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Molecular and Cellular Biology, June 1999, p. 4503-4515, Vol. 19, No. 6
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Nuclear Localization and Formation of
-Catenin-Lymphoid
Enhancer Factor 1 Complexes Are Not Sufficient for Activation of
Gene Expression
Mary G.
Prieve and
Marian L.
Waterman*
Department of Microbiology and Molecular
Genetics, College of Medicine, University of California, Irvine,
Irvine, California 92697-4025
Received 16 December 1998/Returned for modification 18 January
1999/Accepted 15 March 1999
 |
ABSTRACT |
In response to activation of the Wnt signaling pathway,
-catenin
accumulates in the nucleus, where it cooperates with LEF/TCF (for
lymphoid enhancer factor and T-cell factor) transcription factors to
activate gene expression. The mechanisms by which
-catenin undergoes
this shift in location and participates in activation of gene
transcription are unknown. We demonstrate here that
-catenin can be
imported into the nucleus independently of LEF/TCF binding, and it may
also be exported from nuclei. We have introduced a small deletion
within
-catenin (
19) that disrupts binding to LEF-1, E-cadherin,
and APC but not axin. This
19
-catenin mutant localizes to the
nucleus because it may not be efficiently sequestered in the cytoplasm.
The nuclear localization of
19 definitively demonstrates that the
mechanisms by which
-catenin localizes in the nucleus are completely
independent of LEF/TCF factors.
-Catenin and LEF-1 complexes can
activate reporter gene expression in a transformed T-lymphocyte cell
line (Jurkat) but not in normal T lymphocytes, even though both factors
are nuclear. Thus, localization of both factors to the nucleus is not
sufficient for activation of gene expression. Excess
-catenin can
squelch reporter gene activation by LEF-1-
-catenin complexes but
not activation by the transcription factor VP16. Taken together, these
data suggest that a third component is necessary for gene activation
and that this third component may vary with cell type.
 |
INTRODUCTION |
Wnt signals initiate at the plasma
membrane of receptive cells when the secreted ligand Wnt binds to
frizzled, a seven-transmembrane receptor on the plasma membrane
(9, 10, 15, 43). For Wnt-1, this binding event activates a
multistep cascade that directs nuclear transport of a cytoplasmic
protein named
-catenin. Wnt-1 directs nuclear localization of
-catenin in part by inhibiting the activity of the serine/threonine
kinase GSK-3
via the action of a cytoplasmic phosphoprotein named
dishevelled (14, 35, 61, 68). Most models portray GSK-3
,
along with another protein called APC (for adenomatous polyposis coli),
as promoting
-catenin degradation via the ubiquitin-proteasome
pathway (1, 52, 57). Under these circumstances,
-catenin
is stable only at the plasma membrane in cell adherens junctions as an
adapter protein between the cytoplasmic tail of E-cadherin receptors
and
-catenin, a cytoskeleton binding protein. A Wnt-dependent
increase in cytosolic
-catenin is soon followed by the appearance of
-catenin in the nucleus, where it cooperates with LEF/TCF (for
lymphoid enhancer factor and T-cell factor) proteins to alter
transcription of gene targets (29, 44, 49, 53). In addition
to these parts of the cascade, additional steps are involved because
proteins such as axin/conductin/axil and GBP have been identified
recently as regulatory components of this pathway (6, 32, 34, 69, 70, 72). The final steps of this cascade, nuclear accumulation and gene activation by
-catenin, are not well characterized.
-Catenin is known to interact with a number of proteins, and the
list of these interactions is growing (conductin/axin/axil, APC,
-catenin, E-cadherin, presenilin, LEF/TCF proteins) (10, 71). These proteins are localized to different subcellular
compartments, such as the plasma membrane (E-cadherin,
-catenin),
the endoplasmic reticulum and Golgi (presenilin and derivative
fragments), the cytoplasm (APC, conductin/axin/axil), and the nucleus
(LEF/TCF transcription factors). Although interaction domains for each individual protein have not been precisely delimited, it appears that
-catenin interacts with these proteins through overlapping portions
of its armadillo repeat array (reference 10 and
references therein). The repeating unit of this array, the armadillo
repeat (referred to here as the arm repeat) is so named because of its sequence similarity to a reiterated, degenerate 37- to 43-amino-acid (aa) motif in the Drosophila melanogaster ortholog armadillo
(41, 50). Multiple arm repeats occur in tandem arrays from
as few as 4 to as many as 13 repeats, and together they function as a protein interaction domain. The crystal structure for the 12 arm repeats of
-catenin was solved recently (28). Structural
analysis has revealed that arm repeats are alpha-helical and pack
against one another to form an elongated superhelix of alpha-helices. The groove created by the twisted superhelical structure is highly charged and proposed to be the interacting surface for
-catenin targets. The structure of a second armadillo repeat protein,
yeast importin
/karyopherin
/Srp1, was recently
determined, and while the amino acid sequence of its 9- or 10-arm
repeat array is only 17% similar to
-catenin, the repeats fold into
a similar twisted structure with a groove (13). Importin
, and a second arm repeat protein, importin
, function together
as receptors for nuclear localization signals (NLSs) in proteins
(26, 47). NLSs are highly basic, whereas
-catenin targets
are enriched in acidic residues. Thus, even though arm repeat arrays
are found in a wide variety of proteins and the amino acid sequences of
their targets vary, there are unifying structural similarities in the
way these proteins bind to their targets.
A major unanswered question is how
-catenin localizes to different
subcellular compartments. For example, since
-catenin does not have
an obvious NLS for importin
/
receptors, it was proposed that
-catenin must enter the nucleus by binding to NLS-containing LEF/TCF
transcription factors in the cytoplasm (29, 33, 43, 44). An
alternative model, in which
-catenin can access the nucleus
independently of LEF/TCF binding (17), has been proposed. This model was derived from a study of nuclear localization of recombinant
-catenin in digitonin-permeabilized cells where
-catenin entered the nucleus on its own, in the absence of
exogenously added extract (17). The inference from this
study was that
-catenin transport was independent of
importin/karyopherins and also of any LEF/TCF protein.
In the nucleus,
-catenin is known to interact with at least one
class of transcription factors, the LEF/TCF family of proteins (36, 44, 62, 64, 66). LEF/TCF factors are sequence-specific DNA binding proteins that function as context-dependent transcription factors, unable to work on their own but requiring the cooperative effort of adjacent DNA binding transcription proteins or non-DNA binding cofactors (11, 21). LEF/TCF proteins have a single HMG (high-mobility-group) DNA binding domain that includes an HMG box,
which functions as a DNA binding-bending motif, and a B box, a very
basic cluster of 8 or 9 aa with dual functions in DNA binding and
nuclear localization (20, 23, 40, 54).
