* Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel; and Department of
Biology, University of Toledo, Toledo, Ohio 43606
-Catenin and plakoglobin (
-catenin) are
closely related molecules of the armadillo family of
proteins. They are localized at the submembrane
plaques of cell-cell adherens junctions where they form
independent complexes with classical cadherins and
-catenin to establish the link with the actin cytoskeleton. Plakoglobin is also found in a complex with desmosomal cadherins and is involved in anchoring intermediate filaments to desmosomal plaques. In addition to
their role in junctional assembly,
-catenin has been shown to play an essential role in signal transduction
by the Wnt pathway that results in its translocation into
the nucleus. To study the relationship between plakoglobin expression and the level of
-catenin, and the
localization of these proteins in the same cell, we employed two different tumor cell lines that express
N-cadherin, and
- and
-catenin, but no plakoglobin
or desmosomal components. Individual clones expressing various levels of plakoglobin were established by
stable transfection. Plakoglobin overexpression resulted in a dose-dependent decrease in the level of
-catenin in each clone. Induction of plakoglobin expression increased the turnover of
-catenin without affecting RNA levels, suggesting posttranslational regulation of
-catenin. In plakoglobin overexpressing cells,
both
-catenin and plakoglobin were localized at cell-
cell junctions. Stable transfection of mutant plakoglobin molecules showed that deletion of the N-cadherin
binding domain, but not the
-catenin binding domain,
abolished
-catenin downregulation. Inhibition of the
ubiquitin-proteasome pathway in plakoglobin overexpressing cells blocked the decrease in
-catenin levels and resulted in accumulation of both
-catenin and plakoglobin in the nucleus. These results suggest that (a)
plakoglobin substitutes effectively with
-catenin for
association with N-cadherin in adherens junctions, (b)
extrajunctional
-catenin is rapidly degraded by the
proteasome-ubiquitin system but, (c) excess
-catenin
and plakoglobin translocate into the nucleus.
CELL adhesion plays a central role in complex biological processes including motility, growth, differentiation and cell survival. The most direct effect of
adhesion is on morphogenesis, i.e., the assembly of individual cells into highly ordered tissues and organs through
cell-cell junctions (Takeichi, 1995 In addition to their function in cell adhesion, To study the relationship between Cell Culture and Transfection
BALB/C 3T3, SVT2 that are BALB/C 3T3 cells transformed by SV40,
MDCK, and HT1080 cells were grown in Dulbecco's modified Eagle medium plus 10% calf serum (GIBCO BRL, Gaithersburg, MD). SVT2 cells
were transfected with a full-length human plakoglobin cDNA (Franke et al.,
1989 Protease Inhibitors
The calpain inhibitor N-acetyl-leu-leu-norleucinal (ALLN, used at 25 µM)
and the inactive analogue N-acetyl-leu-leu-normethional (ALLM), as well
as aprotinin, leupeptin, and pepstatin A (all used at 10 µg/ml), were purchased from Sigma Chemical Co. (St. Louis, MO). Lactacystin A (dissolved in water at 0.4 µg/ml was used at a final concentration of 4 ng/ml)
and MG-132 (used at 10 µM) were purchased from Calbiochem-Novabiochem (La Jolla, CA).
Immunofluorescence Microscopy
Cells were cultured on glass coverslips, fixed with 3.7% paraformaldehyde
in phosphate-buffered saline and permeabilized with 0.5% Triton X-100.
A mAb recognizing the COOH terminus of human plakoglobin (PG5.1;
Cowin et al., 1986 Protein Labeling and Immunoprecipitation
Cells (on 35-mm dishes) were incubated for 30 min in methionine-free
medium, followed by 30-min incubation in the presence of 250 µCi/ml of
[35S]methionine (pulse). The cells were washed extensively with medium containing excess non-radioactive methionine and incubated in fresh medium (chase). At different times after initiation of the chase, the cells were
harvested in RIPA buffer (50 mM Tris HCl, pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.5% deoxycholate, and 0.1% SDS). 5 × 106 cpm of radioactive
total cell extract was incubated at 4°C for 1 h with 40 µl of monoclonal
11E4 anti-plakoglobin antibody, followed by incubation for 30 min with
50 µl protein A+G/agarose and then processed as described (Sacco et al.,
1995 Immunoblotting and Polyacrylamide
Gel Electrophoresis
Equal amounts of total cell protein from the different clones were separated by SDS-PAGE, electrotransferred to nitrocellulose and incubated
with the monoclonal anti-plakoglobin antibodies, or with monoclonal antibodies against human N-cadherin (13A9; Knudsen et al., 1995 In some experiments, cells were fractionated into Triton X-100-soluble
and -insoluble fractions as described (Rodríguez Fernández et al., 1992).
Cells cultured on 35-mm dishes were incubated in 0.5 ml buffer containing
50 mM of MES, pH 6.8, 2.5 mM EGTA, 5 mM MgCl2, and 0.5% Triton-X-100, at room temperature, for 3 min. The Triton X-100-soluble fraction
was removed and the insoluble fraction, enriched in membrane-cytoskeletal complexes, was scraped into 0.5 ml of the same buffer. Equal volumes
of the two fractions were analyzed by SDS-PAGE followed by immunoblotting with anti-plakoglobin antibodies.
