefficient
R indicates the performance of regression analyses. Values
close to 1 indicate a good correlation; values close to 0 indicate that
there is no correlation. The values were obtained from 3 to 5 experiments per protein concentrations. Error bars indicate
S.E. B, quantitative bandshift studies were done as
described in A except for using an oligonucleotide derived
from the fibronectin promoter shown in Fig. 3B. Note the
different binding behavior of XTCF-3 and XTCF-4C with respect to the
siamois oligonucleotide. In these cases, Kd is
estimated only by the first plotting procedure. C,
truncation of the C-terminal domain of XTCF-3 shifts the binding
behavior from XTCF-3 on the FN-WntRE toward the one observed in case of
mLEF-1.
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The most obvious outcome of these experiments is the fact that all
tested fusion proteins have a significant higher affinity toward the
sia-WntRE than the FN-WntRE. However, the absolute difference in
binding affinity was dependent on the fusion protein used. We found for
mLEF-1 a 2-fold higher affinity toward the siamois-derived target site
in comparison to the FN-derived sequence, whereas the fold difference
in binding affinity for the others were as follows: XTCF-4A, 2-fold
higher; XTCF-4C, 5-fold higher; and XTCF-3, 5-fold higher (Fig. 4,
A and B). One major difference between the two
target oligonucleotides became obvious by comparing the binding
affinities for XTCF-3. Whereas in case of the FN-WntRE XTCF-3 displays
sigmoidal binding with comparable low affinity, it exhibits a
hyperbolic binding to the sia-WntRE with a significant higher affinity.
A similar behavior was observed for XTCF-4C. XTCF-4A showed sigmoidal
binding to both oligonucleotides. In these cases we were not able to
plot linear Scatchard plots but obtained graphs with a maximum, which
is an indication for positive cooperative binding behavior (32). This
lower binding affinity toward the FN-WntRE is most likely not due to
protein misfolding or degradation as this experiment was done with
different protein preparations, and the same batches of proteins showed
a higher binding affinity toward the sia-WntRE. These data raise the
possibility that the two different bases in the core sequence as well
as the flanking regions influence the binding behavior of a given HMG box transcription factor. In addition, the extended C termini of XTCF-3
and XTCF-4 might influence the binding characteristics on the
FN-derived oligonucleotide. Indeed, we found that truncation of the C
terminus of XTCF-3 significantly shifts the binding behavior toward the
mLEF-1-derived one (Fig. 4C).
Most strikingly, although bacterially expressed XTCF-4C binds to the
FN-WntRE in vitro with much lower affinity than mLEF-1, it
is a more potent in vivo activator of the FN promoter than mLEF-1. We conclude that although members of the TCF/LEF family differ in their in vitro DNA binding affinities toward
different promoter target sites, this difference does not correlate
with their transactivation potentials.
Members of the TCF/LEF Family Differentially Bind
-Catenin When
Bound to DNA--
We next asked whether there might be differences in
complex formation mediated by the analyzed TCF/LEF factors in
vivo. We therefore transfected epithelial A6 cells with expression
constructs encoding for the transcription factors. After 3 days we
prepared cell lysates under mild conditions and analyzed the behavior
of the overexpressed proteins in gel shift assays. Western blot
analysis of these lysates revealed that all overexpressed proteins were expressed at comparable levels (Fig.
5C). Although XTCF-4A and XTCF-4C are of nearly identical size, the formed DNA-protein complexes show different migrating behavior (Fig. 5A). The XTCF-4C
induced DNA-protein complex was of larger size compared with the
XTCF-4A one. Whereas the mLEF-1-DNA complex behaved similar to the
XTCF-4C-DNA complex, the XTCF-3-induced complex migrates as fast as the
XTCF-4A one. Thus, the ability of these transcription factors to
trigger target gene activation is paralleled by the migration behavior of the corresponding DNA-protein complexes.

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Fig. 5.