-Catenin binds to
a region within the first 56 aa of all known LEF/TCF proteins (7,
29, 44). The highly conserved
-catenin binding domain in
LEF/TCF proteins is separate and independent from the HMG DNA binding
domain, which is located in the middle or near the C terminus. A model
has been proposed whereby the tethering of
-catenin to promoters or
enhancers by LEF/TCF factors enables a transcription activation domain
in
-catenin, perhaps in cooperation with LEF/TCF sequences, to
regulate transcription of Wnt-responsive genes (27, 63).
Reporter plasmids containing only multimerized LEF/TCF binding sites
are activated by this complex in a context-independent manner that
apparently does not require additional factors binding to flanking
regulatory elements (27, 29, 36, 44, 45, 53, 59, 63).
However, whether there is a requirement for additional factors that do
not bind DNA is not known. We have constructed a deletion mutant that
does not bind LEF/TCF proteins and have used this mutant protein to explore the mechanisms by which
-catenin accumulates in nuclei and
activates gene expression. We show that
-catenin localizes to nuclei
independently of its interaction with LEF/TCF. We also show that in the
nucleus
-catenin cooperates with LEF-1 and perhaps at least one
other factor to activate reporter gene expression.
 |
MATERIALS AND METHODS |
Cell culture, DNA transfections, luciferase and
-galactosidase
assays.
Cos-1 cells were cultured in high-glucose (4.5-g/l)
Dulbecco's modified Eagle's medium (Gibco). 293 cells were cultured
in Eagle's minimum essential medium with nonessential amino acids (Irvine Scientific). Jurkat cells were cultured in RPMI 1640 (Irvine Scientific). All media contained 10% fetal bovine serum (Irvine Scientific), 100 U of penicillin per ml, 100 µg of streptomycin per
ml, 250 ng of amphotericin B (Gibco) per ml, and 50 µM
-mercaptoethanol. Cell lines were grown in a humidified atmosphere
containing 5% CO2 at 37°C. Cos-1, 293, and Jurkat cells
were transiently transfected by electroporation. Normal human
peripheral blood mononuclear cells (PBMCs) were prepared and
transfected as previously described (30). For reporter gene
expression, 1 µg of a reporter gene plasmid containing five tandemly
repeated Gal4 recognition elements upstream of a luciferase reporter
gene (generous gift of P. Carlsson, Göteborg University,
Göteborg, Sweden) was cotransfected with various amounts of
G4LEF-1,
-catenin, and control expression plasmids as indicated in
the figure legends. One microgram of the reporter plasmid Top TK
(generous gift of H. Clevers, University of Utrecht) was cotransfected
with 10 µg each of wild-type
-catenin and wild-type LEF-1
expression plasmids for reporter gene expression in PBMCs. Cells were
harvested at 15 to 24 h posttransfection, and cell lysates were
analyzed for luciferase activity as described previously
(42). To normalize between transfections, 0.5 µg of LacZ
expression plasmid was included in each sample and
-galactosidase activity was determined with the Galacto-Light/Plus kit (Tropix, Inc.).
Plasmid construction.
c-Myc epitope-tagged
Xenopus full-length
-catenin from pCS2 (generous gift of
B. Gumbiner, Memorial Sloan-Kettering Cancer Center, New York, N.Y.)
was cloned into pBluescript at the BamHI and
HincII sites. An internal deletion of aa 346 to 364 of
-catenin (
19
-catenin) was constructed by digesting
-catenin in pBluescript with AflII and
HindIII. Blunt ends were generated with Klenow enzyme,
and the ends were religated.
19
-catenin was then transferred to
pCS2 in the same orientation as
-catenin in pCS2. An N-terminal deletion of the first 148 aa of
19
-catenin (
N
19
-catenin) was constructed by digesting MT10
-catenin in pCS2 (aa
149 to 525) (generous gift of B. Gumbiner) with XhoI and
SnaBI and replacing this fragment with a fragment from
19
-catenin digested at the identical sites. All mutations were
sequenced to ensure that no second-site mutations were introduced
during the cloning procedure. Construction of G4LEF, G4LEF (aa 80 to
256), FL LEF, G4VP16, and
NLEF (aa 67 to 399) is mentioned elsewhere
(11).
GST fusion protein purification.
Glutathione
S-transferase (GST) and full-length GST-hLEF-1 were
purified as described previously (54). GST-importin-
(generous gift of S. A. Adam, Northwestern University) was
purified as above in the presence of 0.1 mM ZnSO4.
In vitro binding and immunoprecipitation assays.
Radiolabeled full-length
-catenin,
19
-catenin, the C-terminal
tail of E-cadherin (generous gift of P. Polakis, Onyx
Pharmaceuticals), full-length pendulin/Rch1/importin-
2, and an
N-terminal deletion of pendulin (aa 62 to 529 [
N importin-
2])
(55) were prepared with a coupled in vitro
transcription/translation system (Promega) and
[35S]methionine (DuPont-NEN). The in vitro binding assay
was performed as previously described (55). For the
coimmunoprecipitation assay, 293 cells were transiently transfected by
electroporation with 10 µg of the plasmids described in the figure
legends. At 24 h after transfection, cells were harvested and
washed once in 1× phosphate-buffered saline (PBS). Each cell pellet
was resuspended in ice-cold IP buffer, which contains 20 mM Tris (pH
8.0), 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, 10%
glycerol, 50 mM NaF, 1 mM NaVO4, 20 µg of soybean trypsin
inhibitor per ml, 10 µg of aprotinin per ml, 4 µg of leupeptin per
ml, and 1 µg of pepstatin A per ml. Lysates were microcentrifuged at
14,000 rpm for 10 min at 4°C. Two microliters of an anti-c-Myc
monoclonal antibody (MAb) (9E10) (Santa Cruz Biotechnology) was added
to each cleared lysate and incubated on a rotating platform for 1 h at 4°C. Forty microliters of a 50% slurry of protein G-Sepharose
beads (Pharmacia) diluted in IP buffer was added to each lysate and
incubated for 1 h at 4°C. The precipitates were centrifuged for
5 min at 2,000 rpm, washed three times with 1 ml of IP buffer for each
wash, and boiled in 20 µl of 2× sodium dodecyl sulfate (SDS) sample
buffer. The samples were analyzed by Western blotting with protein
A-purified hLEF-1 polyclonal antiserum and a secondary horseradish
peroxidase-conjugated goat anti-rabbit antibody (Amersham) and
detection by enhanced chemiluminescence (ECL) (Amersham). In samples
with additional hLEF-1 recombinant protein, 5 µg (100 pmol) of
recombinant, purified full-length LEF-1 protein (46a) was
added and the samples were incubated for 30 min on a rotating platform
at 4°C prior to incubation with anti-c-Myc antibody. For normalizing
levels of
-catenin, the immunoblot was stripped and reblotted with
anti-c-Myc MAb, followed by incubation with horseradish
peroxidase-conjugated goat anti-mouse antibody (Amersham) and detection
with the ECL kit.
