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INTRODUCTION |
-Catenin together with Plakoglobin and the more distantly
related p120ctn (where ctn is catenin) belong to a large
family of proteins that are involved in diverse cellular processes (1).
The members of this protein family are characterized by the presence of
multiple copies of a 42-amino acid motif, the so-called Armadillo
(Arm)1 repeat,
which was named after a founding member of the family, the product of
the Drosophila Armadillo gene (1). In vertebrates,
-catenin and Plakoglobin were first described as components of cadherin-catenin cell adhesion complexes, where they link cadherin transmembrane proteins to
-catenin and the actin filaments of the
cytoskeleton (2). Meanwhile, the dual involvement of
-catenin and
its invertebrate homolog Armadillo in cell adhesion and cell-cell signaling is well established. These proteins are central components of
the Wnt/wingless signal transduction cascade which for example functions to specify anterior-posterior segment polarity in
Drosophila larvae or to determine the embryonic
dorso-anterior body axes in Xenopus laevis (3-5).
Wnt/wingless growth factors are secreted glycoproteins that utilize
members of the Frizzled family of seven transmembrane domain proteins
as receptors (5, 6). Stimulation of Frizzled receptors by Wnt activates
a signaling pathway which includes Dishevelled, GSK-3
, Axin, or
Conductin and the adenomatous polyposis coli tumor suppressor protein.
GSK-3
, Axin, and adenomatous polyposis coli are thought to form a
multiprotein complex that regulates the stability of
-catenin and
thereby its subcellular distribution (5, 7). In cells receiving a Wnt
signal
-catenin is translocated into the nucleus where it interacts
with transcription factors of the TCF family (5, 7-12). Formation of
-catenin-TCF complexes ultimately leads to the activation of
specific target genes such as Ultrabithorax,
siamois, twin, nodal-related-3 or
c-MYC (13-17).
Whereas the importance of the
-catenin-TCF complex during activation
of Wnt target genes is well established, the specific function of
-catenin and its mechanism of action are less clear. TCF
factors are sequence-specific DNA-binding proteins with a high mobility
group domain recognizing a common consensus motif but which lack
classical transactivation domains (12, 18). Instead, LEF-1, for
instance, performs architectural functions or relies on interactions
with accessory factors such as TLE/groucho co-repressors or a
transcriptional coactivator termed ALY to modulate gene expression
(19-21).
-Catenin could therefore act either by inducing changes in
promoter structure (8), by alleviating repression through displacement
of the TLE/groucho factors, or by providing a TAD. In fact, a TAD has
been identified at the C terminus of
-catenin (22). This domain is
both necessary and sufficient for the signaling activity of
-catenin
in early Xenopus development (23), and in the fly the
absence of the corresponding region from Armadillo causes diverse
developmental defects (24, 25). Thus, the currently prevailing view is
that docking of
-catenin to TCF factors primarily serves to deliver the C-terminal TAD to promoter elements of Wnt target genes
and thereby to attract the basal transcription machinery (5, 12, 22).
Therefore, it is important to seek evidence for an interaction between
-catenin and the general transcription apparatus.
-Catenin shares 68 and 71% sequence similarity with Plakoglobin and
Armadillo, respectively (1). All three proteins can interact with TCFs
(7, 9) and possess transactivation domains at their C termini (22, 26).
Nonetheless neither
-catenin nor Plakoglobin can rescue the
signaling defects of Armadillo mutant flies (27), even
though
-catenin can interact with Pangolin/dLEF-1 and murine LEF-1
can function as a downstream effector of Armadillo in
Drosophila (15, 28). Moreover, Plakoglobin cannot replace
-catenin in mouse embryonic development (29), and it is dispensable during early Xenopus development, whereas
-catenin is
absolutely required to establish Spemann organizer activity (30, 31). Although the basis for these differences is unknown, it is likely that
they are associated with
-catenin, Plakoglobin, and Armadillo themselves, and further characterization of their transactivation properties might provide insight into their distinct functions.
To understand better the signaling activities of
-catenin and its
relatives, we have systematically investigated their transactivation properties. Since analyses of catenin-dependent
transcriptional activation in vertebrate cells is frequently biased by
the presence of endogenous
-catenin (26, 32, 33), we have used the
yeast Saccharomyces cerevisiae as a model system that does
not possess cadherins, catenins, TCFs, or homologs of any of the
components of the Wnt signaling pathway except GSK-3
. Our results
indicate that the overall activity of
-catenin stems from the
functional cooperation of multiple transactivating elements distributed
over broad regions at both its C terminus and N terminus, as well as parts of the Arm repeat region. Interestingly,
-catenin differs from
Plakoglobin and Armadillo with respect to the transactivation capacities of its N terminus. This could, at least in part, explain their distinct signaling characteristics. We also show that
transactivating elements of
-catenin can specifically interact with
the TATA-binding protein in vitro. This further supports the
model that a major function of
-catenin during Wnt signaling is to
recruit components of the basal transcription machinery to promoter
regions of
-catenin/TCF target genes.
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EXPERIMENTAL PROCEDURES |
Yeast Strains--
The strains used in this study were EGY48
(MATa, his3, trp1, ura3-1,
leu2::pLEU2-LexAop6) (34) and its derivative AYH50
that carries a single chromosomal copy of the LexAop8-
Gal1-lacZ::URA3 cassette in the ura3
locus. Yeast were grown under standard conditions in rich or synthetic
minimal media (35).
Plasmids--
All plasmids were constructed and purified using
standard procedures (36). Sequences of plasmids generated with
polymerase chain reaction fragments were verified using the BigDye
dideoxy cycle sequencing kit (Applied Biosystems) on an Applied
Biosystems 310 sequencer.