TCF- -catenin complex
formation on the fibronectin oligonucleotide. A,
epithelial A6 cells were transfected with expression constructs as
indicated, and cell lysates prepared in the presence of phosphatase
inhibitors were assayed for their ability to bind specifically the
fibronectin-derived oligonucleotide. The unspecific competition was
performed as described under "Experimental Procedures." The data
for XTCF-3 C are from an independent experiment in comparison to the
other shown results. B, bandshifts were done as in
A in presence of phosphatase inhibitors, but antibodies
against TCF-4 or -catenin were added. Note that XTCF-4B and XTCF-4C
display two distinct signals in the absence of antibodies. The faster
migrating signal can be supershifted by adding TCF-4-specific
antibodies. Formation of the slower migrating complex is prevented by
adding -catenin antibodies indicating the presence of -catenin in
the slower migrating complex. XTCF-4A transfectants never showed this
slower migrating complex. C, immunoblot of A6 cells
transfected as indicated and probed for expression of TCF-4. All
constructs are expressed at similar levels. Note that XTCF-4B and
XTCF-4C clearly show two distinct bands (arrowheads),
whereas the second signal is barely visible for XTCF-4A. D,
immunoblot for -catenin protein (arrowhead) in
transfected A6 cells showing no significant differences in expression
level of -catenin after transfection.
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These differences are probably not due to differences in DNA bending as
we never observed such differences when using bacterially expressed
fusion proteins (see Fig. 3 as example). This raises the possibility
that the observed differences in migration behavior reflect
differential complex formation with transcriptional coactivators, namely
-catenin, rather than corepressors. We aimed to study this
possibility by supershift analyses. We focused on the different TCF-4
variants as antibodies are commercially available that specifically recognize XTCF-4 and
-catenin. Our attempt to include mLEF-1 and
XTCF-3 into this study was hampered by the lack of antibodies against
these proteins that function under native conditions.
For the supershift experiments the buffer and gel conditions were
slightly altered compared with the experiments shown in Fig.
5A to achieve a higher resolution for the slower migrating complexes (Fig. 5B). This led to the detection of a faster
migrating complex in XTCF-4B- and XTCF-4C-transfected cells that runs
at the level of the complex also found in parental and
XTCF-4A-transfected cells (for reasons described below and labeled as
TCF/DNA in Fig. 5B). Whereas upon XTCF-4A
transfection and in untransfected cells only this faster migrating
complex was seen, XTCF-4B and XTCF-4C transfection revealed an
additional slower migrating complex that corresponds to the main signal
seen in Fig. 5A. One additional faster migrating band was
also observable in untransfected cells indicating that this signal is
of endogenous origin and not due to the overexpressed TCFs (labeled as
non TCF in Fig. 5B).
For all tested XTCF-4 variants, addition of an antibody against TCF-4
supershifted the faster migrating band, indicating that this band
represents an XTCF-4-DNA complex. However, the slower migrating upper
band was not supershifted by addition of TCF-4 antibody indicating that
most probably the binding epitope for the TCF-4 antibody may be blocked
by an additional protein. On the other site, adding an antibody against
-catenin interfered with formation of the upper band and resulted in
a strong increase in the TCF-4/DNA band (Fig. 5B)
identifying
-catenin as the additional protein in the slower
migrating band. Thus, the slower migrating band that we observed in
XTCF-4B and XTCF-4C cells, but not in those transfected with XTCF-4A,
indicates the presence of a XTCF-4-
-catenin-DNA trimer. This
difference in complex formation with
-catenin is not due to an
up-regulation of the overall amount of
-catenin as judged by Western
blot studies of transfected cells (Fig. 5D). As the TCF-4
variants used differ in the LVPQ and SFLSS motifs, this indicates that
the complex formation of the analyzed XTCF-4 variants with
-catenin
is directly or indirectly dependent on the two described amino acid
motifs. Another striking observation obtained by these experiments
using cell lysates was that in vivo expressed XTCF-4A
apparently binds with lower affinity to the FN-WntRE than XTCF-4B and
-C, although all proteins were expressed at comparable levels (see Fig.
5, B and C).
TCF/LEF-
-Catenin Complex Formation Is Dependent on the
Phosphorylation of TCF--
By comparing the expression level of TCF-4
variants in cell lysates, we made the observation that XTCF-4B and
XTCF-4C appeared in Western blots as a double band (Fig.
5C). This raises the question whether there are
differentially phosphorylated forms of XTCF-4. Indeed, treating the
lysates with calf intestine alkaline phosphatase, as well as acidic
potato phosphatase, resulted in an increased appearance of a faster
migrating band in all XTCF-4 variants (Fig. 6A). We conclude from these
data that members of the TCF family are the subject of phosphorylation
in vivo. As we never observed an XTCF-4A-
-catenin-DNA
complex in our bandshift experiments (Fig. 5B) or the faster
migrating form of XTCF-4A in the absence of phosphatase activities
(Fig. 6A), we reasoned that the complex formation of XTCF-4
with
-catenin might be dependent on the existence of a
dephosphorylated form. By having established the identity of different
retarded bands in our mobility shift studies, we therefore repeated
these studies with XTCF-4A transfectants in the presence or absence of
exogenously added phosphatase activity. These experiments revealed two
important observations.

