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Apoptosis-induced Cleavage of -Catenin byCaspase-3 Results in Proteolytic Fragments withReduced Transactivation Potential
Ulrike Steinhusen1, Volker Badock2, Andreas Bauer3, Jürgen Behrens4,
Brigitte Wittman-Liebold2, Bernd Dörken5 and Kurt Bommert1,6
1Max-Delbrück-Center for Molecular Medicine, Department of Medical Oncology
and Tumorimmunology, 2Department of Protein Chemistry, 4Department of
Epithelial Differentiation, Invasion, and Metastasis, Robert-Rössle-Str. 10, D-13122
Berlin, Germany
3Max-Planck Institute of Immunobiology, Department of Molecular Embryology,
Stübeweg 51, D-79108 Freiburg, Germany
5Humboldt University of Berlin, University Medical Center Charité, Robert-
Rössle-Klinik, Department of Hematology, Oncology and Tumrimmunology,
D-13122 Berlin, Germany
6Corresponding author
Kurt Bommert
Telephone: +49-30-9406-3817
Telefax: +49-30-9406-3124
E-mail: [email protected]
This work was supported by the Deutsche Forschungsgemeinschaft (SFB366)
RUNNING TITLE
β-Catenin Proteolytic Fragments in Apoptotic Cells
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SUMMARY
β-catenin is a member of the Armadillo repeat protein family with a dual cellular function as a
component of both the adherens junction complex and the Wnt/wingless signaling pathway.
Here we show that β-catenin is proteolytically cleaved during anoikis and staurosporine-
induced apoptosis. Cleavage of β-catenin was found to be caspase dependent. Five
cleavage products of β-catenin were identified in vivo and after in vitro cleavage by
caspase-3. Amino acid sequencing and mass spectrometry analysis indicated two caspase-3
cleavage sites at the C-terminus and three further sites at the N-terminus, while the central
Armadillo repeat region remained unaffected. All β-catenin cleavage products were still able to
associate with E-cadherin and α-catenin and were found to be enriched in the cytoplasm.
Functional analysis revealed that β-catenin deletion constructs resembling the observed
proteolytic fragments show a strongly reduced transcription activation potential when
analyzed in gene reporter assays. We therefore conclude that an important role of the β-
catenin cleavage during apoptosis is the removal of its transcription activation domains to
prevent its transcription activation potential.
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INTRODUCTION
Growth of epithelial and endothelial cells is strictly anchorage dependent, with strong cell-
matrix and cell-cell contacts (1-3). When cultured in suspension, the cells rapidly undergo
apoptosis a process termed anoikis (1). It has been suggested that the biological role of
anoikis is to restrict inappropriate cell growth after loss of matrix attachment (4). Different
signaling pathways are involved in this process. Thus, it was shown that integrin signaling is
necessary for survival and growth of epithelial cells and prevents anoikis (3,5,6). Furthermore,
overexpression of oncogenes like v-Ha-ras, v-src and bcl-2 prevents apoptosis after
detachment (1,7) while activation of the Jun-N-terminal kinase (JNK) pathway appears to be
involved in the induction of anoikis (8-10).
The protein family of caspases plays a major role during the execution phase of apoptosis
(11). Up to now more than 10 different human caspases have been cloned (12-14), these can
be subdivided into 2 groups: initiator caspases whose main functin is to activate downstream
caspases, and executor caspases, which are responsible for dismantling cellular proteins
(14). Caspases recognize tetrapeptide motifs and cleave their substrates behind aspartate. A
growing number of caspase-3 substrates has been identified, including the focal adhesion
kinase (FAK) which is involved in cell matrix adhesion via integrins (15-19) and MEKK-1,
which plays a role in the JNK-pathway (10). Other proteins cleaved by caspases are
involved in structural changes in epithelial cells during apoptosis, like keratin 18 and 19
(20,21), PAK2 (p21-activated kinase 2) (22), Gas2 (growth-arrest-specific gene 2) (23) or
Gelsolin (24). Disruption of cell-cell contacts may also play a role in anoikis, as expression of
a dominant negative N-cadherin in the mouse intestinal epithelium was shown to disturb cell-
cell adhesion and subsequently led to an increased number of apoptotic cells (25,26). Cell-cell
adhesion is predominantly mediated by the cadherin-type of transmembrane proteins,
generating a homophilic interaction within two cells. During recent years further cytoplasmic
proteins were isolated as part of the cadherin cell-cell adhesion complex linking the cadherins
to the actin cytoskeleton, thus termed catenins. Three major catenins are known, α-catenin, β-
catenin and γ-catenin (plakoglobin). β-catenin and γ-catenin directly bind to the cadherin in a
mutually exclusive manner, while α-catenin binds to β-catenin or γ-catenin and additionally
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makes a direct or indirect contact to the actin cytoskeleton (27,28). β-catenin belongs to the
Armadillo repeat protein family, containing 13 Armadillo (Arm) repeat motifs which seem to be
involved mainly in protein-protein interaction. β-catenin shows dual cellular function, being
involved in cell-cell adhesion and Wnt-signaling. Upon Wnt-signaling β-catenin becomes
stabilized in the cytoplasm and subsequently translocates into the nucleus, where it is
involved in the expression of specific target genes such as Xtwn, Xnr3, siamois, c-myc, cyclin
D1, c-jun and fra-1 (29-34) together with transcription factors of the lymphocyte enhancer-
binding factor 1 (LEF-1)/T-cell factor (TCF) family (35-38).