The in vitro coimmunoprecipitation assay represented by Fig. 2C was
performed as described previously (46) except as follows. Five microliters of 35S-labeled in vitro-translated
wild-type or
19
-catenin was incubated with 1.5 µg of purified
recombinant APC2 (aa 1034 to 2130), APC3 (aa 2130 to 2844), or axin (aa
320 to 530) (generous gifts from P. Polakis, Onyx Pharmaceuticals) or
no protein, and the volume of the mixture was brought to 30 µl with
buffer B (20 mM Tris [pH 8.0], 150 mM NaCl, 0.5% Nonidet P-40) plus
0.2% bovine serum albumin (BSA). Two micrograms of Glu-Glu antibody
(gift of P. Polakis) and 10 µl of protein G-Sepharose beads were
added to immunoprecipitate Glu-Glu-tagged APC2, APC3, and axin.
For the in vitro coimmunoprecipitation assay represented by Fig. 2D,
35S-labeled in vitro-translated C-terminal tail of
E-cadherin was incubated with either radiolabeled full-length
-catenin or
19
-catenin in TBST (200 mM NaCl, 10 mM Tris-HCl
[pH 8.0], 0.2% Tween 20) with 0.2% BSA in a 100-µl total volume
for 30 min at room temperature on a rotating platform. One microliter
of anti-c-Myc MAb and 20 µl of a 50% slurry of protein G-Sepharose
beads (Pharmacia) washed in TBST-0.2% BSA were added and incubated
for 1 h at room temperature on a rotating platform. The
precipitates were centrifuged for 5 min at 2,000 rpm, washed three
times with 1 ml of TBST for each wash, and boiled in 15 µl of 2× SDS
sample buffer. The samples were analyzed by SDS-polyacrylamide gel
electrophoresis, and the gel was treated with En3Hance
(DuPont-NEN) and exposed to film overnight.
Fluorescence microscopy.
Full-length
-catenin in pCS2,
MT10 in pCS2 (generous gifts of B. Gumbiner, Memorial Sloan-Kettering
Cancer Center, New York, N.Y.), and
19
-catenin in pCS2 were
transiently transfected into Cos-1 and Jurkat cells, followed by
immunofluorescence, as described previously (54), with
anti-c-Myc MAb and fluorescein isothiocyanate (FITC)-conjugated or
Texas red-conjugated anti-mouse (Amersham) antibodies. Jurkat cells
were washed in 1× PBS and diluted to ~1 × 106
cells/ml, and 100 µl of cells were cytospun onto
poly-L-lysine-coated slides prior to fixation. For
detection of endogenous
-catenin, anti-
-catenin MAb (Transduction
Laboratories) was used. Nuclei were stained with
4',6-diamidino-2-phenylindole (DAPI) prior to being mounted slides with
mounting media. Normal human PBMCs were prepared and transfected as
previously described (30). Four hours after transfection,
PBMCs were treated with media containing 25 ng of
phorbol-12-myristate-13-acetate (PMA) per ml and 1 µg of ionomycin
per ml or with media containing an identical amount of ethyl alcohol
carrier control (untreated) for 3 h at 37°C. For export of
-catenin, 10 µg of actinomycin D per ml and 100 µg of
cycloheximide per ml were added to PBMCs treated with PMA and ionomycin
and cells were incubated for 3 h at 37°C. Cells were washed with
PBS containing identical amounts of PMA and ionomycin for treated
cells, PBS alone for untreated cells, and PMA, ionomycin, actinomycin
D, and cycloheximide for export conditions and then resuspended to
~1 × 106 cells/ml. One hundred microliters of this
cell preparation was cytospun onto poly-L-lysine-coated
slides. Cells were fixed in 3.7% formaldehyde-1× PBS, and
immunofluorescence was performed as described above. Slides were
examined as described previously (55).
 |
RESULTS |
An internal deletion of arm repeat 6 destroys LEF/TCF binding in
vitro and in vivo.
In determining which region of the
-catenin
arm repeat array recognizes LEF/TCF proteins, we found that a deletion
of part of the sixth arm repeat could destroy binding. Figure
1A depicts the location and scale of the
deletion mutation (
19). Based on the arrangement of alpha-helices in
the arm repeat array, the 19-aa deletion removes the final four
residues of helix 3 of the fifth arm repeat and 15 aa (helix 1 and 2)
of the sixth arm repeat (Fig. 1B). This region is well conserved
between all
-catenin orthologs as well as catenin-related proteins
such as plakoglobin. Mutant
19 was tested for binding to human LEF-1
in a GST pull-down experiment. Wild-type
-catenin and
19 were
labeled with [35S]methionine by in vitro
transcription/translation procedures and incubated with either
GST-LEF-1 or GST recombinant fusion protein. Figure
2A shows that while wild-type
-catenin
binds specifically to GST-LEF-1, the
19
-catenin protein does
not bind. Neither the wild type nor
19 binds nonspecifically to the GST control fusion protein. The loss of LEF-1 binding was examined in
vivo in transfected 293 embryonic kidney cells. A eukaryotic expression
vector producing Myc-tagged wild-type or
19
-catenin protein was
cotransfected with an expression vector producing wild-type LEF-1.
Extracts from these transfected cells were prepared and anti-Myc MAb
was added to coimmunoprecipitate
-catenin and interacting proteins.
Using LEF-1 polyclonal antisera, a Western analysis was performed to
determine if LEF-1 protein was present in these coimmunoprecipitates.
Figure 2B shows that LEF-1 interacts with wild-type
-catenin in vivo
but not with
19
-catenin (compare lanes 1 and 2). Five micrograms
(~1 × 10
10 mol) of recombinant, full-length LEF-1
protein was added to cell extracts to determine whether a weak
interaction between LEF-1 and
19 could be detected, but even an
excess amount of LEF-1 protein was unable to reveal any binding to
19 (lanes 5 and 6). This total lack of an interaction is not due to
differential stability of the
19 mutant in 293 cells, because
reprobing of the same Western blot with anti-Myc antibody showed that
the expression levels of wild-type and
19
-catenin proteins are
nearly the same (compare lanes 1 and 2 and lanes 5 and 6). Western
analysis of total cell lysates from transiently transfected cells also showed that the expression levels of wild-type and
19
-catenin vary by less than twofold (data not shown).

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FIG. 1.
Amino acid sequence and structure of the arm repeat
array of wild-type -catenin and deletion mutants. (A) Schematic of
wild-type -catenin, a 19-aa deletion mutant of -catenin ( 19);
MT10 -catenin, which contains only the first nine arm repeats and no
flanking sequences; and N 19, which is identical to 19 but
contains a deletion of the first 148 aa. (B) Amino acid alignment of
the arm repeats of -catenin. According to the recently solved
structure for -catenin, each arm repeat consists of three
alpha-helices, which are denoted by H1, H2, and H3 (28). The
sequence deleted in the 19 -catenin mutant is in boldface type
and underlined.