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Fig. 6.
XTCF-4 variants are differentially
phosphorylated in A6 cells. A, treatment of TCF-4
transfectants with exogenous phosphatase. In the absence of phosphatase
activity, XTCF-4B and -C appear as a double band, whereas XTCF-4A
appears as a single slower migrating band. The faster migrating signal
is strongly enhanced by phosphatase treatment in all three samples.
B, lanes 1 and 2, addition of
exogenous phosphatase to lysates prepared from XTCF-4A-transfected A6
cells results in the appearance of the slower migrating band
(arrowhead) identified in Fig. 5B to represent a
TCF-DNA- -catenin trimer. Lanes 3-5, formation of
the slower migrating band can also be triggered by endogenous
phosphatases. Addition of phosphatase inhibitors as described under
"Experimental Procedure" prevents formation (lane 3),
whereas this complex is formed in absence of phosphatase inhibitors
(lane 5). In lane 4 only sodium orthovanadate and
sodium fluoride were added. Inset in B, reduced
binding affinity of XCF-4A is not due to reduced protein amounts as a
result of protease contaminations of the added phosphatases. Treatment
of bacterially expressed XLEF-1 or XTCF-4A with phosphatases
(lanes 4) did not result in protein degradation in
comparison to protein samples that were similar treated in the absence
of phosphatases (lanes 3). Lanes 1 are buffer
only samples and lanes 2 are buffer including phosphatases
but in the absence of fusion proteins. Detection of proteins was either
by Coomassie staining (XLEF-1) or immunoblot against the His tag
(XTCF-4A). C, bacterially expressed fusion proteins of
XTCF-3, XTCF-4C, and mLEF-1 are phosphorylated in the presence of
casein kinase II. D, phosphorylation of bacterially
expressed TCF/LEF transcription factors by casein kinase II enhances
the DNA binding affinity. Suboptimal amounts of proteins were used in
quantitative bandshift studies, and the results are presented as
bars. For each protein equal amounts of protein were used in
the presence or absence of CKII. Three independent experiments were
performed for each sample, and error bars indicate
S.E.
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First, the overall binding affinity of XTCF-4A decreased after
phosphatase treatment because we needed comparable longer exposure times to detect signals. This loss in binding is most probably not due
to any protease contaminations since all experiments were done in the
presence of protease inhibitors. Furthermore, we tested the added
phosphatases negative for any protease contaminations by treating
purified TCF/LEF fusion proteins under identical experimental conditions (inset, Fig. 6B). Therefore, this
observation suggests that the binding behavior of TCF/LEF factors is
dependent on pre-phosphorylation. A similar effect has recently been
described for Drosophila HMG1 proteins,
non-sequence-specific, DNA-binding proteins containing several HMG
boxes (33). Pre-phosphorylation has been shown to be casein kinase II
(CKII)-dependent for HMG1 proteins. As all TCF/LEF
transcription factors have multiple CKII target sites and CKII is a
ubiquitously found enzyme, we tested whether these factors are
phosphorylated by CKII and whether phosphorylation influences the
binding behavior of TCF/LEF factors. In fact, all tested TCF/LEF
factors were found to be phosphorylated by CKII (Fig. 6C),
and this phosphorylation was accompanied by an increased binding
affinity (Fig. 6D).
Second and more important, these experiments indicate that the
formation of a TCF-4-
-catenin-DNA complex is dependent on the
presence of a partially dephosphorylated form of the transcription factor (Fig. 6B). Treatment of cell lysates with
phosphatases did not only result in dephosphorylation of the proteins
but also in formation of the slower migrating complex indicating
entrance of
-catenin into the complex.
To compare the results shown in Fig. 6B and in Fig.
5B, and to test whether this dephosphorylation might also
occur in vivo, we prepared lysates of TCF-4A transfectants
in the presence or absence of added phosphatase inhibitors. As shown in
Fig. 6B, addition of a phosphatase inhibitor mixture to the
lysis buffer (as in Fig. 5B) prevented formation of
TCF-4-
-catenin-DNA complexes, whereas a lack of phosphatase
inhibitors resulted in formation of the slower migrating band. This
experiment clearly indicates that endogenous phosphatases are able to
dephosphorylate TCF/LEF factors and that most probably this
dephosphorylation results in the recruitment of
-catenin into the
TCF-DNA complex.