The complete coding regions for mouse
-catenin from pGEX4T1MMBC
(37), human Plakoglobin from pGEX4T1HPG (37), and human p120ctn from pGEXp120 (38) were inserted, respectively,
into the SmaI/XhoI sites,
NcoI/EcoRI sites, and EcoRI site of
p570.1, which was made by cloning a ScaI/SalI
fragment carrying the ADH1 promoter/terminator cassette from pACT2
(CLONTECH) without the GAL4 activation domain, into
pRS424 (39) cut with ScaI and XhoI. The parental
plasmid for expression of the LexA fusions was p573.2, which is a
derivative of pEG202 (34) based on the pRS423 backbone (39). To
generate the LexA-LEF-1 expression vector, the entire coding region of murine LEF-1 (9) was cloned into p573.2. From this construct an
EcoRI/EcoNI fragment or an
EcoNI/XhoI fragment was deleted to obtain
LexA-LEF
N or LexA-LEF
C. To make constructs coding for
LexA-
-catenin, LexA-p120ctn, LexA-Plakoglobin, and
LexA-Armadillo, an EcoRI/BamHI fragment from
pGEX4T1MMBC, a NcoI/XhoI fragment from
pGEX4T1HPG, an EcoRI fragment from pGEXp120, or a
BamHI/NotI fragment from pGEXArmadillo was
inserted into p573.2. Mutants with N-terminal deletions of LexA-
-catenin, LexA-Plakoglobin, and LexA-Armadillo were generated by in-frame insertion of suitable restriction fragments into p573.2. C-terminal deletions of LexA-
-catenin, LexA-Plakoglobin, and LexA-Armadillo were obtained by cutting of the corresponding
full-length constructs with appropriate restriction enzymes within
coding regions and downstream thereof and religating. Deletion
constructs for fine mapping of transactivating elements in
-catenin,
Plakoglobin, and Armadillo were made by polymerase chain reaction
amplification of the desired fragments and insertion into the
EcoRI/BamHI sites of p573.2. To obtain plasmids
for the expression of GAL4-
-catenin fusion proteins in mammalian
cells, we excised EcoRI/NotI restriction fragments coding for the desired
-catenin portions from the
appropriate yeast expression vectors and cloned them into the
EcoRI/Bsp120I sites downstream of the GAL4 DBD in
pCMVGAL4 (40). The luciferase reporter construct pG5E1bLuc was made by
inserting a PvuII/BamHI fragment from pG5E1bCAT
(41) into the SmaI/BglII sites of pGL3Basic (Promega).
Expression plasmids for GST fusions with
-catenin residues 1-781,
1-284, 1-183, 1-119, or 683-781 and WT or histidine-tagged human
TBP (TBP-His6) have been described (37, 42). All other GST
fusions were generated by inserting appropriate DNA fragments from the
LexA-expression plasmids or by inserting polymerase chain reaction-derived DNA fragments into the
EcoRI/XhoI sites of pGEX-2TK with the polylinker
region from pGEX-4T-1 (Amersham Pharmacia Biotech).
Yeast Transformation and
-Galactosidase Assay--
Yeast
strains were transformed by the lithium acetate method (43) and plated
on appropriate selective media. To determine
-galactosidase activity
in EGY48, individual transformants were pregrown to saturation in
selective media, diluted into YEPD to an A600 of 0.2, and
regrown until mid-log phase. One ml from each culture was transferred
into a 1.5-ml Eppendorf tube. Cells were pelleted, washed once in 1 ml
of Z buffer (0.1 M sodium phosphate, pH 7.0, 10 mM KCl, 1 mM MgSO4), resuspended in
200 µl of Z buffer, and permeabilized by two cycles of freezing and
thawing. Of the final cell suspension 20 µl was used to determine
-galactosidase activity as described (23). Values given represent
the average from at least three independent measurements with several
isolates for each plasmid combination and are expressed as relative
light units (RLU) per A600 of cells. To
determine
-galactosidase activity after transformation of AYH50,
samples corresponding to one-tenth of the transformation reaction were
grown to saturation in selective media. The cultures were then diluted
to an A600 0.2 and analyzed for
-galactosidase activity as above. Each measurement from these pool
cultures averages the activity of approximately 1000 independent primary transformants.
Preparation of Whole Cell Extracts from Yeast and Western Blot
Analyses--
For preparation of whole cell extracts the same cultures
were used as for determining
-galactosidase activity. From each culture aliquots with 2 A600 units of cells were
pelleted, washed once in sterile water, and resuspended in 50 µl of
50 mM HEPES/NaOH, pH 7.4, 300 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Nonidet P-40, and 0.4%
"Complete" protease inhibitor mix (Roche Molecular Biochemicals). An equal volume of glass beads was added, and cell lysis was carried out by vortexing for 5 min on an IKA Vibrax VXR mixer at 4 °C. After
addition of SDS-PAGE loading buffer 0.2 A600
units were separated by SDS-polyacrylamide gel electrophoresis and
transferred onto nitrocellulose. Western blotting experiments were
performed as described (23) using polyclonal antisera against LexA
(Invitrogen) or mouse monoclonal antibodies against
-catenin,
Plakoglobin, and p120ctn (Transduction Laboratories).
Transient Transfections and Reporter Gene Assays--
Human 293 kidney cells (ATCC number CRL-1573) were transfected by the
calcium-phosphate coprecipitation method as described (23) using 1.0 µg of luciferase reporter plasmid, 0.5 µg of expression vector for
the GAL4-
-catenin fusions, and 1.0 µg of pRSVLacZ as internal
control. Cell lysis occurred in 100 µl of 50 mM Tris
phosphate, pH 7.8, 250 mM KCl, 0.1% Nonidet P-40, 10% glycerol, on ice for 20 min. Luciferase activities were determined as
before (23) and normalized against
-galactosidase activities. Average values from at least three independent transfection experiments are given.
Preparation of Nuclear Extracts--
Nuclear extracts from HeLa
cells grown to confluence in eight 15-cm tissue culture dishes were
prepared as described (44) except that nuclei were extracted in 1 volume of elution buffer (20 mM HEPES/KOH, pH 7.5, 420 mM NaCl, 25% glycerol, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM
dithiothreitol, and Complete protease inhibitor mix). After elution the
nuclei were removed by centrifugation at 25,000 × g
for 30 min, and the nuclear extract was stored in small aliquots at
80 °C without dialysis. For use in pull-down assays the crude
nuclear extracts were diluted 1:5 in elution buffer without NaCl.