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FIG. 2.
Deletion of 19 aa within the arm repeat array of
-catenin disrupts interaction with LEF-1, APC, and E-cadherin but
not axin. (A) Wild-type (wt) -catenin and a 19-aa deletion mutant of
-catenin ( 19) were produced by a coupled
transcription/translation system (Promega) in the presence of
[35S]methionine. These proteins were incubated with
recombinant GST-LEF-1 or GST alone in a GST pull-down binding assay
(see Materials and Methods). The first two lanes indicate the amount of
-catenin protein added (10% input). Glutathione beads are able to
cosediment wild-type -catenin with GST-LEF-1 but not GST alone. In
contrast, 19 -catenin is unable to bind to GST-LEF-1. (B)
Whole-cell extracts were prepared from 293 cells that had been
transiently transfected with the indicated expression plasmids (see
Materials and Methods). Anti-Myc MAb was used to coimmunoprecipitate
-catenin-associated proteins. A Western blot of the
immunoprecipitates was probed with LEF-1 antisera. LEF-1 can be
detected in coimmunoprecipitates with wild-type (lane 1) but not 19
-catenin (lane 2). Purified recombinant LEF-1 protein (5 µg) added
to cell extract prior to immunoprecipitation (lanes 5 and 6) is able to
interact only with wild-type -catenin and not even weakly with 19
-catenin. In lanes 3 and 4, a control eukaryotic expression plasmid
was cotransfected with either LEF-1 (lane 3) or wild-type -catenin
(lane 4) to show that LEF-1 coimmunoprecipitates specifically by
association with -catenin and not nonspecifically with the anti-Myc
MAb. The same Western blot was stripped and reprobed with anti-Myc MAb
to determine that the protein levels of wild-type and 19 -catenin
were equivalent. (C) In vitro-translated wild-type or 19 -catenin
was incubated with two purified APC fragments (APC2 and APC3), an axin
fragment (Axin), or no protein (Cnt). Anti-Glu-Glu antibody and protein
G-Sepharose beads were added in a coimmunoprecipitation assay. Ten
percent of the amount of -catenin protein added is shown (Tln). APC2
and axin are coimmunoprecipitated with wild-type -catenin but only
axin is coimmunoprecipitated with 19 -catenin. (D) In
vitro-translated wild-type or 19 -catenin and the C-terminal tail
of E-cadherin were incubated with anti-Myc MAb and protein G-Sepharose
beads in a coimmunoprecipitation assay. E-cadherin can be detected in
coimmunoprecipitates with wild-type but not 19 -catenin. The
number of additional bands is due to partial translation products of
-catenin which are coimmunoprecipitated with anti-Myc MAb.
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Mutant
19 binds to axin but is defective for binding to APC and
the cytoplasmic tail of E-cadherin.
Large deletions of the arm
repeat region have been shown to destroy binding to APC, axin, and
E-cadherin (18, 31, 32, 48). We tested whether the much
smaller
19 deletion might also disrupt interactions with these three
proteins (Fig. 2C and D). Wild-type
-catenin and
19 mutant
proteins were in vitro translated and tested for binding to two
Glu-Glu-tagged protein fragments of APC (APC2 and APC3) and one
Glu-Glu-tagged protein fragment of axin (Fig. 2C). APC2 (aa 1034 to
2130) and axin (aa 320 to 530) each contain a
-catenin binding
domain, while APC3 (aa 2130 to 2844) does not and serves as a negative
control (58). Anti-Glu-Glu antibody and protein G-Sepharose
beads were added to immunoprecipitate Glu-Glu-tagged protein. Wild-type
-catenin coimmunoprecipitated with APC2 and axin but not with APC3
or protein G-Sepharose beads alone.
19 mutant protein
coimmunoprecipitated with axin at levels that were significantly
greater than that observed with wild-type
-catenin. In contrast,
19 did not bind to APC2. Next, wild-type
-catenin or
19 mutant
protein was incubated with the cytoplasmic tail of E-cadherin (Fig.
2D). Anti-Myc antibody and protein G-Sepharose beads were added to
immunoprecipitate Myc-tagged
-catenin protein. E-cadherin
coimmunoprecipitated with wild-type
-catenin but not with
19.
Thus, sequences within or near the sixth arm repeat are required for
binding to three of the four proteins tested. These sequences could be
directly involved in interactions with E-cadherin, APC, and LEF/TCF.
Alternatively, because tandem arm repeats are interdependent for
correct folding, removal of these sequences may have caused structural
distortions in neighboring regions of the arm repeat array. However,
this region must not be grossly misfolded because removal of half of
the sixth arm repeat does not disrupt axin recognition of arm repeats 3 through 7 (6, 32).
Nuclear localization of
-catenin is LEF/TCF independent.
Since
19 is unable to bind to LEF/TCF proteins, this mutant allowed
us to test the model that
-catenin can move into the nucleus
independent of LEF/TCF binding in vivo. Immunofluorescence detection of
transiently expressed
-catenin protein in Cos-1 cells was carried
out to determine patterns of subcellular localization. Overexpressed
-catenin was detected with an anti-Myc MAb. Localization of
transiently expressed protein was similar to that of endogenous
-catenin (Fig. 3A and B); the protein
localized to plasma membrane and cytoplasmic sites. A mutant
-catenin protein (MT10), missing the flanking NH2- and
COOH-terminal regions as well as arm repeats 10 through 12, was also
observed to localize to membranes and cytoplasm (Fig. 1A and 3C)
(16). In marked contrast, most of the
19 protein was
present in the nucleus (Fig. 3D). This surprising pattern of nuclear
localization directly shows that
-catenin can move into the nucleus
independently of LEF/TCF binding. The mechanism by which
-catenin
gains entry to the nucleus is not understood. Nuclear localization of
recombinant
-catenin in digitonin-permeabilized cells does not
require cytoplasmic extracts which contain importin
/
NLS
receptor complexes (2, 25). While these data suggest that
-catenin is capable of entering nuclei independently of importins, a
direct test for recognition of
-catenin by an NLS receptor complex
has not been reported.

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FIG. 3.
19 -catenin localizes constitutively to the
nucleus. Plasmids encoding Myc epitope-tagged wild-type -catenin
(A), MT10 (C), and 19 (D) were transiently transfected into Cos-1
cells. Subcellular localization was determined 48 h later by
immunofluorescence with anti-Myc MAb to detect transfected -catenin
expression plasmids or anti- -catenin MAb to detect endogenous
-catenin. In some cases, FITC antimouse or Texas red antimouse
secondary antibody was used. (A to C) Wild-type -catenin, endogenous
-catenin, and MT10 localize to various sites in the cytoplasm and
the plasma membrane. Background levels are higher for MT10, since
immunofluorescence of MT10 was weak and exposure times were longer than
for the other -catenin proteins. (D) In contrast to wild-type and
MT10 -catenin, 19 -catenin localizes predominantly to the
nucleus. DAPI staining of DNA indicates the locations of nuclei.