In this study we could show that β-catenin is proteolytically cleaved during apoptosis. The
cleavage was inhibited by the caspase-3 family specific peptide inhibitor Z-DEVD-fmk
demonstrating that caspase-3 is the major protease involved in this process. The
determination of the exact cleavage sites revealed that β-catenin is exclusively cleaved in the
N- and C-terminal regions at multiple positions while the central core region was not affected.
Consistently, an interaction of all cleavage products was detected in the E-cadherin/α-catenin
complex but additionally to a high extent in the cytoplasm not associated with E-cadherin. β-
catenin deletion constructs resembling the apoptotic cleavage products of β-catenin showed a
strongly reduced transactivation potential in reporter gene assays.
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MATERIALS AND METHODS
Cell Culture and Induction of Apoptosis
The human breast epithelial cell line H184A1 was cultured in DMEM HAMs F12 (Biochrom,
Berlin, Germany) supplemented with 5 % fetal calf serum, 10 µg/ml transferrin (Life
Technologies, Karlsruhe, Germany), 10 µg/ml insulin (Biochrom), and 1.8 µg/ml hydrocortisol
(Sigma, Steinheim, Germany). MDCK cells (Madine Darby canine kidney cells), the human
breast carcinoma cell line MCF-7.3.28 (stably transfected with a caspase-3 expressing
plasmid), MCF-7/Vector (39), and the human HEK293 kidney epithelial cell line were cultured in
DMEM supplemented with 10 % fetal calf serum.
Apoptosis was induced by maintaining cells in suspension as follows. Cells were grown to
confluency and harvested by trypsination. Approximately 1x107 cells were transferred to
175 cm2 culture flasks coated with 1 % polyHEMA (poly(2-hydroxyethylmethacrylate))
(Sigma) to keep them in suspension as described (1). After different times suspended cells
were harvested and washed twice with PBS. Cell pellets were frozen in liquid nitrogen and
stored at -80 °C for further analysis. Alternatively, apoptosis was induced by incubating 80 %
confluent cells in medium containing 1 µM staurosporine for up to 20 h. The percentage of
apoptotic cells was determined with APO-BRDUTM kit (Pharmingen, Hamburg, Germany) by
nick-end BrdUTP labeling of single- and double-strand DNA breaks with BrdUTP by terminal
transferase. Incorporation of the nucleotide analog was detected with FITC-conjugated
antibodies and measured by FACS-analysis (FACSort Becton Dickinson, Heidelberg,
Germany).
Antibodies and Reagents
Monoclonal anti-β-catenin (clone 571-781), anti-α-catenin and anti-E-cadherin antibodies were
purchased from Transduction Laboratories (Dianova, Hamburg, Germany). Monoclonal anti-
HA antibody was purchased from His (Freiburg, Germany), monoclonal anti-caspase-3
antibody from Pharmingen, polyclonal anti-caspase-6 and -7 antibodies from Santa Cruz
(Heidelberg, Germany). Recombinant caspases-3 and -6 were purchased from Pharmingen,
recombinant caspase-7 was kindly provided by R. Beyaert (40). Z-DEVD-fmk was
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purchased from Calbiochem (Bad Soden, Germany) and staurosporine and polyHEMA were
purchased from Sigma. Tropix Dual-Light chemiluminescence kit was purchased from Perkin
Elmer (Weiterstadt, Germany).
Immunoblotting and Immunoprecipitation
Immunoblotting was essentially done as described previously (41). In short, cells were lysed
by boiling in 10 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1 % SDS, and 30-50 µg protein was
separated by SDS-PAGE and transferred to nitrocellulose membrane (Schleicher & Schuell,
Dassel, Germany). Primary antibodies were diluted 1:1000, anti-β-catenin (clone 571-781)
1:3000, HRP-conjugated secondary anti-mouse or anti-rabbit antibody (Promega, Mannheim,
Germany) 1:10,000. Proteins were visualized using the ECL-system (Amersham,
Braunschweig, Germany).
Immunoprecipitation was done as described (42). In short, 3x106 cells were harvested and
proteins were extracted in lysis buffer (140 mM NaCl, 4.7 mM KCl, 0.7 mM MgSO4, 1.2 mM
CaCl2, 10 mM Tris, pH 8.0, 1 % Triton X-100, 1 mM PMSF, 10 µg/ml aprotinin, 5 µg/ml
leupeptin) on ice. After centrifugation at 12,000 x g, the supernatant was collected as the
detergent soluble fraction. The detergent insoluble fraction was washed twice with lysis buffer
and solubilized by boiling in SDS-sample buffer. Appropriate antibodies and protein A-
Sepharose were added to the supernatant. Precipitates were washed with 500 mM NaCl,
5 mM EDTA, 50 mM Tris-HCl, pH 8.0, 1 % Triton X-100. Western blot analysis was
performed with monoclonal anti-β-catenin antibody (clone 571-781), anti-α-catenin and anti-E-
cadherin antibody. Western blots were quantified by using the NIH Image program version
1.59 (NIH, Bethesda).