Northern Blot Hybridization
Total RNA was extracted from cells by the guanidinium thiocyanate
method. Northern blots containing 20 µg, per lane, of total RNA were
stained with methylene blue to determine the positions of 18S and 28S rRNA
markers, and then hybridized with plakoglobin (Franke et al., 1989 To study the effect of plakoglobin overexpression on
To study the dynamics of the relationship between plakoglobin expression and
When HT1080 cells were stably transfected with plakoglobin under an inducible promoter,
To determine if the decrease in
We examined the possibility that Expression of Since plakoglobin and
NH2-terminal deletions of plakoglobin that retained both
We have also analyzed the association of the various
plakoglobin mutants with a Triton X-100-insoluble membrane-cytoskeletal complex. Interestingly, COOH-terminal
plakoglobin mutants that were unable to confer a decrease
in
We have also determined the distribution of
Expression of To compare the ability of ectopically expressed Stabilization of A recent study has demonstrated that the turnover of
To determine the localization of plakoglobin and
Regulation of the expression of We have shown for the first time that stable or inducible
overexpression of plakoglobin leads to a decrease in Inhibition of the ubiquitin-proteasome protein degradation system resulted in the stabilization of Immunofluorescence staining revealed that in wt plakoglobin overexpressing cells the remaining Our results reinforce the view that nuclear translocation
of Another well documented posttranscriptional regulation
of Wnt-induced signaling that involves The accumulation of The challenge for future studies is to determine whether
elevated ; Gumbiner, 1996
; Larue
et al., 1996
). The specific adhesive interactions between
cells involve transmembrane cell adhesion receptors of the
cadherin family (Takeichi, 1991
, 1995
; Geiger and Ayalon, 1992
; Kemler, 1992
), but effective adhesion and junction
formation requires an association of the receptors with the
cytoskeleton which is mediated by junctional plaque proteins (Kemler, 1993
; Knudsen et al., 1995
; Rimm et al.,
1995
). Cadherin-mediated cell-cell junctions are linked to
either actin filaments in adherens junctions (via catenins,
- ,
- , and
-catenin, or plakoglobin; reviewed in Takeichi, 1991
; Kemler, 1992
; Knudsen and Wheelock, 1992
;
Wheelock et al., 1996
), or to intermediate filaments in desmosomes (via plakoglobin, desmoplakins, plakophilins
and other molecules; Schmidt et al., 1994
). Plakoglobin is a
common plaque component to both types of cell-cell junctions (Cowin et al., 1986
; Franke et al., 1989
; Cowin and
Burke 1996
; Wahl et al., 1996
) that is essential for the sorting out of desmosomes and adherens junctions in the embryonic heart. Its elimination by gene disruption results in the collapse of this segregation in mouse embryos, and the
development in the heart of extended adherens junctions
that contain desmosomal proteins, which is lethal in the
embryo (Bierkamp et al., 1996
; Ruiz et al., 1996
).
-catenin
and plakoglobin are highly homologous to Drosophila armadillo (Peifer and Weischaus, 1990) and belong to the armadillo family (Peifer et al., 1994a
). Armadillo in Drosophila and
-catenin in Xenopus have been shown to play a
role in the transduction of transmembrane signals initiated
by the extracellular glycoprotein wg/Wnt that regulates cell
growth, differentiation and fate (Peifer et al., 1994b; Peifer
and Wieschans, 1990, Peifer, 1995
; Gumbiner, 1995
, 1996
;
Huber et al., 1996a
; Miller and Moon, 1996
). Activation of
this pathway results in the elevation of
-catenin levels
and its nuclear localization in a complex with the TCF/LEF
family of transcription factors (Behrens et al., 1996
; Huber
et al., 1996b
; Molenaar et al., 1996
), suggesting that
-catenin may have a role in regulating gene expression (Riese
et al., 1997
; van de Wetering et al., 1997
). In the absence of
wg/Wnt signaling,
-catenin is degraded in mammalian
cells by a process involving the adenomatous polyposis
coli (APC)1 tumor suppressor protein (Powell et al., 1992
)
and the ubiquitin-proteasome degrading pathway (Aberle
et al., 1997
). Mutations in the APC gene that constitute the
major genetic defect in inherited colon cancer and certain
melanoma result in the accumulation of
-catenin (Munemitsu et al., 1995
, 1996
; Papkoff et al., 1996
; Peifer, 1996
; Rubinfeld et al., 1996
; Yost et al., 1996
), and most probably cause inappropriate activation of target genes by the
-catenin-LEF/TCF complex (Korinek et al., 1997
; Morin
et al., 1997
; Rubinfeld et al., 1997
). In contrast, the involvement of plakoglobin in suppressing tumorigenesis was inferred from studies showing loss of heterozygosity of the
plakoglobin gene in certain types of tumors (Aberle et al.,
1995
), its reduction in several tumor cell types (Sommers et al., 1994
; Navarro et al., 1994; Simcha et al., 1996
), and by demonstrating that plakoglobin overexpression can
suppress the tumorigenicity of mouse and human cells,
while localized in the nuclei of such cells (Simcha et al.,
1996
). The regulation of
-catenin and plakoglobin level
may therefore be a key element in their nuclear localization and signal transduction. In addition, plakoglobin and
-catenin bind in a mutually exclusive manner to cadherins, APC, and transcription factors (Butz and Kemler, 1994
; Hülsken et al., 1994
; Nathke et al., 1994
; Rubinfeld et al., 1995
; Huber et al., 1996b
).