Expression and Purification of Recombinant Protein--
GST
fusion proteins were expressed in Escherichia coli BL21
(DE3) and purified with GSH-Sepharose beads (Amersham Pharmacia Biotech) as described (9, 37). After elution from the GSH-Sepharose fusion proteins were dialyzed against 50 mM Tris/HCl, pH
8.0, concentrated using Centricon ultrafiltration cartridges where necessary, frozen in liquid nitrogen, and stored at
80 °C until use. To express TBP-His6 in E. coli the plasmid
pQE60-TBP was transformed into E. coli M15/pREP4 (Qiagen)
and TBP-His6 was purified by ion exchange and metal
affinity chromatography as described (42, 45).
GST Pull-down Assays--
Approximately 10 µl bed volume of
GSH-Sepharose beads (Amersham Pharmacia Biotech) was preincubated in
200 µl of pull-down buffer (20 mM HEPES/KOH, pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 0.05% Nonidet P-40, 1 mM
dithiothreitol, 0.02% bovine serum albumin, and Complete protease
inhibitor) mix together with the various GST fusion proteins. For
pull-down assays from nuclear extracts 5 µg GST or GST fusion protein
was used; for pull-down assays from reticulocyte lysates we used 1 µg
of GST or GST fusion proteins with C-terminal portions of
-catenin
and 5 µg of GST or GST fusion proteins with N-terminal portions of
-catenin, respectively. After 30 min of preincubation binding
reactions were supplemented with 20 µl of the Ni-NTA eluate
containing approximately 500 ng of TBP-His6 or with 1 µl
of reticulocyte lysate containing human TBP that had been transcribed
from pCS2+TBP and translated in vitro in the presence of
[35S]methionine as described by the manufacturer (SP6 TNT
system, Promega). For pull-down experiments from nuclear extracts, the prebinding solution was removed and replaced with 500 µl of diluted nuclear extract (approximately 500 µg of protein) from HeLa cells. Binding reactions were done on a shaker platform for 2 h at
4 °C. The GSH-Sepharose beads were pelleted; the supernatant with unbound material was removed; and the beads were washed extensively in
pull-down buffer with 200 mM KCl. Bound proteins were
eluted from the GSH beads with SDS-PAGE loading buffer, resolved by
electrophoresis on 10% SDS-polyacrylamide gels, and analyzed by
Western blotting with mouse monoclonal anti-TBP antibodies
(Transduction Laboratories, Promega) or by fluorography after soaking
the gels in Amplify solution (Amersham Pharmacia Biotech).
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RESULTS |
-Catenin and Plakoglobin Can Function as Transcriptional
Coactivators of LEF-1 in Yeast--
To determine whether it was
possible to analyze transactivation by
-catenin and other Arm family
members in the yeast S. cerevisiae, we inserted the coding
regions for
-catenin, Plakoglobin, and p120ctn into a
multicopy yeast expression vector. Full-length LEF-1, the N-terminal
CBD of LEF-1 (8, 9), or a C-terminal portion lacking the CBD (Fig.
1A) were fused to the LexA
DNA-binding domain and also inserted into a multicopy yeast expression
plasmid. Combinations of the resulting plasmids were transformed into
the yeast strain EGY48 carrying a plasmid-borne lacZ
reporter gene driven by the GAL1 minimal promoter and the LexA operator
(34). Individual transformants were isolated and analyzed for
expression of the LexA-LEF-1 fusion proteins,
-catenin, Plakoglobin,
and p120ctn. All factors were properly expressed as shown
by Western blotting experiments performed on whole cell extracts that
were probed with anti-LexA antibodies to detect the different
LexA-LEF-1 fusions (Fig. 1B, lanes 1-4) or monoclonal
antibodies against
-catenin, Plakoglobin, and p120ctn
(Fig. 1B, lanes 5-13).

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Fig. 1.
Transactivation by
-catenin and Plakoglobin but not
p120ctn in yeast cells. A, schematic
representation of -catenin, Plakoglobin, p120ctn, and
the LexA-LEF-1, LexA- -catenin, LexA-Plakoglobin, and
LexA-p120ctn fusions. Numbers of residues present in each
protein, the CBD, and the high mobility group domain in LEF-1 and the
Arm repeats (dark stippled boxes) in -catenin,
Plakoglobin, and p120ctn are shown. Hatched
bars, LexA. B, Western blot analyses to determine
expression of -catenin, Plakoglobin, or p120ctn and the
various LexA fusions. High copy number expression plasmids for the
constructs as indicated were transformed into the yeast strain EGY48
(34). Individual transformants were isolated, and whole cell extracts
were prepared and separated on 10 (lanes 1-4) or 7.5%
SDS-polyacrylamide gels (lanes 5-13). After transfer onto
nitrocellulose Western blots were probed with antibodies against LexA
(lanes 1-4), -catenin (lanes 5-7),
Plakoglobin (lanes 8-10), or p120ctn
(lanes 11-13). Material migrating faster than the
full-length proteins in lanes 10, 12, and 13 represents degradation products of LexA-Plakoglobin,
p120ctn, and LexA-p120ctn, respectively.
Lane 10 shows a shorter exposure of the same blot as for
lanes 8 and 9. C, EGY48 was
transformed with the indicated combinations of expression plasmids and
the lacZ reporter plasmid pSH18-34 (34), and
-galactosidase activity was determined in individual transformants.
Values are given as relative light units
(RLU)/A600 of cells and represent the
average from at least three measurements with two independent isolates
for each factor or factor combination. Standard deviations are
given.
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Next, we analyzed expression of the lacZ gene (Fig.
1C). As expected, no activation of the lacZ
reporter gene was seen in control experiments with the catenins alone
(Fig. 1C, lines 1-4) or with the LexA-LEF-1 fusion proteins
in the absence of the catenins (Fig. 1C, lines 5, 9 and
12). Also, the combination of LexA-LEF-1 and
p120ctn had no effect on reporter gene activity (Fig.