Purification of 6His -Catenin
His-tagged β-catenin was expressed in E. coli and purified to homogeneity by nickel-chelate
chromatography (38).
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In Vitro Cleavage of -Catenin with Recombinant Caspases
Cleavage reactions with recombinant caspase-3, -6 or -7 were performed in 20 µl caspase-
buffer (50 mM HEPES, pH 7.4, 0.1 % CHAPS, 5 mM DTT, 1 mM PMSF, 50 µM leupeptin,
200 µg/ml aprotinin) at 37°C for 2 to 4 h using 0.5 µg affinity-purified recombinant 6His-β-
catenin as a substrate.
Identification of Cleavage Sites of -Catenin
To identify caspase-3 cleavage sites within β-catenin, 5 µg recombinant β-catenin was
digested with 100 ng recombinant caspase-3. Cleavage products were separated by SDS-
PAGE and transferred to a PVDF membrane (ProBlott, Applied Biosystems, Foster City, CA)
using a semi-dry blotting apparatus (Bio-Rad Laboratories, Hercules, CA) and 100 mM
CAPS, 10 % methanol as blotting buffer. Proteins transferred to the membrane were stained
with Coomassie Blue R-250 (Serva, Heidelberg, Germany) for 1 min. Coomassie-stained
protein bands were excised, soaked in 60 % acetonitrile for destaining and analyzed by
Edman sequencing. Edman sequencing was performed on a Procise sequencer (Applied
Biosystems).
The two carboxy-terminal β-catenin fragments were identified by chromatography of in vitro
cleavage products using high-pressure liquid chromatography (HPLC) (Smart System,
Pharmacia, Freiburg, Germany) on a C4 reversed-phase column (2.1 mm ID, 300 Å, 5 µm)
obtained from Vydac (Hesperia, CA). Collected fractions were screened for the carboxy-
terminal fragment by MALDI-mass spectrometry (VG Tof Spec, Fisons, Manchester, UK). The
sequences of the C-terminal fragments were obtained by ESI-MS/MS using a Q-Tof
(Micromass, Manchester, UK) equipped with a nano-electrospray ion source.
GST-E-Cadherin Affinity Precipitation Assay
A recombinant gluthatione S-transferase (GST)-tagged cytoplasmic domain of mouse E-
cadherin (kindly provided by O. Huber, Berlin) was adsorbed on glutathione S-transferase-
agarose beads. Approximately 5 µg of GST-E-cadherin was used for the affinity precipitation
of β-catenin as described (43).
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-catenin Expression Constructs
β-catenin FL (bp 215-2557), ∆C1 (bp 215-2506), ∆C2 (bp 215-2467), ∆N (bp 560-2557),
∆N∆C1 (bp 560-2506) and ∆N∆C2 (bp 560-2467) were amplified by PCR using a plasmid
containing the full-length β-catenin sequence (pQE32β415) as template (38). 5‘ primers
contained a NotI site and corresponding 3‘ primers contained a XbaI site. The amplified
fragments were cloned into pGEMTeasy (Promega) sequenced, excised by NotI/XbaI
digestion and inserted into the pGEMT-HAX vector containing the sequence for a triple HA-tag
at the 3‘ end. For expression all constructs were excised with NotI and inserted into the
eukaryotic expression vector pcDNA6 B (Invitrogen).
Reporter Assays
Approximately 2x105 HEK293 or H184A1 cells were transiently transfected by the calcium
phosphate precipitation method using 1 µg of luciferase reporter pS01234 or its negative
control containing mutated TCF sites (pS) (31), 0.25 µg hTCF-4 (44), 0.5 µg β-catenin
constructs and 1 µg of pCH110 β-Gal expression vector as internal control. 48 h after
transfection cells were harvested and resuspended in 100 mM potassium phosphate buffer,
pH 7.2. Cell lysis was carried out by three cycles of freezing and thawing. The lysate was
cleared by centrifugation and luciferase and β-galactosidase activities were measured with a
Tropix Dual Light Chemiluminescence kit according to the manufacturer’s instructions.
Luciferase activity was normalized to β-galactosidase activity.
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RESULTS
Anoikis-Induced Cleavage of -Catenin in the Human Mammary Epithelial Cell Line
H184A1 is Mediated by Caspase-3-like Proteases
To investigate if components of the cadherin-mediated cell-cell adhesion complex are impaired
in apoptotic cells we analyzed the protein level of E-cadherin, α-catenin and β-catenin in the
human mammary epithelial cell line H184A1. Anoikis was induced as described in Materials and
Methods. Western blot analysis revealed lower molecular mass cleavage products of β-
catenin while E-cadherin and α-catenin remained apparently unaffected. The different β-
catenin cleavage products appeared in a time dependent manner. Already 4 h after induction
of anoikis, truncated β-catenin fragments were detectable at approximately 90 kDa
(fragment A), 76 kDa (fragment B) and 72 kDa (fragment C). A fourth 70 kDa cleavage product
(fragment D) became visible after 8 h of induction (Fig. 1A). A 85 kDa fragment (fragment E*)
was already observed in uninduced cells, thus representing, an apotosis-unspecific β-catenin
degradation product. The amounts of the three lower molecular mass polypeptides (fragments
B, C, D) increased during the time course of apoptosis in correlation with the increasing
proportion of apoptotic cells as determined by TUNEL-assay. 24 h after induction 96 % of the
cells were found apoptotic (Fig. 1B). The total amount of the β-catenin cleavage products was
comparable to the amount of full-length (FL) β-catenin as determined by densitometric
measurement of the band intensities (data not shown). Low levels of E-cadherin at time points
0 and 4 h were due to the trypsination process of anoikis induction (Fig. 1A).