-catenin and plakoglobin expression and their localization in the same cell,
we have employed cell lines that express
-catenin, but
very low, or undetectable levels of plakoglobin, and do not
form desmosomes. We present data indicating that overexpression of plakoglobin affects
-catenin stability, we
identify the plakoglobin domains that are responsible for
conferring
-catenin instability, and demonstrate that when the ubiquitin-proteasome protein degradation system is
inhibited, plakoglobin and
-catenin translocate into the
nucleus.
Materials and Methods
) cloned into the EcoRI site of the polylinker of the pJ4
expression
vector (Rodríguez Fernández et al., 1992) that consists of the Mo-MuLV
LTR promoter-enhancer sequence, the SV40 small t-antigen intron, and
the SV40 large T polyadenylation signal in the pBR322 plasmid. The neomycin resistance gene (neor), which was cotransfected with the pJ4
construct, was subcloned into the pSVL expression vector. Transfection was
carried out by the calcium phosphate precipitation method and colonies resistant to 800 µg/ml G418 (Geneticin; GIBCO BRL) were isolated. HT1080 cells were transfected with full-length or mutant plakoglobin cDNA constructs inserted into the pLKneo plasmid, driven by the dexamethasone-inducible MMTV promoter, as described (Sacco et al., 1995
; Wahl et al.,
1996
). The construction and isolation of mutant plakoglobin cDNAs, as well
as their ability to complex with either N-cadherin or
-catenin, by coimmunoprecipitation, were previously described (Sacco et al., 1995
; Wahl et al.,
1996
). HT1080 clones transfected with plakoglobin cDNA were grown in the presence of 1 mg/ml G418 and the expression of plakoglobin was induced using 10
7 M dexamethasone. Mutant
-catenin lacking the NH2-terminal 57 amino acids (
N57) was prepared by isolating a 2.6-kb HincII
fragment of mouse
-catenin cDNA (Butz et al., 1992
) from the bluescript plasmid. This cDNA fragment lacking the first 57 NH2-terminal amino
acid sequences was subcloned into the SmaI site of the pECE-Flag plasmid. The Flag epitope is localized at the NH2 terminus of the
N57 construct.
) was obtained from Dr. W.W. Franke. A mAb recognizing an epitope at the NH2 terminus of human plakoglobin was previously
described (11E4; Sacco et al., 1995
; Wahl et al., 1996
). The secondary antibody was rhodamine-labeled goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). A polyclonal antiserum against
-catenin (Sigma, Holon, Israel) was employed and the secondary antibody was FITC-labeled goat anti-rabbit IgG. The cells were examined by
epifluorescence with a Zeiss Axiophot microscope.
). The immune complexes were recovered by boiling in Laemmli sample buffer and resolved by SDS-PAGE (Laemmli, 1970
). The radioactivity
associated with the plakoglobin band was determined using a Fujix Bas
1000 PhosphorImager.
),
-catenin, (1G5; Johnson et al., 1993
), or
-catenin, (5H10; Johnson et al., 1993
).
The antigens were visualized by the ECL method (Amersham, Buckinghamshire, UK), and the density of each band quantitatively determined by
laser densitometry using the ImageQuant software. Affinity-purified rabbit antibodies to APC (APC2), generated against a protein fragment containing amino acid residues 1034-2130 (Rubinfeld et al., 1993), were generously provided by Dr. P. Polakis (ONYX Pharmaceuticals, Richmond,
CA). For APC resolution, 6% SDS-PAGE was employed.
) or
-catenin (Butz et al., 1992
) cDNAs, which were labeled with [32P]dCTP
by the random priming technique, as described (Glück et al., 1993
).
Results
-Catenin Expression in Plakoglobin-transfected Cells
-catenin levels, we employed a human fibrosarcoma cell line
(HT1080) and an SV40-transformed 3T3 cell line (SVT2),
neither of which express detectable levels of plakoglobin
(Sacco et al., 1995
; Simcha et al., 1996
), but express N-cadherin and
- and
-catenin (Fig. 1 A). Both cell lines were
transfected with full-length plakoglobin and G418-resistant colonies were isolated that express varying levels of
plakoglobin (Fig. 1, B and C). Immunoblot analysis revealed an inverse correlation between plakoglobin and
-catenin expression showing a more dramatic reduction
of
-catenin in clones expressing higher levels of the transfected plakoglobin (Fig. 1, B and C). In addition, 3T3 fibroblasts that contained low levels of endogenous plakoglobin expressed high levels of
-catenin (Fig. 1 C, lane 7).
Fig. 1.