1C, line 8). In contrast, coexpression of
-catenin or
Plakoglobin and LexA-LEF-1 strongly activated the lacZ gene
(Fig. 1C, lines 6 and 7). The addition
of the LEF-1 CBD to the LexA DNA-binding domain was sufficient to allow
activation of the lacZ gene by
-catenin or Plakoglobin, whereas no transactivation was seen with LexA-LEF
N lacking the CBD
(Fig. 1C, lines 10, 11, 13, and 14). Thus, as in
vertebrate cells,
-catenin and Plakoglobin can interact with LEF-1
through the CBD and function as transcriptional coactivators of LEF-1 when expressed in yeast.
Both
-catenin and Plakoglobin harbor transactivation domains that
can act independently from LEF-1 when added to a heterologous DNA
binding moiety (22, 26). To test whether these TADs also functioned in
yeast, we fused the entire coding region of
-catenin or Plakoglobin
directly to the LexA DNA-binding domain. We also constructed a
LexA-p120ctn fusion since we could not rule out that lack
of transactivation by coexpressing LexA-LEF-1 and p120ctn
was due to a lack of interaction between the two factors. The different
LexA fusions were expressed in EGY48 (Fig. 1B), and stimulation of reporter gene expression was determined. The
LexA-p120ctn fusion protein did not increase expression of
the lacZ reporter (Fig. 1C line 18) even though
its expression was clearly detectable (Fig. 1B, lane 13).
Apparently, p120ctn has no transactivation capacity. In
contrast, high levels of
-galactosidase activity were measured in
cell lysates from transformants expressing LexA-
-catenin and
LexA-Plakoglobin (Fig. 1C, lines 16 and
17). Thus, the transactivation domains present in
-catenin and Plakoglobin can act independently from LEF-1 also in
yeast cells. In summary, transcriptional stimulation by the
LexA-
-catenin and LexA-Plakoglobin fusions or the
LexA-LEF-1·
-catenin and LexA-LEF-1·Plakoglobin complexes in
yeast appears very similar to their activities in vertebrate or insect
cells. This indicates that the mechanism of transactivation by the
catenins is conserved throughout eukaryotic organisms and that S. cerevisiae can be used as a model system to characterize further
the transactivation properties of
-catenin and related proteins.
-Catenin Contains Multiple Transactivating Elements within Its N
and C Termini--
A systematic analysis of the regions within
-catenin that contribute to its activity as transcriptional
activator has not previously been undertaken. Therefore, we generated a
series of C-terminal and N-terminal deletion mutants of
-catenin
which were fused to the LexA DNA-binding domain. To assess their
transactivation capacities, the various deletion mutants were
introduced into the yeast strain AYH50 which contains a single,
chromosomal copy of the LexA-operator-GAL1-driven lacZ gene.
Thus, variations in expression levels of the LexA-
-catenin fusions
are less likely to influence the evaluation of their transactivation potential.
First, the N-terminal TAD was analyzed (Fig.
2A). A construct with residues
1-159 was chosen as reference, because preliminary tests had revealed
that the highest transactivating capacity was associated with this
region. The presence of additional sequences from the Arm repeat region
in some constructs reduced or even abolished the activity of the
N-terminal TAD (data not shown). Expression of the various LexA fusions
was examined by Western blotting (Fig. 2C). LexA fusions
with
-catenin residues 1-159 and 47-159 were equally potent in
reporter gene stimulation and achieved about 50% of the activity of
-catenin (Fig. 2A, lines 2, 3, and 5). The
fusion with residues 47-117 also transactivated but to a lesser extent
(30% activity of
-catenin) (Fig. 2A, line 6). No
transactivation potential was associated with residues 1-52 and
108-159 (Fig. 2A, lines 4 and 7). Comparison of
reporter gene activation by constructs with residues 47-159, 47-117,
and 108-159, however, indicates that residues 108-159 augment
transactivation by elements within residues 47-117. Thus, it appears
that the full activity of the N-terminal TAD is conferred by the
cooperation of multiple elements present within residues 47-159.

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Fig. 2.
Fine mapping of transactivating elements at
the N terminus and within the Arm repeat region of
-catenin. Fragments of -catenin coding for
portions of the N terminus (A) or of the Arm repeat region
(B) as schematically shown were fused to the LexA
DNA-binding domain and expressed in AYH50. lacZ reporter
gene activity in pools of stably transformed cells was determined as
described under "Experimental Procedures." Stippled
bars, -catenin sequences. Dark stippled boxes, Arm
repeats. End points of -catenin sequences are given. C
and D, Western blot analyses to monitor expression of
LexA- -catenin fusion proteins in whole cell extracts with antibodies
against LexA. M, molecular weight markers. C,
N-terminal fusions. D, fusions from the Arm repeat region of
-catenin.
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Next, we examined the activity of LexA-
-catenin fusions with
residues from the Arm repeat region, all of which were properly expressed when introduced into AYH50 (Fig. 2D). A construct
with
-catenin residues 108-683 reached approximately 42% of the
activity of
-catenin (Fig. 2B, line 3). All additional
deletion mutants lacking portions from the C-terminal end of the Arm
repeat region were unable to stimulate reporter gene expression (Fig.
2B, lines 4-7). Analyses of the complementary set of
mutants showed that the transactivation capacity of the remaining
-catenin sequences was abolished upon removal of residues 159-281
(Fig. 2B, lines 8 and 9). However, even shorter
LexA-
-catenin fusions containing residues 400-683 or 536-683
regained some activity, reaching about 10.5 and 6.5% of the activity
of full-length
-catenin (Fig. 2B, lines 10 and
11). From this we conclude that aside from the elements between residues 47-117 and 108-159, additional transactivating regions of
-catenin reside between residues 159-281 and 536-683. Although the elements between residues 159-281 and 536-683 have no or
only little capacity to stimulate expression of the lacZ reporter gene by themselves, when present together they can achieve considerable activity. Again, this implies that overall activity of
-catenin results from the combined action of multiple
transactivating elements.
Finally, we analyzed the C-terminal TAD. A LexA fusion with
-catenin
residues 536-781 was nearly as active as
-catenin (Fig. 3A, lines 2 and 3).