As caspase-3 family members have recently turned out to be important effector molecules in
apoptosis, we tested whether β-catenin cleavage could be inhibited by incubating H184A1
cells with the irreversible caspase-3 family specific inhibitor Z-DEVD-fmk. Cells incubated with
100 µM Z-DEVD-fmk and harvested 24 h after induction of anoikis were analyzed by
Western-blotting for β-catenin and the rate of apoptosis was determined by TUNEL-assay.
Appearance of β-catenin fragments B, C and D was completely abolished by the inhibitor but
not appearance of the apoptosis-unspecific degradation product (fragment E*) (Fig. 1C). The
rate of apoptotic cells was reduced to control level (Fig. 1D).
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To analyze if the apoptosis-induced, proteolytic cleavage of β-catenin is cell type specific or
was dependent on the apoptotic stimulus used, we further tested the β-catenin fragmentation
in the epithelial Madin Darby Canine Kidney (MDCK) cell line. Apoptosis was induced by cell
detachment or by incubation with the protein kinase inhibitor staurosporine (STS) which was
described as a potent inducer of the apoptotic program (45). Identical in vivo cleavage
patterns of β-catenin were observed for both cell lines and for both apoptotic stimuli (Fig. 2A).
A significant proportion of cells detached from the culture plate after staurosporine treatment.
We thus analyzed the adherent and detached cell populations separately (Fig. 2B). β-catenin
was almost entirely cleaved 20 h after STS treatment in the detached (sus) cell population,
while 35 % of the full-length β-catenin remained in the adherent cell fraction. However, just
37 % of the adherent cells compared to 89 % of the detached cells were found apoptotic as
determined by TUNEL assay (data not shown).
In vitro Cleavage of -Catenin with Recombinant Caspase-3, -6 and -7
In order to identify the caspase which is directly involved in cleavage of β-catenin, we
digested recombinant His-β-catenin with recombinant caspase-3, -6 and -7 in vitro. β-catenin
was cleaved by all caspases, resulting in different cleavage products, and this could be
blocked in each case by addition of the caspase-specific inhibitor Z-DEVD-fmk (Fig. 3A). The
apparent molecular masses of β-catenin fragments obtained after in vitro cleavage by
caspase-3 and cleavage products from H184A1 cells treated with STS were identical (Fig. 3A,
C3 and STS), indicating that caspase-3 might be the main caspase involved in cleavage of β-
catenin in vivo. Western blot analysis of apoptotic H184A1 cell lysates for caspases-3, -6
and -7 with antibodies recognizing the activated enzymes showed that only caspase-3 was
detectable in its activated form (data not shown). The involvement of caspase-3 in the
cleavage of β-catenin was further supported by analysis of MCF-7 cells, which have been
shown previously to be deficient in caspase-3 (39). After STS treatment the three main β-
catenin cleavage products B, C and D were detected only in MCF-7 cells stably transfected
with caspase-3 (Fig. 3B, Casp3) but not in control MCF-7 cells transfected with the vector
alone (Vec).
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Mapping of the -Catenin Cleavage Sites
Two consensus motifs corresponding to a potential caspase-3/-7 cleavage site (DXXD) are
found at positions Asp764 and Asp459 in the β-catenin amino acid sequence. Three further
putative caspase-6 cleavage sites (XEXD) are present in the N-terminal region at positions
Asp11, Asp17 and Asp58 (46). However, the calculated molecular masses of such putatively
truncated β-catenin molecules did not correlate with the apparent molecular masses of the
observed β-catenin cleavage products except for a potential cleavage at Asp764. We
analyzed apoptosis-induced cleavage products of β-catenin by Western-blotting with
antibodies directed against the N- and C-terminal domains of β-catenin, respectively. Both
antibodies detected only full-length β-catenin but none of the degradation fragments (data not
shown). Therefore, we presume that the N- and C-terminal regions of β-catenin were cleaved
off, and that the cleavage products were too small (< 7.5 kDa) to be detected in our assay
system.
For identification of the cleavage sites we digested recombinant His-β-catenin with
recombinant caspase-3 and sequenced the resulting β-catenin fragments A, B, C, D and E by
N-terminal Edman degradation (Fig. 4A). Sequencing of fragment A revealed a complete, non-
truncated N-terminal region of β-catenin indicating that cleavage had occurred at the C-
terminus. We identified the potential corresponding 1.8 kDa β-catenin cleavage product by
HPLC separation. Analysis of this peptide by ESI-MS/MS revealed the sequence
GLPPGDSNQLAWFDTDL, which corresponds to the amino acids 765-781 in the C-terminus
of β-catenin. The cleavage therefore had occurred at position Asp764, a potential caspase-3
consensus sequence DLMD (Fig. 4B). The N-terminal cleavage sites for fragments B, C and
E were determined by Edman degradation, revealing cleavage at position Asp32 (SYLD) for
fragment E, Asp83 (ADID) for fragment B, and Asp115 (TQFD) for fragment C (Fig. 4B).