Expression of -catenin in HT1080 and SVT2 cells
overexpressing plakoglobin. (A) Equal amounts of total cell protein from HT1080 (HT), SVT2 (SV), and MDCK (M) cells were
analyzed by immunoblotting with antibodies against plakoglobin
(PG),
-catenin (
-CAT),
-catenin (
-CAT), and N-cadherin
(N-CAD). (B) HT1080 cells were transfected with plakoglobin
cDNA and equal amounts of total cell protein from individual
clones stably expressing varying levels of plakoglobin and (cultured in the presence of dexamethasone) were analyzed by immunoblotting with antibodies against plakoglobin and
-catenin. The bars represent densitometer tracing of the intensity of plakoglobin (dotted bars) and
-catenin (filled bars) bands. C, SVT2
cells were stably transfected with plakoglobin and clones expressing varying levels of the transgene (lanes 1-6) were analyzed as in B. Lane 7, nontransfected 3T3 cells; lane 8, control SVT2 cells.
[View Larger Version of this Image (32K GIF file)]
-catenin levels, we used HT1080
cells stably expressing plakoglobin under the dexamethasone-inducible MMTV promoter. Upon stimulation with
dexamethasone, plakoglobin expression was induced reaching a peak between 6 and 8 h (Fig. 2 A). Simultaneous analysis of plakoglobin (Fig. 2 A) and
-catenin (Fig. 2 B)
after induction with dexamethasone revealed a three- to
fivefold decrease in
-catenin levels 8-12 h after plakoglobin induction (Fig 2 C). By 12 h after induction,
-catenin
was barely detectable (Fig. 2 B).
Fig. 2.
Regulation of -catenin levels in HT1080 cells stably
transfected with plakoglobin under an inducible promoter. (A)
HT1080 cells expressing plakoglobin under a dexamethasone-
inducible MMTV promoter were stimulated with dexamethasone, and at different time points after stimulation equal amounts
of total cell protein were analyzed for plakoglobin expression by
immunoblotting with antibodies against plakoglobin (PG). (B)
Simultaneous analysis, on the same protein blot, of
-catenin
(
-CAT) expression at different times after induction with dexamethasone. (C) Densitometer tracing of the levels of plakoglobin
(filled bars) and
-catenin (hatched bars) expressed in A and B. M, MDCK cell lysates.
[View Larger Version of this Image (38K GIF file)]
-Catenin Localization and Degradation in Cells
Overexpressing Plakoglobin
-catenin (but not plakoglobin) was observed in intercellular junctions before
induction with dexamethasone (Fig. 3, A and B). In cells
stimulated with dexamethasone for 12 h, when
-catenin
was reduced by about fourfold and plakoglobin was at its
peak (Fig. 2), plakoglobin displayed very prominent junctional staining (Fig. 3 D). The
-catenin still expressed in
these cells was apparently organized in the same junctions (Fig. 3 C), but displayed a much reduced intensity of staining. This suggests that the remaining
-catenin in plakoglobin overexpressing clones was not displaced from the
junctions by the exogenous plakoglobin.
Fig. 3.
Organization of
-catenin and plakoglobin in
HT1080 cells before and after induction of plakoglobin
expression. HT1080 cells expressing inducible plakoglobin (as described in Fig. 2)
were doubly stained with
polyclonal anti-
-catenin (A
and C) and monoclonal anti-plakoglobin (B and D) antibodies, before (A and B) and
12 h after (C and D) induction with dexamethasone
(Dex). The secondary antibodies were FITC-anti-rabbit antibody and rhodamine
anti-mouse-IgG. Bar, 10 µm.
[View Larger Version of this Image (113K GIF file)]
-catenin levels resulted
from a reduction in
-catenin RNA, Northern blots from
uninduced and dexamethasone-induced HT1080 cells were
hybridized with cDNAs to
-catenin and plakoglobin (Fig.
4 A). Dexamethasone stimulation resulted in a dramatic increase in plakoglobin RNA levels (Fig. 4 A, compare lanes
2 with 3), but did not effect the level of
-catenin RNA
(Fig. 4 A). This suggests that the decrease in
-catenin levels of plakoglobin overexpressing cells does not result
from a loss of
-catenin mRNA.
Fig. 4.
Expression of -catenin RNA and stability of
-catenin
protein in cells overexpressing inducible plakoglobin. (A) RNA
was extracted from MDCK cells (lane 1), untransfected HT1080
cells (lane 4), and plakoglobin-transfected HT1080 cells before
(lane 3), and 12 h after stimulation with dexamethasone (lane 2).
Equal amounts of total cell RNA were analyzed by Northern blot
hybridization with plakoglobin (PG) and
-catenin (
-CAT)
cDNAs. The levels of 18 and 28S rRNA are shown for comparison. (B) Uninduced (
Dex) and cells induced for 12 h (+Dex)
were pulse labeled with [35S]methionine for 30 min, followed by
chase with fresh medium. Equal amounts of radioactive cellular
proteins were immunoprecipitated with anti-
-catenin antibody
and analyzed by SDS-PAGE. (C) The radioactivity in the
-catenin band in B and in an identical independent experiment was determined by a phosphorimager, and the values ±SD are presented
as percent of the values obtained after 30 min pulse labeling.