Further removal of residues up to positions 630, 662, and 695 reduced
the activity of the resulting LexA fusions to 68, 30, and 32% of
maximum levels (Fig. 3A, lines 4-6). Slightly higher
activity was seen with residues 728-781 (50% activity of
-catenin)
(Fig. 3A, line 7). Weak, but significant,
reporter gene activation was mediated even by a fusion construct with
only the C-terminal 31 residues (7.5% activity of
-catenin) (Fig. 3A, line 8). Analyses of two complementary
deletion mutants showed that a LexA fusion with residues 536-661 was
unable to stimulate reporter gene expression, whereas residues 536-683
weakly activated expression (Fig. 3A, lines 9 and
10, and Fig. 2). If one takes into account that LexA fusions
with residues 662-781 and 695-781 are expressed at very low levels
(Fig. 3B), it appears likely that the C-terminal
transactivating elements are largely contained within residues 662-781
and that there is probably only a minor contribution of residues
536-661 to the overall activity of the C-terminal TAD.

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Fig. 3.
Fine mapping of transactivating elements at
the C terminus of -catenin. A,
fragments of -catenin (stippled bars) coding for portions
of the C terminus of -catenin as schematically shown in the
left part of the figure were fused to the LexA DNA-binding
domain and expressed in AYH50. lacZ reporter gene activity
in pools of stably transformed cells was determined as before. End
points of -catenin sequences are given. B, expression of
the LexA fusion proteins was analyzed by Western blotting experiments
performed on whole cell extracts with antibodies against LexA.
M, molecular weight markers. Asterisks in
lanes 6 and 7 denote the position of weakly
expressed LexA fusions with -catenin residues 662-781 and 695-781,
respectively.
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Additional analyses of LexA fusions with
-catenin residues 662-756
and 662-729 revealed that both constructs reached approximately 36%
of maximum activity (Fig. 3B, lines 11 and 12). The finding that each of the LexA fusions with
-catenin residues 662-729 and 728-781 strongly activated the
lacZ reporter gene identifies at least two independent
transactivating subdomains at the C terminus of
-catenin. To define
further the transactivating element(s) within residues 662-729, we
generated constructs containing residues 662-698 and 681-729. Despite
extensive overlap both fusions had little or no transactivation
capacity (Fig. 3B, lines 13 and 14). Most likely,
this region, too, contains two or more elements that together generate
the transactivating properties of the parental fragment. Interestingly,
residues 662-683 on their own do not transactivate (Fig. 3A,
lines 9, 10, and 13), yet their presence confers some
activity on the LexA fusion with residues 536-683. This result implies
that a transactivating element is located around position 662 and
therefore may have been destroyed in constructs with this end point.
Alternatively, an additional transactivating element, without
autonomous activity but with the ability to synergize with other
elements, may be located more N-terminally of position 662. At present,
we cannot distinguish between these two possibilities. Nonetheless, it
appears that the C-terminal TAD of
-catenin is a complicated
assembly of multiple subdomains that are distributed over the entire
region encompassing at least residues 662-781 and that functionally
cooperate to generate its full transactivation capacity.
Functional Testing of
-Catenin Transactivating Elements in Human
Cells--
To determine whether the transactivating elements
identified in yeast were also functional in vertebrate cells, we fused
various portions of
-catenin to the GAL4 DNA-binding domain. The
resulting constructs were transfected into human 293 kidney cells
together with a luciferase reporter construct containing five GAL4 DNA binding motifs in front of the adenovirus E1b promoter (41). Western
blotting experiments with an anti-GAL4 antibody showed that all
GAL4-
-catenin fusions were properly expressed and present at roughly
similar levels (data not shown). Stimulation of the luciferase reporter
gene by the various GAL4-
-catenin fusions in 293 cells is shown in
Fig. 4. For comparison, the
transactivation potential of the corresponding LexA fusions in yeast is
included. Qualitatively, the results obtained in yeast and in 293 cells are very similar, although quantitatively we observed differences. Compared with full-length
-catenin the N-terminal fragment with residues 1-159 is considerably more active in 293 cells. However, as
in yeast cells, the only autonomous transactivating activity is
associated with residues 47-117 (20-30% of full-length
-catenin). As before, residues 1-52 and 108-159 have no transactivation
potential above control levels. Apparently, the function of the
N-terminal TAD depends on cooperation of its subelements even more in
293 cells than in yeast. Although the C-terminal fragment with residues 536-781 displayed approximately the same relative activity in yeast
and 293 cells, fragments with residues 695-781 and 728-781 turned out
to be much more potent in the human cell line and exceeded the activity
of full-length
-catenin by 15- and 2.5-fold, respectively. Residues
662-729, on the other hand, reached only 16% of the activity of
full-length
-catenin in 293 cells compared with 36% in yeast. The
reduced activity of this fragment indicates that at least one of the
elements, which is active in yeast, is not functional in 293 cells. In
accordance with this assumption we found that residues 536-683, which
weakly transactivated in yeast (see Fig. 2B), are completely
inactive in the human cell line. Given that in the absence of this
portion of
-catenin the activity of residues 695-781 vastly
increases, it is even possible that a negative regulatory function
resides within residues 536-683. The lack of transactivation by this
region of
-catenin also explains why residues 159-683 failed to
activate the reporter gene in 293 cells since transcriptional
activation by this domain in yeast required the simultaneous activity
of transactivating elements at both of its ends. In summary, with the
exception of the transactivating element located between residues 536 and 683, all other elements identified in yeast are also functional in
293 cells. Although some of these elements appear to operate more
efficiently in human cells, this result further validates the use of
the yeast system to analyze transactivation by
-catenin.

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Fig. 4.
Comparison of transactivation by N- and
C-terminal domains of -catenin in yeast and in
human cells. Human 293 kidney cells were transfected with
pG5E1bLuc, pRSVLacZ, and expression vectors for GAL4(DBD) and
GAL4(DBD)- -catenin fusions, as indicated. Luciferase activities were
determined 40 h post-transfection as described under
"Experimental Procedures" and are expressed as percentages relative
to full-length -catenin fused to GAL4(DBD). For comparison, the
relative activities of the various -catenin fragments in yeast cells
as determined in Figs. 2 and 3 are included. Values given are derived
from at least three independent experiments. Standard deviations are
shown. Note the different scales of the top and
bottom panels.