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DISCUSSION |
We report herein that different, highly similar XTCF-4 variants
show divergent behavior in target gene activation assays. By using
different biochemical approaches, we provide evidence that these
differences are neither due to preferential interaction with
corepressors of the groucho family or CtBP nor to differential binding
of the transcriptional activator SMAD4. We demonstrate that the
formation of a TCF-
-catenin-DNA complex and the activation of the
target gene fibronectin depends on two short amino acid motifs. These motifs, LVPQ and SFLSS, are involved in regulating posttranslational modifications of XTCF-4. Since they are not present
within all variants of TCF/LEF factors (e.g. mLEF-1
versus XTCF-3), and since cofactors of the TCF/LEF complex
are expressed in a tissue-specific manner, a complex pattern of
regulation is emerging that has to be considered when studying Wnt signaling.
Implications on TCF/LEF-mediated Complex Formation--
In our
attempts to elucidate the molecular mechanism responsible for the
different transactivation properties of TCF/LEF factors, we here
present data that support multiple levels of regulation of
TCF/LEF-mediated transcription factor complex formation.
First, there is no correlation between DNA binding affinities of
TCF/LEF factors and target gene activation. The transcriptional activator XTCF-4 C has a significant lower DNA binding affinity than
mLEF-1 but activates the expression from the FN promoter stronger than
mLEF-1.
Second, although the oligonucleotides used for quantitative band shift
analyses both contain the canonical TCF/LEF target site,
(C/G)TTTG(A/T)(A/T), they show significant differences in complex
formation with TCF/LEF. All studied transcription factors showed a
higher affinity toward the sia-WntRE than toward the FN-WntRE. These
differences in binding affinity and behavior of TCF/LEF transcription
factors might indicate that the nucleotides flanking the conserved core
region of the target site influence the binding affinity by providing
additional contacts. These putative interactions do not only modify DNA
binding strength but also the type of binding behavior. XTCF-3 displays
hyperbolic binding behavior on the sia-WntRE but sigmoidal binding
behavior on the FN-WntRE indicating additional protein/DNA
interactions. In accordance with this hypothesis is the observation
that deleting the C-terminal domain of XTCF-3 shifts the DNA binding
curve of this mutant toward the mLEF-1-derived one. Further experiments
have to confirm whether these additional, promoter-specific protein/DNA
interactions mediated by the C-terminal part of XTCF-3 can be proven
and, if so, whether this holds true for XTCF-4C as well. This is of
general interest, since it has recently been shown that
LEF-
-catenin-SMAD4 complexes are involved in regulating the
twin promoter (11, 12), although the same factors are not
involved in regulating the c-myc gene (11). Thus, with
respect to activation of Wnt target genes for future promoter analysis,
the fine structure of the analyzed promoter region has to be taken into account.
Third, we found, that at least in vitro, all TCF/LEF
transcription factors are able to interact with ESG-1, a member of the groucho family. This is of general interest as it is in contrast to an
earlier hypothesis suggesting that differences in TCF/LEF-mediated transactivation is a biased ability of these factors to interact with
transcriptional corepressors. Although we acknowledge that there still
might be differences in the ability to interact in vivo, our
results clearly show that additional levels of complexity with respect
to TCF/LEF-mediated complex formation have to be postulated. The only
tested TCF/LEF-groucho protein interaction using Xenopus
proteins described so far showed that XTCF-3 can interact with Xgrg-5.
However, this result was only based on two-hybrid analyses (21). The
same study further provides indirect evidence for an interaction of
XTCF-3 with Xgrg-5 and Xgrg-4, since both are translocated to the
nucleus in XTCF-3-transfected COS-1 cells. Further experiments are
required to analyze the in vivo interactions of all known
TCF/LEF and groucho proteins from one species (e.g. Xenopus or mouse) under different cellular conditions.
Fourth, another important and striking observation is the fact that
complex formation of XTCF-4B with DNA and
-catenin is not sufficient
for target gene activation. This is in agreement with a previous
observation in a different cell culture system (34). In this case,
-catenin and LEF-1 can activate target gene expression in
transformed Jurkat T-cells but not in normal T-lymphocytes despite the
nuclear localization of both factors. Thus, additional tissue-specific
components of TCF/LEF-mediated transcription factor complexes or
additional posttranslational modifications have to be postulated. Of
the known components of TCF/LEF-mediated transcription factor
complexes, neither p300/CBP nor TBP are expressed in a tissue-specific
manner. However, in Xenopus we recently described that
Xpontin and Xreptin are expressed in a tissue-specific manner (18).