[View Larger Version of this Image (35K GIF file)]
-catenin degradation
was enhanced in plakoglobin overexpressing HT1080 cells,
by labeling with [35S]methionine for 30 min and chasing
for increasing periods of time in fresh medium with excess
nonradioactive methionine, containing plakoglobin under
the dexamethasone inducible MMTV promoter. Immunoprecipitation of
-catenin from equal amounts of radioactive whole cell lysates showed that the level of newly synthesized
-catenin decreased significantly faster (more than
threefold) in dexamethasone induced cells than in control,
uninduced cells (Fig. 4, B and C). Dexamethasone had no
effect on the degradation of
-catenin in control HT1080 cells (results not shown). These results suggest that the decrease of
-catenin in plakoglobin overexpressing cells resulted from a faster turnover of
-catenin.
-Catenin in Cells Overexpressing
Mutant Plakoglobin
-catenin are localized in the submembrane plaque where they form mutually exclusive complexes with cadherins, we examined the possibility that the
overexpressed plakoglobin competes with
-catenin for
N-cadherin binding, and this competition leads to displacement and degradation of uncomplexed
-catenin. HT1080
cell lysates, prepared before and after plakoglobin induction, were immunoprecipitated with anti-N-cadherin antibody followed by Western blotting with anti-
-catenin
and plakoglobin antibodies (Fig. 5 A). When plakoglobin
expression was induced with dexamethasone, the complexes with N-cadherin contained more plakoglobin than
-catenin (Fig. 5 A, compare lanes 3 and 4 with lane 1). To
examine the possibility that N-cadherin binding with plakoglobin is responsible for conferring the decrease in
-catenin levels, we employed HT1080 cells stably transfected with mutant plakoglobin constructs that included or
lacked either the N-cadherin or the
-catenin binding domains (see Fig. 9; Sacco et al., 1995
). Expression of full-length (FL) plakoglobin (Fig. 5 B, lanes 1 and 2), and deletions that left the armadillo repeats intact (Fig. 5 B,
C727, lanes 3 and 4) or removed part of the last armadillo repeat (Fig. 5 B,
C632, lanes 5 and 6), but were still
capable of associating with N-cadherin, reduced the level
of
-catenin when compared to uninduced cells (Fig. 5 B,
lanes 1, 3, and 5, compare with 2, 4, and 6, respectively). In
contrast, larger COOH-terminal deletions in plakoglobin
that disrupt association with N-cadherin, but retain
-catenin binding (Fig. 9), leaving 458 amino acids or less (Fig. 5
C;
C458,
C414,
C375,
C161, and
C114), were unable to affect
-catenin levels when overexpressed (Fig. 5
C, lanes 2, 4, 6, 8, and 10, compare with 1, 3, 5, 7, and 9, respectively).
Fig. 5.
-Catenin and plakoglobin levels in complexes with
N-cadherin in cells transfected with COOH-terminal deletion
mutant plakoglobin under control of an inducible promoter. (A)
Equal amounts of cellular protein from unstimulated HT1080
cells (lane 1) and cells induced with dexamethasone to express
plakoglobin driven by the MMTV promoter for: 3 (lane 2), 8 (lane 3) and 12 h (lane 4) were immunoprecipitated with anti-
N-cadherin antibody and the protein blot was reacted with anti
-catenin (
-CAT), plakoglobin (PG), and anti-N-cadherin (N-CAD) antibodies. (B) Cells expressing full-length (FL), or COOH-terminal-truncated (
C) plakoglobin retaining 727 (
C727) and 632 (
C632) amino acids. (C) Cells expressing COOH-terminal plakoglobin deletions containing 458 (
458), 414 (
414), 375 (
375), 161 (
161), and 114 (
114) amino acids. Uninduced (
)
and cells induced (+) with dexamethasone, to express the different plakoglobin constructs, were analyzed by immunoblotting
with anti-plakoglobin (PG) and
-catenin (
-CAT) antibodies on
the same blots.
[View Larger Version of this Image (26K GIF file)]
Fig. 9.
Schematic representation of plakoglobin functional domains involved in reducing -catenin levels and in complexing with
-catenin and N-cadherin. Full-length human plakoglobin (745 amino acids) is shown with the 13 armadillo repeats and the different deletion mutants from the NH2 (
N) and COOH terminus (
C) used in this study. The binding studies to
-catenin and N-cadherin of the various plakoglobin mutants were described (Sacco et al., 1995
; Wahl et al., 1996
). The binding sites for the monoclonal antibodies 11E4
and PG 5.1 used in this study are also indicated.
[View Larger Version of this Image (18K GIF file)]
-catenin and N-cadherin binding sites (Fig. 9;
N686), or
removed armadillo repeat 1 and part of armadillo repeat 2 (see Fig. 9;
N554), and thus deleted
-catenin binding but
retained N-cadherin binding, were able to reduce
-catenin levels when overexpressed in HT1080 cells (Fig. 6 A,
compare lanes 3 with 4 and 5 with 6). However, more extensive NH2-terminal truncations that removed armadillo
repeat 3, which is important for N-cadherin binding (see
Fig. 9, Sacco et al., 1995
), did not affect
-catenin levels (Fig. 6 A, compare lane 7 with 8). The expression of other
partners that associate with both
-catenin and plakoglobin, such as N-cadherin,
-catenin, and APC, was not affected by the overexpression of full-length or mutant plakoglobin (results not shown).
Fig. 6.