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Transactivating Properties of the Plakoglobin and Armadillo N
Termini Differ from Their Counterpart in
-Catenin--
To test
whether the signaling differences between
-catenin, Plakoglobin, and
Armadillo might be related to their transactivating properties, we
constructed LexA-Plakoglobin and LexA-Armadillo fusions containing
amino acid sequences that correspond to the N-terminal and C-terminal
transactivation domains in
-catenin. As expected, full-length
LexA-Plakoglobin and LexA-Armadillo fusions strongly activated the
lacZ reporter gene. LexA-Plakoglobin or LexA-Armadillo
fusions including the C-terminal one and a half-Arm repeat as well as
the non-conserved C-terminal extension stimulated
-galactosidase
expression at levels even higher than the full-length proteins (Fig.
5A, lines 2 and 5 and lines 8 and 11). Slightly shorter LexA
fusions that consisted primarily of the C-terminal extension exhibited
somewhat lower activities concomitant with a drop in expression levels
of the respective constructs (Fig. 5A, lines 6 and
12, and Fig. 5, B and C). This is
highly reminiscent of the results obtained with the corresponding
LexA-
-catenin fusions and suggests that functional differences
between Armadillo,
-catenin, and Plakoglobin probably are not based
on particular properties of their C-terminal transactivating
regions.

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Fig. 5.
Analyses of the transactivation potential of
N- and C-terminal regions in Plakoglobin and Armadillo.
A, full-length Plakoglobin (light gray bars) or
Armadillo (dotted bars) and N-terminal or C-terminal
fragments as shown in left part of the figure were fused to
the LexA DNA-binding domain and expressed in AYH50. The Arm repeat
regions are highlighted (dark boxes), and end points of
Plakoglobin and Armadillo sequences are given. lacZ reporter
gene activity was determined as before. B and C,
expression of the LexA fusion proteins was analyzed by Western blotting
experiments performed on whole cell extracts with antibodies against
LexA. M, molecular weight markers. B,
LexA-Plakoglobin fusion proteins. C, LexA-Armadillo fusion
proteins. Lane 7 shows a longer exposure of the Western blot
to visualize weakly expressed full-length LexA-Armadillo.
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In contrast, a striking difference between
-catenin, Armadillo, and
Plakoglobin was revealed when we examined LexA fusions with N-terminal
portions of the factors. Plakoglobin residues 1-149 (equivalent to
residues 1-159 in
-catenin) possess no transactivation capacity,
and a construct with Plakoglobin residues 39-108 (equivalent to
-catenin 47-117) was also completely inactive for reporter gene
stimulation (Fig. 5A, lines 3 and 4).
On the other hand, we found that LexA fusions with Armadillo residues
1-167 strongly activated the lacZ gene, as did their
counterpart in
-catenin (residues 1-147) (Fig. 5A, lines
9). However, the relative importance or distribution of
transactivating elements at the Armadillo N terminus seems to differ
because Armadillo residues 58-125, which correspond to the main
activating element at the
-catenin N terminus, confer only 4.5% of
the maximum activity of the Armadillo N-terminal TAD as opposed to 56%
of the same region in
-catenin (compare Figs. 2A and
5A, line 10). These results show that there are functional differences between the N termini of
-catenin, Plakoglobin, and Armadillo and suggest that the distinct signaling properties of these
factors partly arise from the presence or absence of specific transactivating elements at their N termini.
Transactivating Elements of
-Catenin Interact with the
TATA-binding Protein--
The fact that the transactivation domains of
-catenin function in yeast and vertebrate cells suggests that an
evolutionarily conserved component of the transcription apparatus may
be one of the targets of
-catenin during the process of gene
activation. TBP is highly conserved among various organisms and is
known to interact with a variety of transcription factors (46). We
therefore wished to determine whether
-catenin could interact with
TBP. Full-length
-catenin, a C-terminal portion with residues
536-781, and a N-terminal fragment with residues 1-284 were expressed
as GST fusion proteins in E. coli and used in pull-down
assays with nuclear extracts from HeLa cells. Proteins bound to the GST
fusions were recovered from the binding reactions and analyzed for the presence or absence of TBP by Western blotting experiments with specific antibodies. As shown in Fig. 6, TBP specifically associates with the fusion proteins containing
-catenin sequences but not with
GST alone. The highest amount of TBP was isolated with the C-terminal
fusion construct, whereas the interaction between TBP and the
N-terminal region was weakest (Fig. 6,
left panel). A similar binding pattern was also seen with
radioactively labeled TBP that was transcribed and translated in
vitro (Fig. 6, center panel). We also tested the
interaction between the GST fusion proteins and recombinant,
histidine-tagged TBP-His6. In these experiments
-catenin
residues 536-781 again exhibited the highest level of TBP binding,
whereas the N-terminal fragment showed an increased ability to interact
with TBP compared with the full-length
-catenin fusion (Fig. 6,
right panel). Although the basis for the differences in
relative binding between TBP from nuclear extracts and recombinant TBP
from E. coli is not clear at the moment, these results show
that
-catenin and fragments harboring the transactivating elements
of
-catenin can directly interact with TBP in vitro.

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Fig. 6.
The TATA-binding protein interacts with
-catenin in vitro. GST or GST
fusion proteins with -catenin sequences as shown were purified from
E. coli, bound to glutathione-Sepharose beads, and incubated
with nuclear extracts from HeLa cells (left panel), TBP,
transcribed and translated in vitro in the presence of
[35S]methionine (center) or
TBP-His6 purified from E. coli (right
panel). Material bound to the GSH beads was recovered from the
binding reactions, washed extensively, and analyzed by SDS-PAGE and
Western blotting with antibodies against TBP or by fluorography. In
pull-down experiments with [35S]methionine-labeled TBP an
aliquot of the reticulocyte lysate corresponding to 10% of the
material used for the assay was analyzed in parallel (center,
lane 5, Input).