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To test for a direct interaction between
-catenin and the importin
/
NLS receptor,
-catenin and importin
2 (also known as
pendulin/Rch1) were translated in vitro and incubated with recombinant
GST-importin
(Fig. 4). An N-terminal
deletion mutant of importin
2 defective for GST-importin
binding
was also generated by in vitro translation and used as a control for
binding specificity in the assay. Full-length importin
2 but not the
N-terminal deletion (
N importin
2) bound specifically to
GST-importin
, as expected.
-Catenin did not bind to importin
2 or GST-importin
. There was no interaction even if all three
proteins were incubated together. A test for binding of
-catenin
alone with importin
2 was also negative (data not shown). In
vitro-translated
-catenin is capable of binding to LEF-1 protein,
indicating that the in vitro-translated protein product is functional
in other well-known binding activities (Fig. 2A). These data suggest
that
-catenin does not interact with classic NLS receptor complexes
such as importin
2-importin
. It is possible that
-catenin
could interact with another importin
or
subtype not tested
here. However, a lack of an interaction is consistent with a model that
-catenin enters the nucleus via a mechanism independent of the NLS
receptor pathway (17).

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FIG. 4.
-Catenin does not interact with importin 2 or
importin . A GST pull-down experiment with 35S-labeled,
in vitro-translated wild-type -catenin, importin 2, and
recombinant GST-importin was performed. An N-terminal deletion
mutant of importin 2 that removes the importin interaction
domain ( N importin 2) was included as a negative control for
nonspecific binding. The first three lanes indicate the amount of
importin 2 and -catenin proteins added (10% input). While
full-length (FL) importin 2 readily interacts with GST-importin ,
-catenin does not, either in the presence or absence of importin
2.
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Localization of
-catenin in normal peripheral blood lymphocytes
and Jurkat T lymphocytes.
Many studies that have examined
-catenin activation of gene transcription have used the B-lymphocyte
cell line IIA1.6 (36, 37, 44, 63). Likewise, T-lymphocyte
cell lines have often been used to study gene activation by LEF-1 and
TCF-1 (11, 21, 22, 62, 64). In contrast to transcription
activation, most studies of
-catenin localization have used adherent
cell lines or normal cells in whole animal model systems. Therefore, we
wished to determine the subcellular localization of
-catenin in
normal, untransformed T lymphocytes as well as a T-lymphocyte cell
line. Preparations of PBMCs from normal human blood can be transiently transfected with plasmid DNA (30). The overwhelming majority of cells that are transfected in this procedure are T lymphocytes (>98%), and we used this method to assess the localization of
-catenin in nontransformed T cells. Both wild-type
-catenin and
MT10 localized to the plasma membrane or cytoplasm, while
19 was
localized to the nucleus (Fig. 5A)
(lymphocytes have very little cytoplasm compared to nucleus, making it
difficult to distinguish between plasma membrane and cytoplasm). The
localization patterns of wild-type and mutant
-catenins in these
cells were similar to those in Cos-1 cells (Fig. 3).

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FIG. 5.
Localization of -catenin in normal human peripheral
blood lymphocytes and Jurkat T lymphocytes. (A and B) PBMCs were
transfected with the indicated expression constructs. Four hours after
transfection, cell preparations were treated with PMA and ionomycin or
treated with ethanol as a mock control (untreated) for 3 h. (C)
Jurkat cells were transiently transfected with the indicated
-catenin constructs, and subcellular localization was determined
24 h later. Transiently expressed -catenin proteins were
detected with anti-Myc MAb and FITC-antimouse secondary antibody. DAPI
staining of DNA indicates the locations of nuclei. (A) In untreated
cells, both the wild type and MT10 are cytoplasmic while 19 is
nuclear. (B) When cells are treated with PMA and ionomycin, both
wild-type -catenin and MT10 localize to the nucleus. (C) Wild-type,
MT10, and 19 -catenin all localize to the nucleus in Jurkat
cells.
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Activation of T lymphocytes through T-cell receptor stimulation or by
PMA-ionomycin treatment can mimic some of the effects of activating the
Wnt signal transduction pathway. For example, GSK3-
, the
serine/threonine kinase that negatively regulates
-catenin, is
inhibited upon T-cell stimulation (67). This causes at least
one transcription factor (NF-AT) to accumulate in the nuclear
compartment, in part because GSK-3
-dependent nuclear export of NF-AT
is inhibited (4, 5). We tested whether activation of
transfected human T lymphocytes by PMA-ionomycin treatment would cause
a shift in the localization pattern of transiently expressed
-catenin. Transiently transfected PBMC preparations were treated
with PMA and ionomycin for 3 h prior to fixation; within this
time, there was a quantitative shift in localization of both wild-type
-catenin and MT10 protein from the plasma membrane or cytoplasm to
the nucleus in all transfected cells (Fig. 5B). The nuclear
localization of
19 remained unchanged.
Jurkat T cells are transformed T lymphocytes; these cells behave
similarly to activated T lymphocytes in that they synthesize and
secrete the cytokine interleukin 2, a marker of T-cell activation (60). Transient expression of wild-type, MT10, and
19
-catenin protein in these cells exhibited a pattern of subcellular
localization nearly identical to that of activated normal T cells (Fig.
5C). All three forms were constitutively nuclear; there was no dramatic difference between wild-type and
19
-catenin as there was in the
Cos-1 cells. These results again confirmed that nuclear localization of
-catenin is independent of LEF/TCF binding (
19 is nuclear) and
that a nuclear localization domain or determinant is located within the
first nine arm repeats of the protein (MT10 is nuclear). However, these
results also suggested that the ability of
-catenin to accumulate in
the nucleus differs between cell types.
The PMA-ionomycin-directed shift in subcellular localization of
-catenin in normal T cells suggested either that the ability of
-catenin to access its nuclear transport pathway is regulated or
that the transport pathway used by
-catenin is itself under tight
restriction. Another possibility is that
-catenin might be capable
of both nuclear import and export. In a manner similar to that observed
for NF-AT, PMA-ionomycin treatment may cause nuclear accumulation of
-catenin by shifting the kinetic balance to favor import over
export. If this were true, the constitutive nuclear accumulation of
19 might be due to an inability of the
19 mutant protein to be
exported from the nucleus.
Export of
-catenin from nuclei in normal human peripheral blood
lymphocytes.