Levels of -catenin in cells expressing inducible
NH2-terminal deleted plakoglobin and
-catenin mutants. (A)
Levels of
-catenin in cells expressing dexamethasone-inducible
plakoglobin mutants with NH2-terminal deletions retaining 686 (
N686), 554 (
N554), or 512 (
N512) amino acid, were determined as in Fig. 5. (B) Levels of
-catenin in individual clones
stably expressing an NH2-terminal deleted
-catenin lacking the
first 57 amino acids (
N57, lanes 4-6) or neor controls (lanes 1-3).
[View Larger Version of this Image (36K GIF file)]
-catenin levels were mostly detected in the Triton
X-100-soluble fraction (Fig. 7 A, compare lanes 9 with 10,
and 11 with 12; Fig. 7 B, compare lanes 3 with 4, 5 with 6 and 7 with 8), while plakoglobin deletions that conferred a
decrease in
-catenin were found mostly in the Triton
X-100-insoluble fraction (Fig. 7 A, compare lane 3 with 4 and 13 with 14), similar to full-length plakoglobin expressing HT1080 (Fig. 7, A-C, lanes 1 and 2) and MDCK cells
(Fig. 7 A, lanes 7 and 8). Analysis of NH2-terminal deletions of plakoglobin gave similar results, with
N512 being
completely Triton X-100-soluble (Fig. 7 C, lanes 7 and 8),
in contrast to
N686 (Fig. 7 C, lanes 3 and 4) that was mostly in the Triton X-100-insoluble fraction, like full-length plakoglobin (Fig. 7 C, lanes 1 and 2). This distribution
of the plakoglobin mutants between the Triton X-100-soluble and -insoluble fractions most likely reflects their ability or inability to associate with N-cadherin.
Fig. 7.
The solubility in Triton X-100 of full-length and plakoglobin mutants. (A) HT1080 cells expressing full-length plakoglobin (FL), untransfected HT1080, MDCK, and cells expressing
COOH-terminal deletions of plakoglobin containing 727 (727),
632 (
632), 161 (
161), and 114 (
114) amino acids were separated into Triton X-100-soluble (S) and -insoluble (I) fractions.
Equal volumes of each fraction were analyzed for plakoglobin
levels by immunoblotting. (B) HT1080 expressing COOH-terminal deletions that contain 458 (
458), 414 (
414), and 375 (
375)
amino acids were analyzed as described in A. (C) Analysis of the
Triton X-100 solubility of NH2-terminal plakoglobin deletions
containing 686 (
686), 554 (
554), and 512 (
512) amino acids.
In A and B the 11E4 antibody was used, while in C antibody PG
5.1 was used (see Fig. 9 for the plakoglobin domains recognized
by these antibodies).
[View Larger Version of this Image (25K GIF file)]
-catenin
between the Triton X-100-soluble and -insoluble fractions
in HT1080 cells expressing full-length plakoglobin and in
COOH-terminal deleted plakoglobin expressing cells (
C161)
where the truncated plakoglobin could not confer a decrease in
-catenin levels (see Fig. 9). The results summarized in Fig. 8 show that
-catenin levels were reduced in
both Triton X-100-soluble and -insoluble fractions upon
plakoglobin induction, and a lower molecular mass product of
-catenin (probably a degraded form) was apparent
at later times after plakoglobin induction (Fig. 8 A, lanes
7-9). In
C161-expressing cells, no significant changes in
-catenin levels and detergent solubility were apparent
when the mutant plakoglobin (mainly found in the soluble
fraction) was induced (Fig. 8 B).
Fig. 8.
Triton X-100 solubility of -catenin in cells expressing
full-length and mutant plakoglobin. HT1080 cells transfected with full-length plakoglobin (A), or with the COOH-terminal deletion mutant
C161 (B) were stimulated to express plakoglobin by
dexamethasone, and at various times after induction the levels of
plakoglobin and
-catenin were determined in the Triton X-100-
soluble and -insoluble fractions as described in Fig. 7.
[View Larger Version of this Image (59K GIF file)]
-Catenin in Cells Overexpressing
Mutant
-Catenin
-catenin
to that of plakoglobin in conferring a decrease in the level
of cellular
-catenin, HT1080 cell lines stably expressing
an epitope tagged NH2-terminal deleted
-catenin were
isolated (
N57). This mutant, lacking the first 57 amino
acids, but containing the
-catenin and N-cadherin binding
domains (Fig. 9), was shown to be more stable than wt
-catenin (Munemitsu et al., 1996
; Yost et al., 1996
). Individual clones expressing this mutant
-catenin (Fig. 6 B)
contained a lower level of endogenous
-catenin. The mutant
-catenin was localized at cell-cell junctions, and its
distribution in the cell was indistinguishable from that of
the endogenous wt protein (data not shown).
-Catenin and Nuclear Translocation
of Plakoglobin and
-Catenin after Inhibition
of the Ubiquitin-dependent Proteasome System
-catenin is mediated by the ubiquitin-proteasome degradation system (Aberle et al., 1997
). When this proteolytic
pathway is inhibited by specific inhibitors of the proteasome-mediated proteolysis such as Lactacystin, MG-132
and the peptide aldehyde ALLN, it leads to the stabilization and accumulation of ubiquitinated forms of
-catenin
(Aberle et al., 1997
). To examine if stabilization of
-catenin against proteasome-mediated degradation will block the decrease in
-catenin levels of HT1080 cells induced to
express plakoglobin, we treated cells with MG-132 or ALLN
and determined the levels of
-catenin and plakoglobin.