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To gain information about the importance of the TBP interaction for
transactivation by
-catenin, we localized the TBP-binding elements
at the
-catenin N and C termini. GST fusions with various subfragments derived from
-catenin residues 1-284 or 536-781 were
prepared and used for binding experiments with TBP-His6
(Fig. 7). Fusion proteins with
-catenin residues 1-183, 1-119, and 120-302 all bound TBP but
with reduced efficiency compared with
-catenin residues 1-284 (Fig.
6A, compare lanes 4-6 and 12). These
findings indicate that at least two binding sites for TBP are present
at the N terminus of
-catenin. One of these binding regions most
likely is contained within residues 183-284. The other, which appears
to localize more N-terminally, was examined more closely. Analyses of
complementary sets of
-catenin fragments show that the presence of
residues 47-107 is absolutely required for the interaction with TBP
(Fig. 7A, lanes 5-7 and 9-11), even though
residues 47-117 by themselves bound TBP only weakly and preferentially
associated with a particular degradation product of
TBP-His6 (Fig. 7A, lane 13). Yet, a fragment
with residues 39-108 from Plakoglobin corresponding to
-catenin
residues 47-117 did not interact with TBP at all (Fig. 7A, lane
14).

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Fig. 7.
Mapping of TBP interaction domains in
-catenin. A, GST or GST fusions
with N-terminal portions of -catenin (5 µg) as indicated were used
in pull-down assays with bacterially expressed TBP-His6.
For comparison, GST fusions with residues 536-781 from the C terminus
of -catenin (1 µg) (lane 3) or with residues 39-108 of
Plakoglobin (lane 14) equivalent to residues 47-117 of
-catenin were also used. In lane 1 (Input) an
aliquot corresponding to 10% of the total amount of
TBP-His6 used for the assay was loaded. B,
mapping of TBP interaction domains at the C terminus of -catenin.
GST or GST fusions with C-terminal portions of -catenin (1 µg) as
indicated were used in pull-down assays with bacterially expressed
TBP-His6. In lane 1 (Input) an
aliquot corresponding to 10% of the total amount of
TBP-His6 was loaded. C, summary of the results
from the binding experiments shown in A and B and
the transactivation studies. Elements in -catenin that transactivate
autonomously are denoted by hatched bars, and elements that
transactivate only when combined with autonomous elements or with each
other are marked lightly hatched. Regions in -catenin
which can interact with TBP are shown above -catenin.
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Analyses of fusion proteins with residues derived from the C terminus
of
-catenin demonstrated that constructs with residues 536-781,
630-781, 536-755, and 536-729 all bound TBP with equal efficiency
(Fig. 7B, lanes 3 and 4 and 8 and
9), whereas a fusion with residues 536-675 displayed a much
reduced TBP binding activity (Fig. 7B, lane 10). GST fusions
with
-catenin residues 683-781, 728-781, 750-781, and 536-630
were not able to interact with TBP (Fig. 7B, lanes 5-7 and
11). Also, a
-catenin fragment with residues 662-729 did
not interact with TBP (data not shown). From this we conclude that the
interaction between TBP and the C terminus of
-catenin is mediated
by residues 630-729 and that residues 630-675 critically contribute
to this binding activity.
A comparison of TBP-binding elements and transactivating elements in
-catenin reveals that of the three TBP-binding regions TI, TII, and
TIII, which we have identified, TII located within Arm repeats 2-4
correlates with a domain that was unable to transactivate on its own
but that was found to play an auxiliary role during transactivation by
-catenin or
-catenin derivatives (Fig. 7C). TIII
partially overlaps with an autonomous transactivating element at the C
terminus of
-catenin but also requires residues located more
N-terminally in a region that does not transactivate by itself. TI
between residues 47 and 107 coincides with the main stimulatory element
in the N terminus of
-catenin. Taken together, our results suggest
that the interaction with the TATA-binding protein may be involved in
various aspects of
-catenin function and is likely to be of
particular importance for the activity of the N-terminal transactivation domain.
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DISCUSSION |
Functional Differences among Arm Family Members--
The
importance of
-catenin and Armadillo as transcriptional coactivators
of TCF family members is well documented, and there is considerable
evidence that transactivation is the primary function that is required
for the signaling activity of
-catenin (22, 23, 47). Yet the
molecular basis for the functional differences among the otherwise
highly related catenins and their precise mode of action are poorly
understood. Here we have used the yeast S. cerevisiae as a
model system to analyze catenin-mediated transcriptional activation. As
p120ctn shares certain features with
-catenin,
Plakoglobin, and Armadillo, including the presence of multiple Arm
repeats, binding to cadherins, and being a target for tyrosine kinases
(48), one might have expected that p120ctn also acts as
transcriptional activator. However, the absence of any transactivation
capacity of p120ctn in our test system is in good agreement
with its apparent lack of signaling activity upon overexpression in
Xenopus embryos (38). On the other hand this underscores the
significance of transactivation by
-catenin, Armadillo, and
specifically Plakoglobin. Although to date there is no evidence that
Plakoglobin can have signal transducing activity, its ability to
associate with LEF-1 and its transactivation potential nonetheless
argue that Plakoglobin, too, may perform a function similar to
-catenin and Armadillo. A possible signaling activity of Plakoglobin
may become important later in development or in the adult organism,
when an involvement of Plakoglobin in transcriptional regulation is
more difficult to investigate and might be obscured by the presence of
-catenin (49).