Analysis of fragment D resulted in a sequence identical to the one obtained for fragment C,
suggesting that β-catenin has a second C-terminal cleavage site. This inference was proven
by identification of a corresponding 1.4 kDa peptide after HPLC separation and subsequent
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ESI-MS/MS sequencing. The resulting sequence GLPDLGHAQDLMD matched amino acids
752-764 of β-catenin, indicating that cleavage had occurred at Asp751 (YPVD).
The time course of β-catenin cleavage showed that, in vitro, β-catenin was first cleaved at the
C-terminus, generating fragment A as the main cleavage product after 10 min, followed by
cleavage at the N-terminus (fragments E, B and C). Finally, β-catenin was further truncated at
the second C-terminal cleavage site, resulting in the appearance of fragment D after 1 h
(Fig. 4A). A β-catenin cleavage product of similar size as fragment E at about 85 kDa was
already detectable under in vivo conditions. However, this fragment (E*) was already
observed in uninduced cells and was found to be apoptosis-unspecific (Fig. 1A, 0h).
Appearance of this fragment was not blocked by caspase-specific inhibitors, revealing that it
is not created in a caspase-dependent manner (Fig. 1C). We thus conclude that fragments E
and E* might be independent of each other and that the generation of fragment E under in vivo
conditions is too small to be detected in relation to the unspecific product (E*).
Binding of N-terminal Truncated -Catenin to -Catenin and E-Cadherin
We then determined whether truncation of β-catenin impairs its association with α-catenin or E-
cadherin, because the identified caspase-3 cleavage sites in the N-terminus of β-catenin are in
close proximity to the α-catenin-binding domain located to amino acids 129 - 143 of β-catenin.
After staurosporine-induced apoptosis, cell lysates from MDCK cells were subjected to
immunoprecipitation with monoclonal antibodies to E-cadherin and β-catenin and a polyclonal
antibody to α-catenin. Western blot analysis of the E-cadherin, α-catenin and β-catenin
immunocomplexes with anti-β-catenin antibodies showed association of the β-catenin
cleavage products B, C and D with E-cadherin (Fig. 5A, STS/E-cad) as well as with α-catenin
(Fig. 5A, STS/α-cat). Subsequent re-probing with anti-α-catenin and anti-E-cadherin
antibodies showed that both proteins were detectable in all immunoprecipitates (Fig. 5A).
The overall amount of all components of the cadherin/catenin complex is significantly lower in
the detergent-insoluble, cytoskeleton-associated protein fraction (P) than in the soluble (S)
protein fraction. After induction of apoptosis a reduction in the overall amount of β-catenin was
observed in both protein fractions but not for α-catenin or E-cadherin. The β-catenin cleavage
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products were, however, mainly detectable in the detergent-soluble protein fraction (Fig. 5B,
S/STS). Just small amounts were visible in the insoluble fraction after longer exposure (data
not shown). This suggests that the β-catenin proteolytic fragments might still be able to
interact with E-cadherin and α-catenin but might not be able to compensate for full-length β-
catenin in the functional cadherin/catenin cell-adhesion complex.
We next determined if the amount of the uncomplexed signaling-competent pool of β-catenin
was changed in apoptotic cells. Soluble β-catenin was precipitated with a recombinant GST-
E-cadherin fusion protein containing the cytoplasmic tail of E-cadherin (GST-ECT). Just small
amounts of cytoplasmic, uncomplexed β-catenin were observed in uninduced cells. A
significant, 5-fold increase in the amount was observed 2 h after induction of apoptosis as
determined by densitometric measurement (Fig. 5C). 8 h after induction no full-length β-catenin
was detectable anymore while the cytoplasmic β-catenin cleavage products were enriched to
a significant extent with a 7-fold excess compared to the initial full-length β-catenin at 0 h. The
overall amount of β-catenin did not change detectably as shown by Western blot analysis of
whole cell lysates (Fig. 5C).
Deletion Constructs of -Catenin Show Reduced Transactivation Potential in Gene
Reporter Assays
To investigate the consequence of β-catenin cleavage for its signaling function, we designed
different β-catenin deletion constructs corresponding to the identified caspase-3 cleavage sites
(Fig. 6A). All constructs showed comparable expression levels after transfection into HEK293
and H184A1 cells (Fig. 6B). Subsequently, we analyzed their transactivation potential in a
gene reporter assay. The pS01234 reporter plasmid containing the TCF/β-catenin-dependent
siamois promoter in front of the luciferase gene was co-transfected with a hTCF-4 expression
plasmid together with the different β-catenin constructs. The activation of the luciferase reporter
gene by the various β-catenin constructs in HEK293 and H184A1 cells is shown in Fig. 6C.