The results summarized in Fig. 10 show that while
-catenin levels were reduced in cells treated with an inactive
peptide analogue (ALLM) (Fig. 10 A, lanes 1 and 2), they
were even higher than the controls in the presence of the
active proteasome inhibitors ALLN and MG-132 (Fig. 10
A, lanes 3-8, compare to lanes 1, 2, and 9). Furthermore, a
higher molecular mass form of
-catenin, probably representing ubiquitinated
-catenin, could be detected in the
presence of these inhibitors (Fig. 10 B, lanes 3-8), in
agreement with Aberle et al. (1997)
. These results imply
that plakoglobin overexpression cannot confer a decrease
in
-catenin levels when the proteasome degradation pathway is inhibited, which results in
-catenin stabilization against degradation.
Fig. 10.
Expression of -catenin and plakoglobin in cells
treated with inhibitors of the ubiquitin-proteasome system. (A)
HT1080 cells were treated for 2 h with proteasome inhibitors
(MG 132 or ALLN), or with the inactive analogue ALLM, as described in Materials and Methods, and then induced to express
plakoglobin with dexamethasone in the presence of the inhibitors. At different times after dexamethasone stimulation, equal
amounts of total cell lysate were analyzed for
-catenin and plakoglobin expression by immunoblotting as described in Fig. 8.
(B) The immunoblot for
-catenin was overexposed to reveal the
higher molecular mass forms of
-catenin (arrowhead, probably
ubiquitinated) formed in the presence of the proteasome inhibitors.
[View Larger Version of this Image (38K GIF file)]
-catenin when the ubiquitin-proteasome proteolytic pathway is
inhibited, HT1080 cells were treated with ALLN or MG-132 and plakoglobin expression was induced for 9 h. The
cells were immunostained with anti-plakoglobin and anti-
-catenin antibodies (Fig. 11). The results shown in Fig. 11
demonstrate that when proteasome inhibitors were applied, a significant amount of
-catenin translocated into the
nuclei of the cells (Fig. 11, B and C), in contrast to cells treated with the inactive ALLM peptide that showed only
cell-cell junctional staining (Fig. 11 A). Interestingly, under these conditions plakoglobin was not organized in
cell-cell junctions, but was diffusely distributed in the cytoplasm with a significant accumulation in the nuclei of the
cells (Fig. 11 D). Similar results were obtained using Lactacystin, but not with other protease inhibitors including pepstatin A, aprotinin and leupeptin which have no effect
on this degradation pathway (results not shown).
Fig. 11.
The localization of -catenin and plakoglobin in cells treated
with inhibitors of the ubiquitin-proteasome system. HT1080 cells were pretreated with the inactive analogue
ALLM (A), with the proteasome inhibitors ALLN (B and D), or with
MG-132 in the presence of dexamethasone to induce plakoglobin expression
(C). After 9 h, the cells were immunostained for
-catenin (A-C) or plakoglobin (D). Bar, 10 µm.
[View Larger Version of this Image (126K GIF file)]
Discussion
-catenin and plakoglobin
are important in morphogenetic events during embryonic
development (McCrea et al., 1993
; Funayama et al., 1995
;
Karnovsky and Klymkowsky, 1995
; Miller and Moon, 1996
;
Orsulic and Peifer, 1996
; Peifer, 1996
; Schneider et al.,
1996
; Rubenstein et al., 1997
), and in the process of tumorigenesis (Aberle et al., 1994; Inomata et al., 1996
; Simcha
et al., 1996
; Ben-Ze'ev, 1997
; Korinek et al., 1997
; Morin et
al., 1997
; Rubinfeld et al., 1997
). In this study, we have addressed the regulation of
-catenin levels and localization by the overexpression of plakoglobin, and by the inhibition of the ubiquitin-proteasome proteolytic pathway.
-catenin level that is proportional to the level of plakoglobin
expression, and results in increased degradation of
-catenin. Since the cells used in this study lack desmosomal cadherins, it is conceivable that the exogenous plakoglobin
competes with
-catenin for N-cadherin binding, directing
the displaced
-catenin molecules for degradation by the
ubiquitin-proteasome system (Aberle et al., 1997
). This notion is supported by our results showing that when plakoglobin expression was induced, the level of
-catenin in
complex with N-cadherin decreased, and the level of plakoglobin in complexes with N-cadherin increased (Fig. 5 A).
In addition, deletion of the N-cadherin binding domain of
plakoglobin abolished the ability of the mutant plakoglobin
molecules to downregulate
-catenin levels. In contrast,
the binding of plakoglobin to
-catenin was neither necessary nor sufficient to influence
-catenin levels, and deletion of this domain of plakoglobin did not affect
-catenin levels in the cell. Expression of a more stable, NH2-terminal deletion mutant of
-catenin (
N57), also conferred a
decrease in the level of the endogenous
-catenin (Fig. 6
B). This mutant
-catenin, which retained the cadherin and
-catenin binding sites, was localized at cell-cell junctions,
displacing endogenous
-catenin and leading to its degradation. The mutant plakoglobin molecules that could not
confer
-catenin degradation were mostly soluble in Triton
X-100, and thus were unable to compete with the membrane-cytoskeleton-associated N-cadherin adhesion complex that contained the junctional
-catenin.