We tested whether wild-type
-catenin could undergo
nuclear export, and we also tested whether
19 exhibited any
differences with respect to this activity. A common method to test for
nuclear export of proteins is to block nuclear import with actinomycin D treatment (a transcription inhibitor) and to block protein synthesis with cycloheximide such that no newly synthesized protein accumulates in the cytoplasm (51). Therefore, any formerly
nucleus-localized protein that accumulates in the cytoplasm in these
treated cells must have undergone active export from the nucleus. PBMC
preparations were transiently transfected with either wild-type or
19
-catenin expression plasmid and then treated with
PMA-ionomycin to direct all
-catenin protein to the nucleus (Fig.
6A). Actinomycin D and cycloheximide were
added to half of the cells to block import and de novo protein
synthesis (Fig. 6B). After 3 h, cells were processed for
immunofluorescence. Both wild-type and
19
-catenin staining was
observed only in the cytoplasm and not the nucleus, suggesting that
both were actively exported from the nucleus during actinomycin
D-cycloheximide treatment. Overall fluorescence was weaker in cells
treated with these reagents because protein synthesis was completely
inhibited for 3 h, and exported
-catenin may have been subject
to degradation in the cytoplasm. A less likely explanation is that a
low level of cytoplasmic
-catenin remained stable throughout the 3-h
incubation with cycloheximide, and nuclear
-catenin was preferentially degraded. Taken together, these data show that the
differing localization patterns between wild-type
-catenin (cytoplasm) and
19 (nucleus) in unstimulated T lymphocytes are not
due to differences in import or export.

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FIG. 6.
Wild-type and 19 -catenins are exported from the
nucleus in normal human peripheral blood lymphocytes. PBMCs were
transfected with the indicated expression constructs. (A) Six hours
prior to harvest, cell preparations were treated with PMA and ionomycin
(Control). (B) Three hours prior to harvest, cell preparations were
treated with actinomycin D and cycloheximide. Transiently expressed
-catenin was detected with anti-Myc MAb and FITC-anti-mouse
secondary antibody. DAPI staining of DNA indicates the locations of
nuclei. In cells treated with PMA and ionomycin, both wild-type and
19 -catenins are localized to the nucleus. Subsequent treatment
with actinomycin D and cycloheximide inhibits nuclear import and
protein synthesis. With these treatments, wild-type and 19
-catenins are exported to the cytoplasm.
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-Catenin- and LEF-1-dependent activation of transcription varies
in different cell types.
In addition to the dramatic effects that
the
19 mutation has on localization, we also determined the effect
of the mutation on reporter gene activation in the nucleus. We examined
the transactivation potential of LEF-1 and
-catenin in Cos-1 cells,
Jurkat T cells, and normal human T lymphocytes, all of which are
different in some aspect of
-catenin localization (Fig.
7). In order to examine the ability of
only the transiently expressed proteins to work together, a LEF-1
fusion protein, in which the first 256 aa of LEF-1 were fused to the
yeast Gal4 DNA binding domain, was used in place of full-length LEF-1
protein (G4LEF) (Fig. 7). The reporter plasmid contained five Gal4
upstream activating sites (UAS) placed upstream of a minimal promoter
driving luciferase coding sequences (G4 UAS/Luc). Transfection of the
G4LEF expression plasmid with the G4 UAS/Luc reporter plasmid did not
produce significant luciferase expression in Cos-1 cells (Fig. 7A).
However, if a
-catenin expression plasmid was included, a fourfold
increase in reporter gene expression was observed. Cotransfection of
19 with G4LEF yielded only basal levels of expression, confirming
that, in vivo, the
19 mutant does not interact with G4LEF even
though both proteins are concentrated in the nucleus (see Fig. 3 for
-catenin localization). The modest fourfold activation is due to a
bona fide interaction between LEF-1 and
-catenin, because an
N-terminal deletion of LEF-1 missing the
-catenin binding
domain does not activate reporter gene expression [G4LEF(80-256) +
-cat; Fig. 7A]. In Cos-1 cells,
overexpression of G4LEF does not cause a change in the subcellular
distribution of
-catenin protein, similar to what is observed in NIH
3T3 cells (27, 55a). Instead,
-catenin remains mostly
localized to the plasma membrane and cytoplasm, thereby accounting for
the modest levels of activation observed in these cells.

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FIG. 7.
A direct interaction between -catenin and LEF-1 is
necessary but not sufficient to activate gene expression. Wild-type,
19, or MT10 -catenin expression plasmids were cotransfected with
the Gal4 DNA binding domain (DBD) fused to LEF-1 aa 1 to 256 (G4LEF)
and a reporter plasmid containing five tandemly repeated Gal4 UAS
directing luciferase reporter gene (G4 UAS/Luc) expression in Cos-1 and
Jurkat cells and PBMCs. A summary of the -catenin localization in
each of these cell lines is shown in the upper right corner of each
panel. (A) Five micrograms of -catenin, 5 µg of G4LEF expression
plasmids, and 1 µg of reporter plasmid were transfected into Cos-1
cells. (B) Seven hundred fifty nanograms of -catenin and 50 ng of
G4LEF expression plasmids or 750 ng of G4VP16 expression plasmid and 1 µg of reporter plasmid were transfected into Jurkat cells. In the
absence of -catenin, the range of luciferase activity varied from
574 to 825. (C) The indicated expression plasmids (10 µg) were
cotransfected with 10 µg of reporter plasmid into PBMCs. P + I,
treatment of cell preparations with PMA plus ionomycin for 3 h
prior to harvest of cells; *, use of the Top TK reporter plasmid in
place of the G4 UAS/Luc reporter plasmid. A plasmid (0.5 µg)
containing the lacZ gene under the control of the CMV
promoter was cotransfected as an internal control for transfection
efficiency. G4LEF (aa 80 to 256), which does not bind to -catenin,
was used as a negative control (A and B). An empty eukaryotic
expression plasmid was used to equalize the amount of DNA used in each
transfection. Cells were harvested at 15 to 24 h posttransfection,
and cell lysates were analyzed for luciferase and -galactosidase
activity. Wild-type -catenin and LEF-1 transactivated the reporter
gene construct in Cos-1 and Jurkat cells, while neither 19 nor MT10
cooperated with LEF-1 to activate gene expression. In addition, the
level of transcription activation of -catenin-LEF-1 complexes in
Jurkat cells was higher than that observed with G4VP16 (750 ng).
-Catenin and G4LEF-1 or full-length LEF-1 (FL LEF-1) did not
transactivate reporter gene expression in PBMCs, although G4VP16 (10 µg) was able to transactivate the reporter gene in PBMCs. Error bars
were calculated by using the average of duplicate points (A and C) or
triplicate points (B). Values shown are from one of three replicate
experiments.