Very similar results were obtained for both cell lines. Full-length (FL) β-catenin showed an over
12-fold induction of the luciferase reporter in HEK293 and H184A1 cells, when co-transfected
with hTCF-4. Truncation of the C-terminus by 17 amino acids (Fig. 6B, ∆C1), which
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corresponds to fragment A of the apoptotic cleavage products, already showed a slightly
reduced luciferase activity of 8.5- and 10-fold activation in the two cell types (Fig. 6C). The
deletion construct ∆N∆C1, which resembles fragment C of apoptotic cleavage products,
showed only 3- to 4-fold increased luciferase activity and this was even more reduced when
the C-terminus was further truncated as shown for ∆N∆C2. This deletion construct, resembling
the smallest apoptotic cleavage product fragment D, only showed basal reporter gene activity
when compared to luciferase activity of hTCF-4 alone (Fig. 6C). We also wanted to know to
what extent the reduction in the transactivation potential of β-catenin deletion constructs
depended on truncation of either the N- or C-terminus alone. Analysis of constructs deleted at
the N- or C-terminus (Fig. 6A, ∆N or ∆C2) revealed that removal of either one of these
domains reduced the luciferase activity to 50 % of the FL level. Transfection of a reporter
plasmid with mutated TCF-binding sites (pS) instead of pS01234 did not show reporter gene
activity (data not shown).
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DISCUSSION
Apoptosis is an important feature of many epithelial tissues with a high turnover and serves to
balance accurately the rate of new cell production. Death by apoptosis eliminates specific
cells without extensive tissue damage, playing a pivotal role during tissue regeneration and
the elimination of tumor cells. Survival factors required to prevent cells from undergoing
apoptosis may involve cytokine signaling from neighboring cells, extracellular matrix- as well
as cell-cell contacts.
Here we could demonstrate that the cell-cell adhesion molecule β-catenin is proteolytically
cleaved during anoikis or staurosporine-induced apoptosis. Four apoptosis-specific cleavage
products of β-catenin with apparent molecular masses of approximately 90 kDa (fragment A),
76 kDa (fragment B), 72 kDa (fragment C) and 70 kDa (fragment D) were observed after
induction of apoptosis in the human breast epithelial cell line H184A1 and the canine kidney
cell line MDCK. The β-catenin cleavage was almost entirely inhibited by the caspase-3
specific tetrapeptide inhibitor Z-DEVD-fmk indicating that caspase-3-family proteases might
play a dominant role during the cleavage process. This notion was further supported by
cleavage of recombinant β-catenin with recombinant caspase-3 under in vitro conditions. The
resulting cleavage pattern was found to be identical to the already observed cleavage pattern
in vivo. Also consistent with this idea, no apoptosis-specific β-catenin cleavage products
were observed in the caspase-3-deficient MCF-7 human breast carcinoma cell line after STS-
induced apoptosis, but β-catenin cleavage could be restored by re-introduction of a
caspase-3 expressing plasmid.
β-catenin is a 92 kDa protein containing a central core-region of 13 incomplete conserved
Armadillo-repeat motifs (Arm-repeats) and unique N- and C-terminal regions. The core-region
acts as a surface for the association of several interacting proteins, e.g. cadherins, α-catenin,
the APC-protein, Axin/Conductin, Pontin52 or LEF-1/TCF (35,47-52) and is able to mediate
cell-cell adhesion on its own. Both the core-region and the C-terminal domain of β-catenin are
involved in its signaling activity (44,53-56). By identification of the proteolytic cleavage sites
in the β-catenin sequence we observed that the C-terminus as well as the N-terminus of
β-catenin are deleted during apoptosis but the integrity of the core-region was not affected.
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Consistently we found that all proteolytic fragments of β-catenin were able to associate with
E-cadherin and α-catenin under in vivo conditions. This demonstrates that the truncated β-
catenin fragments can substitute for the full-length molecule at least in the detergent-soluble
cadherin-catenin complex. Just small amounts of the cleavage products were detectable in the
insoluble, cytoskeleton-associated cadherin-catenin protein complex. The cytoskeleton-
associated fraction, however, is believed to participate solely in active cell-cell adhesion, while
the soluble cadherin-catenin complex is not directly involved in the cell to cell interaction.
Moreover, we found that the amount of full-length β-catenin was greatly diminished in both
subcellular fractions in staurosporine-induced apoptotic cells. This reduction of the protein level
was found to be specific for β-catenin and was not observed for E-cadherin or α-catenin. We
conclude that the proteolytic cleavage of β-catenin during apoptosis might have a direct
influence on cell-cell adhesion. A reduction of cell-cell contacts during apoptosis was already
described for NIH3T3 cells when grown in serum-free media (57). However, the cleavage
pattern of β-catenin was found to be remarkably different from those that we have found in
H184A1 and MDCK cells. Additionally, no association of the β-catenin proteolytic fragments
with α-catenin was observed in this study. In contrast, the interaction of β-catenin cleavage
products with α-catenin was described in apoptotic endothelial cells (58). In those cells a
similar β-catenin cleavage pattern was identified, as we have observed, but just some of the
cleavage products were able to interact with α-catenin. Due to the missing interaction with
α-catenin an influence on the cell-cell adhesion was speculated. However, the interaction of
the β-catenin truncated forms with E-cadherin has not been demonstrated so far. From our
biochemical analysis and the previous results it is hard to conclude if the β-catenin cleavage
has a strong influence on cell-cell adhesion. This needs to be analyzed in more detail with
more physiological assays to determine cell-cell adhesion. We recently obtained some first
evidence that besides β-catenin E-cadherin also is proteolytically cleaved during apoptosis
under specific conditions (O. Huber, unpublished results) which indicates rather a reduced
cell-cell contact during apoptosis.