Previous studies have established that
-catenin, Armadillo,
and Plakoglobin can activate reporter gene constructs when fused to a
heterologous DNA-binding domain (22, 26) but hitherto mainly the
activities of their C termini have been considered, and here the three
factors do not appear to differ (26). However, this does not explain
why Plakoglobin and
-catenin cannot substitute for each other or
complement Arm mutations in Drosophila. The presence of transactivating elements at the N termini of Armadillo and
-catenin raises the possibility that the N-terminal TADs and
possibly other portions of
-catenin or Armadillo contribute to
efficient target gene induction or to the activation of a particular set of target genes. Cooperativity of multiple transactivation domains
in a single transcription factor as well as cell type-specific and
promoter-specific activity of distinct transactivation domains is also
found among nuclear hormone receptors (50, 51). In support of this idea
the Armadillo mutant Arm
N
with a complete deletion of the N terminus is less potent at rescuing
armadillo mutant flies than the
Arm
S10 allele carrying only a short
deletion of the GSK-3
phosphorylation sites (52). In addition, we
have observed here that LexA fusions with the
-catenin Arm repeat
region are able to activate a reporter gene. This activity is probably
specific since p120ctn, despite the presence of multiple
Arm repeats, lacks any transactivation potential. In earlier
experiments expression of the Arm repeat region of
-catenin induced
ectopic body axes in X. laevis (53). Although this effect
was recently assumed to be mediated by endogenous
-catenin (33), our
findings imply that indeed the Arm repeats themselves may have
functioned as signal transducers. Moreover, whereas either the
C-terminal TAD of
-catenin or a strong heterologous transactivation
domain can elicit the formation of ectopic dorso-ventral body axes in
Xenopus embryos (23), it appears that efficient transformation of fibroblasts involves a
-catenin specific activity and is less related to the potency of the transactivator used (47).
Thus, although transactivation per se is clearly critical for the signaling functions of catenins, some of their activities may
be context-dependent, and
-catenin, Armadillo, and
Plakoglobin may have evolved to perform specific tasks possibly through
distinct properties of their N termini.
When recruited to a promoter by LexA-LEF-1 fusion proteins, Plakoglobin
was somewhat less efficient as a transcriptional coactivator than
-catenin. This could reflect the absence of the N-terminal TAD. Yet,
Plakoglobin is a potent coactivator of LEF-1 in yeast. This is in
contrast to the situation in human 293 embryonic kidney cells where it
was suggested that
-catenin was the preferred interaction partner
for LEF-1 (26). The differential behavior of Plakoglobin in yeast and
in 293 cells implies that the interaction of Plakoglobin with LEF-1,
and possibly of catenins with TCF family members in general, is
differentially controlled in a cell type-specific manner,
e.g. by posttranslational modification. A candidate for such
a modifying activity is the CBP acetylase, which was recently identified as a regulator of Wnt signaling in Drosophila
(54) and which is absent from yeast. In fact, our observations that WT
-catenin and Plakoglobin readily coactivate transcription in yeast,
while in vertebrate cells vast overexpression of WT proteins or the use
of mutant
-catenin is required, also indicate that in S. cerevisiae the catenins are not subject to the negative control
mechanisms that govern their activities in other eucaryotic cells.
Therefore, yeast may be useful for the reconstitution of these control
mechanisms and their detailed mutational analysis.
Structural and Functional Characteristics of Transactivating
Elements in
-Catenin--
The architectural function of
-catenin
in cell adhesion raised the intriguing possibility that
-catenin
performed its task as a transcription factor by an unusual mechanism
(8, 18, 55). However, there is now increasing evidence that
-catenin functions primarily as a conventional transactivator. As is the case
for other well characterized transcription factors, the transactivation domains of
-catenin possess a modular structure, function in combination with different types of DNA-binding domains at natural and
artificial target gene promoters (22, 23, 26, 47, 55), and work in
mammalian as well as yeast cells. Sequence examination of the
transactivating elements in
-catenin reveals that they are highly
enriched for acidic residues and in particular the C-terminal TAD
harbors multiple amino acid sequence motifs with neighboring asparagine
and leucine residues similar to the minimal transactivating modules of
the VP16 and RelA (56, 57). A close relationship between
-catenin
and VP16 is further implied by the fact that the VP16 transactivation
domain can be substituted for
-catenin both in signaling and
oncogenic transformation (23, 47). On the basis of these structural and
functional similarities, we propose that
-catenin, like VP16, p53,
and RelA, belongs to the class of acidic activators.
Interactions between
-Catenin and TBP--
The rate-limiting
step for gene activation appears to be the recruitment of general
transcription factors to the promoter region which can be facilitated
through interactions between GTFs and transactivators (46). By using an
in vitro approach, we have shown that three distinct regions
in
-catenin can directly bind to recombinant TBP or to endogenous
TBP in nuclear extracts. Thus, it is possible that
-catenin indeed
stimulates target gene expression by contacting the basal transcription
apparatus. Although our results suggest that this may occur directly,
additional indirect contacts between
-catenin and TBP may be
established through the recently discovered bridging factor Pontin52
(42). At present, however, it is not clear whether Pontin52 has a
stimulatory function or whether it rather serves to destroy
-catenin-containing transcription factor complexes similarly to the
MOT1 ATPase, which disrupts TBP-DNA complexes (58). Further analysis is
needed to distinguish between these possibilities.
By comparing transactivating and TBP-binding elements, we have
attempted to gain information about the physiological relevance of the
different TBP/
-catenin interactions. We have obtained a good match
for the N-terminal core TAD, which we found to both bind to TBP and to
transactivate. In contrast, the corresponding domain in Plakoglobin
failed to interact with TBP and has no transactivation potential. This
correlation suggests that TBP may well be a relevant target for the
N-terminal transactivating element. The other two TBP-binding domains,
TII and TIII, coincide with auxiliary transactivating elements and may
be involved in transactivation by the Arm repeat domain. However,
unlike TI, TII and TIII have no autonomous transactivation potential.
Multiple, functionally distinct TBP-binding domains have also been
described for the VP16 TAD (59, 60). In this case and also for the p53
tumor suppressor protein, certain mutations that disrupted the
interaction with TBP in vitro turned out to have no impact
on the activity of the VP16 or p53 transactivation domains in
vivo (60, 61). TBP binding itself may not be sufficient to confer
transactivation potential, and possibly additional contacts with the
basal transcription machinery must be made. In fact, acidic activators
are known to have multiple binding partners among the GTFs (59,
61-66). The functional difference between the three TBP-binding
regions in
-catenin therefore could arise from the presence of
additional GTF-binding sites in the vicinity of the N-terminal
autonomously transactivating element. That TBP is not the only target
for
-catenin is also suggested by the observation that C-terminal
transactivating elements, which are functional in vivo, do
not bind to TBP in vitro. Therefore, it appears likely that
additional cofactors exist which assist
-catenin during gene
activation. Experiments are under way to identify these factors.