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In Jurkat T lymphocytes, transiently expressed
-catenin protein is
localized to the nucleus (Fig. 5C). Cotransfection of G4LEF and
-catenin expression plasmids in these cells stimulated a large
increase in reporter gene expression (150-fold) (G4LEF +
cat
[Fig. 7B]), an even greater activation than what can be achieved by
the strong activating fusion protein Gal4-VP16 (G4VP16) (35-fold). As
observed for Cos-1 cells, cotransfection of
19
-catenin did not
activate gene expression at all, nor did MT10 or the G4LEF mutant
missing the
-catenin binding domain, even though all of these
proteins are localized to the nucleus. Treatment of Jurkat cells with
PMA-ionomycin did not change the transactivation potential of LEF-1 and
-catenin. Either
-catenin has a strong costimulatory effect in
Jurkat T cells due to the overall strength of its nuclear localization
or another transcription cofactor or basal factor is abundant in these cells.
We tested whether strong activation could be observed in normal human T
lymphocytes (Fig. 7C). Surprisingly,
-catenin and G4LEF were unable
to activate gene expression, even in the presence of PMA-ionomycin
treatment (which causes a quantitative shift of
-catenin into the
nucleus [see Fig. 5B for localization patterns]). In contrast, an
equivalent amount of G4VP16 was able to produce a 115-fold increase in
reporter gene expression. Thus, activation of reporter gene expression
by the VP16 transcription activation domain is possible in normal T
cells, but
-catenin and LEF-1 are not able to elicit detectable
amounts of luciferase gene expression. This dramatic difference between
the activities of
-catenin and LEF-1 in normal versus transformed
lymphocytes is not due to differential localization or to dramatic
differences in protein stability, as our immunofluorescence data
indicate. To rule out that G4LEF was the cause of this differential
activation, we tested whether full-length
-catenin and full-length,
wild-type LEF-1 could activate gene expression with the Top TK reporter
plasmid (44). This plasmid contains five multimerized
LEF/TCF binding sites and has been used extensively to examine
LEF-1-
-catenin reporter gene activation in many different cell
types (27, 29, 36, 44, 45, 59, 63). Wild-type LEF-1 and
wild-type
-catenin were unable to activate Top TK reporter gene
expression. In addition, the Top TK reporter plasmid was tested in
Jurkat cells. High levels of wild-type LEF-1 and wild-type
-catenin
were able to activate reporter gene expression, which is similar to the
results in Fig. 7B with the G4 UAS/Luc reporter plasmid (data not
shown). Thus the G4 UAS/Luc reporter plasmid functions in a manner
similar to the wild-type LEF/TCF binding site reporter plasmid. One
possibility for the different activities of
-catenin and LEF-1 is
that another cofactor is required to activate gene expression. This
cofactor might be abundant in Jurkat cells but absent or limiting in
normal human T lymphocytes. Such a cofactor may not be an essential
basal RNA polymerase II factor, as G4VP16 fusion protein is capable of
activating reporter gene expression in either cell type.
19 can squelch
-catenin-activated gene expression but not
VP16-activated gene expression.
To test whether another nuclear
component is required for
-catenin-LEF-1 complexes to activate
reporter gene expression, a squelching experiment was designed. It has
been proposed that
-catenin contains transcription activation
domains in the C terminus and the N terminus of the protein (27,
63). Although these regions do not have sequence similarity or
amino acid compositions similar to known transcription activation
domains, they can activate gene expression when fused to heterologous
DNA binding domains such as LEF-1 or Gal4 (27, 59, 63). If
either domain of
-catenin is interacting with a third required
transcription factor, then overexpression of this domain should
sequester the third component from wild-type
-catenin-LEF-1
complexes and inhibit activation of gene transcription. This method of
inhibition is referred to as squelching.
To carry out the experiment, overexpression of the
19
-catenin
mutant was used to test for squelching. Since
19 is completely negative for binding to LEF/TCF proteins in vitro and in vivo (Fig. 2
and 7), no squelching should occur due to simple sequestration of LEF-1
protein. Secondly,
19 protein localizes constitutively to the
nucleus of all cells where squelching of transcription factor
components should occur. Jurkat cells were used because the level of
activation is very high. In the experiment represented by Fig.
8A, G4LEF and
-catenin activated
luciferase reporter gene expression 125-fold. Excess plasmid containing
the strong cytomegalovirus (CMV) promoter was used to control for
inhibitory effects due to competition for basal polymerase II
components. As this balancer plasmid was increased from a 4-fold to a
20-fold excess, a 15% drop in G4LEF/
-catenin-dependent luciferase
activity was observed (Fig. 8A). Thus, large amounts of competitive CMV promoter-containing plasmid may account for a minor inhibitory effect.
There was a 2-fold drop in luciferase activity when a 4-fold excess of
19 plasmid was included, and a 20-fold excess of
19 plasmid
caused a 22-fold drop in activation. This suggests that overexpression
of
-catenin can indeed squelch activation of reporter gene
expression by sequestering a required component. The
19 mutant
-catenin was further modified to remove the hydrophilic N terminus
(
N
19) (Fig. 1). Fourfold and 20-fold excess
N
19 plasmid
also exhibited squelching, albeit to a slightly lesser extent. Finally,
a 60-fold excess of an expression plasmid encoding
NLEF, an
N-terminal deletion mutant of wild-type LEF-1 missing the
-catenin
binding domain, was unable to squelch reporter gene expression,
suggesting that the squelching activity is specific to a region of
-catenin. To determine whether
19
-catenin was squelching by
inhibiting a general step in polymerase II transcription, beyond the
point of initiation, 4-fold and 20-fold excess
-catenin was tested
against G4VP16 activation of the same G4 UAS/Luc reporter plasmid (Fig.
8B). Addition of 20-fold excess balancer plasmid elicited a 20%
decrease in G4VP16 activation from 35-fold activation to 29-fold
activation. Addition of a 4-fold excess of
-catenin plasmid caused a
32% decrease in G4VP16 activation, and a 20-fold excess caused a 31%
decrease, compared to matched controls. This amount of inhibition is
minor compared to the striking effect on G4LEF and
-catenin shown in
Fig. 8A. Furthermore, the level of inhibition does not change when the
excess of
-catenin plasmid is increased from 4-fold to 20-fold but
stays constant at approximately 30%, suggesting that this minor
inhibitory effect is due to reasons other than specific squelching. We
conclude that
-catenin can squelch reporter gene expression because
a domain within the arm repeat array or the C terminus is interacting
with a third component required by
-catenin-LEF-1 complexes to
activate gene transcription.

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FIG. 8.