-catenin (in
agreement with Aberle et al., 1997
), thus blocking the ability
of plakoglobin to confer a decrease in the level of
-catenin (Fig. 10). In such cells, higher molecular weight forms
of
-catenin, probably representing ubiquitinated
-catenin molecules, were apparent.
-catenin was
associated with cell-cell junctions (where it was probably
protected from degradation). These junctions also contained the majority of the plakoglobin. In contrast, when
-catenin degradation by the proteasome system was inhibited,
-catenin accumulated in the cell, and a significant amount of the protein translocated into the nucleus. Plakoglobin was unable to displace
-catenin from cell-cell
junctions under these conditions. It was diffusely distributed in the cytoplasm, and a significant level of plakoglobin was also localized in the nucleus (Fig. 11 D). This implies that
-catenin may have higher affinity for binding to
N-cadherin than plakoglobin, and when its level in the cell
is increased, plakoglobin is no more capable of effectively
competing and replacing the junctional
-catenin. The regulation of
-catenin and plakoglobin contents in the cell therefore determines, to a large extent, their cellular localization, and an additional site where these junctional molecules can accumulate is the nucleus of the cell.
-catenin during signaling, conceivably results from increases in its level in the cell. This increase could be
achieved by influencing the regulated degradation of
-catenin by the ubiquitin-proteasome pathway (as shown
in this study), or by artificially overexpressing very high
levels of
-catenin that can saturate the degradation system, as is the case in transiently transfected cells that often express abnormally high levels of the protein, which is
mainly localized in the nucleus (Simcha, I., B. Geiger, and
A. Ben-Ze'ev, unpublished results).
-catenin level is obtained by binding/sequestration
with cadherins, as was demonstrated in studies overexpressing N- or E-cadherin, that leads to increased
-catenin
levels in these cells (Kowalczyk et al., 1994
; Nagafuchi et al.,
1994
; Simcha et al., 1996
). Similarly, stabilization of plakoglobin was documented in CHO cells transfected with desmosomal cadherins (Kowalczyk et al., 1994
). An increase in
-catenin and plakoglobin levels, without an effect on
their mRNA levels, was also seen in Wnt-transfected mammalian cells (Bradley et al., 1993
; Hinck et al., 1994
; Papkoff et al., 1996
). Thus, modulating the degradation of
-catenin by the formation of complexes between
-catenin and cadherins may constitute an important means to
regulate
-catenin levels and consequently, its extrajunctional function(s) in the cell.
-catenin during development also correlates with changes in
-catenin levels
in the cell, and artificially elevated cadherin expression in
Xenopus can antagonize the propagation of the Wnt signal,
by sequestering free pools of
-catenin into a complex
with cadherin, and thus limiting its function in extra-junctional signaling (Heasman et al., 1994
; Fagotto et al., 1996
;
Yost et al., 1996
). The current results suggest that plakoglobin can serve as an additional regulator of
-catenin
level acting upstream of the APC-GSK-3
step, by competing on the cadherin binding site, and thus releasing
-catenin and exposing it to the degradation fate.
-catenin and its nuclear translocation in complex with transcription factors, its aberrant effect on the transcription of genes during development of
colon cancer and melanoma (Korinek et al., 1997
; Morin
et al., 1997
; Rubinfeld et al., 1997
), as well as the ability of
plakoglobin to influence the tumorigenicity of cells when
overexpressed and localized in the nucleus (Simcha et al.,
1996
), highlight the importance of mechanisms that regulate the level of
-catenin in the cell, as shown in this study.
Interestingly, in tumor cells where plakoglobin overexpression resulted in suppression of the tumorigenic ability (Simcha et al., 1996
), the level of
-catenin was reduced
(this study). This may indicate that plakoglobin confers a
tumor suppressive phenotype on these cells by decreasing
the level of
-catenin, whose abnormally increased level
can be oncogenic (Korinek et al., 1997
; Morin et al., 1997
;
Peifer, 1997
; Rubinfeld et al., 1997
).
-catenin can confer tumorigenicity on nontransformed cells, the physiological conditions that are associated with the regulated expression and translocation of
-catenin and plakoglobin into the nuclei of mammalian
cells, and the target genes whose expression is modulated
by transactivation involving complexes that contain these
junctional plaque proteins.
Address correspondence to Avri Ben-Ze'ev, Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel. Tel.: (972) 8-934-2422. Fax: (972) 8-934-4108. E-mail: lgbenzev{at}weizmann.weizmann.ac.il
.
We thank Dr. Kemler for communicating results prior to their publication and B. Geiger for useful comments.
These studies were supported in part by grants from the USA-Israel Binational Foundation, the Forchheimer Center for Molecular Genetics, the Pasteur-Weizmann Research Program, the German-Israeli Foundation for Scientific Research and Development to A. Ben-Ze'ev, and by National Institutes of Health GM 51188 to M.J. Wheelock and K.R. Johnson.
ALLM, N-acetyl-leu-leu-normethional; ALLN, N-acetyl-leu-leu-norleucinal; APC, adenomatous polyposis coli.
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