Also some groucho members show a distinct expression pattern (35).
Beside the tissue-specific expression of cofactors, tissue-specific
posttranslational modifications have to be taken into account.
Protein Phosphorylation as an Additional Level of Regulation in
TCF/LEF-mediated Complex Formation--
The experiments described here
clearly identify phosphorylation as an additional level of TCF/LEF
regulation. First, we found a strict correlation between the presence
of a faster migrating dephosphorylated band of XTCF-4B and XTCF-4C and
the formation of a ternary complex of DNA, TCF-4, and
-catenin. A
dephosphorylated band in Western blots was never observed for XTCF-4A
which was paralleled by the absence of a DNA supershift. However,
treatment of cell lysates derived from XTCF-4A-transfected cells with
different phosphatases resulted in the appearance of the
dephosphorylated form and, in parallel, in the formation of a slower
migrating band in electrophoretic mobility studies. In other words,
dephosphorylation of XTCF-4 most probably at one or more specific sites
allows complex formation with
-catenin, whereas phosphorylation of
XTCF-4 prevents complex formation. Since complex formation between
TCF/LEF transcription factors and
-catenin is a prerequisite for
target gene activation, signal transduction pathways that lead to a
phosphorylation of TCF/LEF factors thus might function as inhibitors of
Wnt signaling. One candidate kinase that might be responsible for this
effect is NLK (nemo-like kinase) as it phosphorylates TCF-4 and TCF-3 and inhibits TCF-
-catenin complex formation (36, 37). With respect
to these previous publications our results offer an attractive hypothesis. As deletion of SFLSS results in the appearance of the
dephosphorylated form of XTCF-4, this sequence motif might either
represent the target site of NLK phosphorylation or might be involved
in TCF-4/NLK interaction. The observation that the phosphorylated form
of XTCF-4 is also present in those variants that lack the SFLSS motif
excludes the first possibility and strengthens the latter one. We also
note that this motif is serine-rich and thus might also be subject to
phosphorylation events that regulate DNA/protein interactions. Further
supporting the idea that posttranslational modifications regulate
complex formation of TCF/LEF factors with
-catenin in
vivo, we found no differences in interaction when using
bacterially expressed fusion proteins in coimmunoprecipitation or pull-
down studies (not shown). Although the absence of the SFLSS motif
allows dephosphorylation and complex formation with
-catenin, it is
not sufficient to turn XTCF-4B into an efficient activator of
transcription. However, this is achieved when both SFLSS and LVPQ
motifs are deleted indicating another important regulatory role for the
latter. Further experiments will reveal whether this short motif is
involved in protein-protein interactions and how those might influence
the transactivation behavior of TCFs.
Furthermore, we observed that the overall DNA affinity of TCF/LEF
transcription factors decreased when they were dephosphorylated. In a
reverse experiment we were able to increase the DNA binding affinity of
unphosphorylated bacterially expressed fusion proteins by treatment
with casein kinase II. All of the tested TCF/LEF factors have several
CKII target site motifs of (S/T)XXE. Two of these are
located within the HMG box and represent the only positionally
conserved target sites for CKII. Although we have no evidence that this
kind of phosphorylation occurs in vivo, these experiments
clearly implicate an additional possibility to modify the outcome of
Wnt signaling.
Implications for Our Understanding of Wnt Signaling--
In
summary, we show that complex formation of TCF/LEF factors with
-catenin correlates with posttranslational
phosphorylation/dephosphorylation events. Furthermore, we provide
additional evidence that binding of TCF/LEF factors with
-catenin is
not necessarily linked with transcriptional activation. In addition to
an active Wnt signal that leads to accumulation of
-catenin in the
nucleus, additional signals are required to convert TCF/LEF into a
transcriptional activator. Beside the SFLSS motif that is involved in
regulation of TCF-
-catenin complex formation, we provide evidence
that the LVPQ motif is involved in turning TCF/LEFs into a
transcriptional activator and might be involved in interactions with
transcriptional repressors. Thus, the outcome of a Wnt signal is
additionally regulated at the level of transcription factors of the
TCF/LEF family. With respect to TCF/LEF-mediated complex formation, our data also highlight the differences between in vivo and
in vitro experiments. Further studies regarding regulation
of Wnt signaling at the level of transcription factors thus have to
consider the following: (i) which splice variants of the transcription
factors are expressed, (ii) their posttranslational modifications,
(iii) what kind of cofactors are expressed in these cells, and (iv) the
actual components of the finally assembled complex. This regulation might be by far more complex than previously thought.