Excess -catenin squelches transactivation of
-catenin and LEF-1 but not transactivation by VP16. (A) Wild-type
-catenin expression plasmid (750 ng) was cotransfected with 50 ng of
G4LEF (aa 1 to 256), 1 µg of G4 UAS/Luc reporter plasmid, and the
following plasmids used as competitors: 4- and 20-fold (3 and 15 µg,
respectively) excess amounts of 19 -catenin, control plasmid
(empty eukaryotic expression plasmid), and N 19 -catenin (see
Fig. 1) and a 60-fold (3 µg) excess amount of NLEF (hLEF-1 aa 67 to 399). Excess amounts of 19 and N 19 -catenin, but not
control plasmid or NLEF, were able to squelch wild-type
-catenin-LEF-1 reporter gene activation. (B) G4VP16 (750 ng) was
cotransfected with 1 µg of G4 UAS/Luc reporter plasmid and the
following plasmids used as competitors: 4- and 20-fold (3 and 15 µg,
respectively) excess amounts of control plasmid or a wild-type
-catenin construct. Wild-type -catenin does not squelch G4VP16
transactivation. A plasmid (0.5 µg) containing the lacZ
gene under the control of the CMV promoter was cotransfected as an
internal control for transfection efficiency. Error bars were
calculated by using the average of duplicate points. Values shown are
from one of three replicate experiments. Fold activation was calculated
by comparing levels of luciferase activity to the G4 UAS/Luc reporter
alone.
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DISCUSSION |
We have examined nuclear localization of
-catenin and
demonstrate directly that nuclear import is independent of LEF/TCF binding; we also show data to suggest that
-catenin can be exported from nuclei. Below, we discuss a model in which subcellular
localization of
-catenin is shaped by competing cytoplasmic and
nuclear sequestration and the relative rates of nuclear import and
nuclear export.
-Catenin-LEF-1 complexes do not activate reporter
gene expression in normal T lymphocytes even though both factors are
abundantly and predominantly in the nucleus. Thus, gene activation by
-catenin does not always correlate with nuclear localization. This
fact, coupled with our observation that overexpression of
-catenin
can squelch activation by LEF-1-
-catenin complexes but cannot
squelch activation by the herpesvirus VP16 transcription activator,
leads us to propose that an additional transcription component is
required for
-catenin to activate gene expression.
Three models could explain how a small deletion of 19 aa in arm
repeat 6 of
-catenin causes a dramatic change in
subcellular localization. First, the 19-aa deletion could
have unmasked or generated a strong, cryptic NLS. Second, if
-catenin is a cytoplasmic-nuclear shuttling protein with
export capabilities, the 19-aa deletion might prevent export such
that newly imported
19
-catenin protein remains trapped in the
nuclear compartment. Third,
-catenin might be sequestered in the
cytoplasm and plasma membrane, and the 19-aa deletion could disrupt one
or more of these interactions.
The first model is unlikely because no obvious clustered or bipartite
NLS sequence is evident around the deleted region. Furthermore, larger
deletions that remove other portions of the arm repeat array also
create nucleus-localized protein (48). While the exact
region specifying nuclear transport has not been defined, we show that
nuclear import can be carried out by arm repeats 1 through 9 (MT10)
(Fig. 5 and 6). Others have also observed MT10 to be localized to the
nucleus (16). The ability of this region to direct nuclear
transport is remarkably resilient, even when subjected to large
deletions. The subcellular localization of a series of armadillo
deletion mutants was determined in Drosophila embryos
(48). Deletion of arm repeat 5, 8, or 3 through 6 did not
destroy nuclear localization; indeed, no deletion mutation has yet been
reported to impair nuclear localization. Instead, deletion of repeats 3 through 6 created a constitutively nucleus-localized armadillo protein,
much like the constitutively localized
19 mutant described here
(48). Either nuclear import is directed by a discrete signal
motif that has yet to be specifically deleted or it is specified by any
one of a number of redundant determinants present in the arm repeat array.
We have presented data to suggest that wild-type
-catenin is capable
of nuclear export (Fig. 6). While this lends support to the second
model, in which
19 is localized to the nucleus because it has lost
the ability to be exported, we show that there is no difference between
wild-type
-catenin and
19 localization (Fig. 6). Therefore, the
second model is insufficient to explain the contrasting patterns of
localization. Even so, regulated shifts in subcellular localization of
-catenin could involve a modulation of the relative rates of import
and export. Indeed, we observe that localization of
-catenin in
normal T lymphocytes can be shifted from cytoplasm to nucleus with
PMA-ionomycin treatment (Fig. 5). Either this T-cell-activating signal
modulates relative rates of import and export, as it does for NF-AT, or
it somehow causes release of
-catenin from anchoring sites in the
cytoplasm or plasma membrane. Future studies of
-catenin
localization should include a consideration of the ability of this
protein to shuttle in and out of the nucleus.
Our data are most consistent with the third model, in which
19
localizes to the nucleus because it cannot be efficiently sequestered
in the cytoplasm and plasma membrane. We have shown that
19 is
completely defective for LEF/TCF binding, but we have also shown that
it no longer interacts with the cytoplasmic tail of E-cadherin or the
cytoplasmic tumor suppressor protein APC (Fig. 2). Thus,
19 may not
be easily anchored in the cytoplasm, and this might be at least part of
the reason for the predominant nuclear localization of this mutant. In
light of the dramatic localization of
19 protein to the nucleus, it
is surprising that it binds well to axin. Obviously, this ability to
interact with axin does not prevent nuclear localization, but more work
is necessary to determine why this is the case. In their study of
armadillo deletion mutants in Drosophila embryos, Orsulic
and Peifer found that the strongest correlation for nuclear
accumulation of armadillo mutants was a loss of E-cadherin binding;
loss of interactions with other cytoplasmic proteins discovered more
recently might produce the same effect (48). Overexpressed
E-cadherin protein can prevent nuclear localization of
-catenin, and
it has been suggested that cadherins act as regulators of
-catenin
signaling by depleting the soluble cytoplasmic pool (16).
Likewise, cytoplasmic extracts inhibit nuclear accumulation of
-catenin in digitonin-permeabilized cells, again suggesting that an
anchoring activity in the cytoplasmic extract prevents
-catenin from
transporting through nuclear pores. Another explanation for the
localization pattern of
19 would be that this protein is much more
stable because it has lost the ability to interact with
conductin/axin/axil and APC. Indeed, an increase in the stability of
-catenin is one of the major effects of the Wnt signaling cascade,
and this increase correlates with an appearance of the protein in the
nucleus. However, there are no apparent differences between the
expression levels of wild-type
-catenin and
19 (Fig. 2 and data
not shown). This is surprising given that
19 no longer binds to APC.
19
-catenin is able to bind to axin as well as, if not better
than, wild-type
-catenin. Perhaps axin is still capable of mediating
some form of degradation. It is curious that
19 can localize to
nuclei even though it can bind to axin; this would appear to be
inconsistent with a cytoplasmic anchoring model. Perhaps
axin/conductin/axil does not play a strong role in sequestration
compared to E-cadherin. Immunolocalization studies of exogenously
expressed conductin show that the protein localizes throughout the cell
(6). Another possibility is that axin relocalizes with
19
to the nucleus. Taken together, our data support a model in which
varying patterns of subcellular localization are due in part