Beside its involvement in cell-cell adhesion, β-catenin is also involved in cell signaling as a
component of the Wnt/wingless signaling pathway. As a major step of Wnt-signal
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transduction, β-catenin becomes stabilized in the cytoplasm due to the inhibition of protein
degradation (59). It is believed that the amount of the cytoplasmic, uncomplexed β-catenin is
the critical parameter for Wnt-signal transduction. Cells with a high amount of free β-catenin
e.g. Wnt-induced cells or cells from colon carcinoma cell lines show a strong activation potential
for endogenous Wnt-target genes. Here we found that the cytoplasmic, uncomplexed pool of
β-catenin is significantly enriched 2 h after induction of apoptosis. A similar increase in the free
pool of β-catenin was already described in Wnt-induced cells (59). At later stages no full-
length β-catenin could be observed while the cleavage products were found enriched to a high
extent in the cytoplasmic fraction. According to our results, the integrity of the core region of β-
catenin is not affected by proteolytic cleavage during apoptosis, but, the N- and C-terminal
regions are removed. Both regions contain transcription activation domains that mediate the
transactivtion of Wnt-target genes if β-catenin is in the complex together with LEF-1/TCF
transcription factors. Concordantly, in gene reporter assays we were able to demonstrate that
β-catenin deletion constructs resembling the observed in vivo cleavage products had a
reduced transcription activation potential. Deletion of the first 115 N-terminal amino acids of β-
catenin (fragment B) already showed an over 50 % reduced reporter activation compared to
the full-length β-catenin. Additional removal of the C-terminus reduced the activity down to
30 % and the shortest deletion construct ∆N∆C2 had no significant activity anymore compared
to the activation by hTCF-4 alone. This observation is consistent with previous reports
demonstrating that deletions of the N- or C-terminus both lead to a decreased transactivation
potential (53,60,61). We therefore assume that an important role for the β-catenin cleavage
during apoptosis might be the removal of the β-catenin transcription activation domains to
prevent its transactivation potential. Already 8 h after induction of apoptosis the full-length,
transactivation-competent β-catenin was entirely cleaved in the cytoplasmic fraction while the
transactivation-incompetent cleavage products were enriched to a high extent. Increased
levels of β-catenin were found in several melanoma and colon carcinoma cell lines due to
mutations in the APC gene or the β-catenin gene leading to a stabilized protein (62).
Furthermore, wild-type β-catenin was found to contain a neoplastic transformation potential in
NIH3T3 fibroblasts (63). The same result was obtained for a stabilized mutant form of β-
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catenin with a point mutation or deletion of the N-terminal region when transfected into RK3E
cells (64). The transformation potential of the mutant forms of β-catenin was found to be
greatly diminished in deletion constructs with a missing LEF-1/TCF binding site or a deletion of
the C-terminal transactivation domain, indicating that the β-catenin transactivation potential is
important for its transformation capability. In this respect it was recently found that the TCF/β-
catenin complex directly regulates the transcription of both c-myc and the cyclinD1 gene
(32,33). Both gene products are involved in cell proliferation by controlling cell-cycle
progression and thus might be critical target genes for the neoplastic transformation potential of
β-catenin. Removal of the β-catenin transactivation domains by proteolytic cleavage might
thus prevent the cell from overcoming the apototic program by a deregulation of cell
proliferation due to the activation of critical β-catenin target genes like c-Myc or CyclinD1.
During recent years β-catenin has become further implicated in the regulation of apoptosis.
Consistent with our observations it was reported that the over expression of a β-catenin
deletion construct with truncated N- and C-terminal regions leads to an increased rate of
apoptosis in rat hippocampal neurons (65). The over-expression of a dominant negative TCF
had the same effect, indicating that the inhibition of TCF/β-catenin signaling induces apoptosis.
Proteolytic cleavage of β-catenin thus might not be simply an effect of apoptosis rather than
inducing of the apoptotic program. The versatile functions of β-catenin within the interplay of
cell growth and cell death will be an interesting aspect for future experiments.
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ACKNOWLEDGMENTS
This work was supported by the Deutsche Forschungsgemeinschaft, SFB 366. We thank Ina
Krukenberg for excellent technical assistance, A. Porter for providing MCF-7.3.28 and
MCF-7/Vector cells, D. Kimelman for providing the siamois luciferase reporter plasmids, H.
Clevers for providing hTCF-4 plasmid, O. Huber for providing GST-fusion proteins and R.
Cassada for critically reading the manuscript.
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FIGURE LEGENDS
Figure 1. Anoikis induced cleavage of β-catenin and inhibition of caspase activity. (A)
Confluent H184A1 cells were trypsinized and transferred to polyHEMA coated flasks, where
they were maintained in suspension for the indicated time. Cell lysates were separated on a
SDS-PAGE, and Western blot analysis for β-catenin, E-cadherin and α-catenin was
performed with monoclonal antibodies. Full-length β-catenin (FL) and fragments A-E* are
denoted by arrows. (B) The rate of apoptosis was quantified by FACS-analysis after
incorporation of BrdUTP into single- and double-DNA strand breaks as described in Materials
and Methods. (C) As described in (A) H184A1 cells were either kept adherent (ad) or
maintained in suspension (sus) for 24 h in the presence (A+DEVD) or absence (A) of 100 µM
of the specific caspase-3-like inhibitor Z-DEVD-fmk. Cells were harvested and lysates
analyzed by Western-blotting with monoclonal anti-β-catenin antibody. (D) The rate of
apoptosis was determined as described in (B).
Figure 2. Stimuli and cell line independent cleavage patterns of β-catenin during apoptosis.
(A) For comparision of the β-catenin cleavage pattern between different cell lines and different
apoptotic stimuli, H184A1 and MDCK cells were maintained in suspension (A) for 24 h or
confluent H184A1 and MDCK cells were incubated with 1 µM staurosporine (STS) for 16 h.
Cell lysates were separated by SDS-PAGE and analyzed by Western-blotting with an anti-
β-catenin antibody. (B) H184A1 cells were incubated with 0.2 % DMSO (C) or 1 µM STS for
20 h. After STS treatment detached (sus) and still adherent (ad) cells were harvested
separately and analyzed by Western-blotting for β-catenin.
Figure 3. In vitro cleavage of β-catenin with recombinant caspase-3, -6 and -7.
(A) In vitro cleavage of recombinant His-β-catenin with 50 ng recombinant caspase-3 (C3),
caspase-6 (C6) and caspase-7 (C7). As a control, β-catenin was incubated with sample
buffer alone (B). All samples were incubated for 4 h at 37 °C separated by SDS-PAGE and
analyzed by Western-blotting with an anti-β-catenin antibody. 50 µg lysate of STS treated
H184A1 cells (STS) was analyzed for comparison of in vivo and in vitro cleavage products of
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β-catenin. (B) MCF-7 cells stably transfected with caspase-3 (Casp3) or vector (Vec) and
H184A1 were treated with 1 µM STS or 0.2 % DMSO as control (C). Samples were
analyzed by Western-blotting with an anti-β-catenin antibody.
Figure 4. (A) Time course of in vitro cleavage of recombinant 6His-β-catenin (5 µg) by
recombinant caspase-3 (100 ng). Samples were incubated at 37 °C for the indicated time,
separated by SDS-PAGE and stained with Coomassie blue. Arrows with letters indicate the
different lower mass cleavage products of β-catenin. (B) Schematic diagram of β-catenin and
apoptotic cleavage products. Caspase-3 cleavage sites (arrows) and recognition sequences
(italics) are indicated. The lines represent the cleavage products A-E. Dashed lines indicate
that the C-termini were not identified precisely.
Figure 5. Binding of truncated β-catenin to α-catenin and E-cadherin.
MDCK cells were grown in medium containing 0.2 % DMSO (Control) or 1 µM staurosporine
(STS) for 6 h. Cells were harvested and protein extracts divided into the soluble and
insoluble protein fraction. (A) The soluble fractions were immediately prepared for
immunoprecipitation with monoclonal antibodies to E-cadherin (E-cad) and β-catenin (β-cat)
and a polyclonal antibody to α-catenin (α-cat). Immunoprecipitates and (B) aliquots of the
detergent soluble (S) and insoluble (P) protein fraction were resolved by SDS-PAGE and
analyzed by Western-blotting with monoclonal antibodies to α-catenin, β-catenin and E-
cadherin. (C) Affinity precipitation of the uncomplexed pool of β-catenin. MDCK cells were
treated with STS for the indicated time. The detergent-soluble fractions of the cell lysates were
incubated with 5 µg GST-E-cadherin fusion protein (GST-ECT) coupled to GST-agarose
beads. Precipitates and whole cell lysates were separated by SDS-PAGE and analyzed for
β-catenin by Western-blotting.
Figure 6. (A) Schematic diagram of FL β-catenin and deletion constructs tagged with a triple
HA-epitope. (B) Expression of FL β-catenin and deletion constructs in HEK293 and H184A1
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cells was detected with a monoclonal anti-HA antibody. (C) Luciferase reporter gene activity
of FL β-catenin and deletion constructs was performed as described in Material and Methods.
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Arm-repeatsN CSYLD ADID TQFD DLMDYPVD
32 83 115 764751
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Fig. 4B
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HA-TagArm-repeats
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Fig. 6A
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HEK293H184A1
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Wittmann-Liebold, Bernd Dorekn and Kurt BommertUlrike Steinhusen, Volker Badock, Andreas Bauer, Jurgen Behrens, Brigitte
Fragments with Reduced Transactivation Potential-Catenin by Caspase-3 Results in ProteolyticβApoptosis-induced Cleavage of
published online March 9, 2000J. Biol. Chem.
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