* Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie AREA Science Park, 34142 Trieste, Italy; Cold Spring
Harbor Laboratory, Cold Spring Harbor, New York 11724; and § Dipartimento di Scienze e Tecnologie Biomediche, Sezione di
Biologia, Universita' di Udine, 33100 Udine, Italy
Cell death by apoptosis is a tightly regulated process that requires coordinated modification in cellular architecture. The caspase protease family has been shown to play a key role in apoptosis. Here we report that specific and ordered changes in the actin cytoskeleton take place during apoptosis.
In this context, we have dissected one of the first hallmarks in cell death, represented by the severing of contacts among neighboring cells. More specifically, we
provide demonstration for the mechanism that could
contribute to the disassembly of cytoskeletal organization at cell-cell adhesion. In fact, -catenin, a known
regulator of cell-cell adhesion, is proteolytically processed in different cell types after induction of apoptosis. Caspase-3 (cpp32/apopain/yama) cleaves in vitro
translated
-catenin into a form which is similar in size
to that observed in cells undergoing apoptosis.
-Catenin cleavage, during apoptosis in vivo and after caspase-3 treatment in vitro, removes the amino- and
carboxy-terminal regions of the protein. The resulting
-catenin product is unable to bind
-catenin that is
responsible for actin filament binding and organization.
This evidence indicates that connection with actin filaments organized at cell-cell contacts could be dismantled during apoptosis. Our observations suggest that
caspases orchestrate the specific and sequential
changes in the actin cytoskeleton occurring during cell
death via cleavage of different regulators of the microfilament system.
APOPTOSIS is a fundamental cellular process in the development and homeostasis of all multicellular organisms (Raff, 1992 The ced-3 gene is essential for developmentally programmed cell death in C. elegans: it encodes a protein
which shares homology with mammalian interleukin-
1 ICE is a cystein protease that cleaves interleukin-1 Nine additional members of ICE-related proteases have
been identified in mammals (Fernandes-Alnemri et al.,
1994 Indeed, recent observations suggest the existence of a
hierarchically ordered proteolytic cascade during apoptosis. In the case of CD95/FAS/APO-1-induced apoptosis,
an initial activity related to ICE itself (caspase-1), and a
later activity related to CPP32 (caspase-3), have been described using specific inhibitor substrates (Enari et al.,
1996 The central role of the caspase proteases in mammalian
apoptosis is supported by the existence of specific inhibitors. The viral proteins p35 and CrmA (Bump et al., 1995 If cystein proteases play a crucial role in apoptosis, specific target proteins or death substrates become critically
relevant for the execution phase of the apoptotic program.
Specific cleavage by ICE-related cystein proteases of
the DNA repair enzymes poly(ADP-ribose) polymerase
(PARP) and the catalytic subunit of the DNA-dependent
protein kinase (DNA-PK), is possibly related to the inactivation of the DNA repair pathway as a choice of economy
(Lazebnik et al., 1994 Two interesting substrates for caspases are the protein
kinase C Apoptotic morphological features are generally similar
in all systems where they have been studied: the nucleus
condenses and the cell shrinks and often fragments into
apoptotic bodies that are rapidly phagocytosed by neighboring cells (Kerr et al., 1972 The present study addresses the roles and changes of the
microfilament system during apoptosis. By a detailed confocal analysis on apoptotic NIH3T3 cells, we have identified specific sequential changes in the microfilament system. Coordinately with actin filament reorganization in
the perinuclear area, the cell retracts from the adhesion
substrate and severs contacts with neighboring cells.
Germane to the above mentioned changes, we also observed a specific modulation of the cell-cell contacts during apoptosis through proteolytic processing of In summary, the complex reorganization of cell morphology during apoptosis seems to be achieved by specific
proteolytic processing of different regulators of actin cytoskeleton.
Cell Lines and Culture Conditions
NIH3T3 and MDCK cells were grown in DME supplemented with 10%
FCS, penicillin (100 U/ml), and streptomycin (100 µg/ml). In each experiment 2.5 x 104 cells/ml were seeded in 35-mm Petri dishes.
For serum starvation, medium was changed to 0.5% FCS when cells
were subconfluent and left in this medium for 48 h. For density-dependent inhibition, cells were plated at 104/cm2 in 10% FCS. 24 h after plating the
medium was changed every 2 d. For induction of apoptosis the culture medium of arrested cells was replaced with serum-free DME. Nonadherent
and adherent cells were harvested, washed in serum-free medium, and solubilized in SDS-PAGE buffer. In the case of genotoxic-dependent apoptosis, density-inhibited cells were treated with 20 µg/ml of cisplatin for 4 h;
nonadherent and adherent cells were harvested separately 24 h later. For
UV treatment, culture medium was removed, dishes were washed once
with PBS, UVC irradiated (180 J/m2 in PBS), and fresh medium, containing 10% FCS, was added to the cells. Adherent and nonadherent cells
were harvested separately 24 h later. NIH3T3 cells were transfected as
previously described (Brancolini et al., 1995 Plasmid Construction
Mouse A site-directed mutagenesis method by overlap extension through PCR
as previously described (Brancolini et al., 1995 All constructs generated were sequenced using an automated (ALF)
system to check for the respective introduced mutations, deletions, and
translating fidelity of the inserted PCR fragments.
Immunofluorescence Microscopy
NIH3T3 cells were grown under the described conditions and then fixed
with 3% paraformaldehyde in PBS for 20 min at room temperature. Fixed
cells were washed with PBS/0.1 M glycine, pH 7.5, and then permeabilized
with 0.1% Triton-X100 in PBS for 5 min. The coverslips were treated with
the anti- Cells were examined with a laser scan microscope (LSM 410; Carl
Zeiss, Inc., Thornwood, NY) equipped with a 488 Two-dimensional Gel Electrophoresis
NIH3T3 cells grown for 36 h in 0.5% FCS were labeled for 12 h in 1 ml of
DME methionine-free 0.5% FCS, containing 400 mCi/ml [35S]methionine
(Amersham Int'l., Little Chalfont, UK). Serum-free medium containing a
cold methionine chase, (0.5 mM final concentration) was then added for 6 h.
Samples of total cellular proteins were prepared from adherent and
nonadherent NIH3T3 cells after washing with cold PBS. Cells were lysed
in hot (100°C) lysis solution (0.3% SDS, 200 mM dithiothreitol, 50 mM
Tris-HCl, pH 8). Samples were then treated with 1:10 vol of nuclease solution (50 mM MgCl2, 50 mM Tris-HCl, pH 7, 1 mg/ml DNaseI, 0.25 mg/ml
RNaseA) for 10 min on ice, precipitated by adding ice-cold acetone to
80% vol/vol, and centrifuged at 14,000 g for 10 min. Air-dried pellets were
resuspended in a mixture of 1 vol lysis solution and 4 vol of sample buffer
(9.9 M urea, 4% NP-40, 2.2% Millipore ampholytes, 100 mM dithiothreitol). Two-dimensional gels were run using the investigator two-dimensional electrophoresis system (Millipore Corp., Waters Chromatography,
Milford, MA) with pH 3-10 and pH 4-8 Millipore ampholytes in the first
dimension and 10% acrylamide in the second dimension.
Immunoblotting
For Western blotting, proteins were transferred to 0.2-µm pore-size nitrocellulose (Schleicher & Schuell Inc., Keene, NH) using a semidry blotting
apparatus (Bio-Rad Laboratories, Hercules, CA) (transfer buffer: 20%
methanol, 48 mM Tris, 39 mM glycine, and 0.0375% SDS). After staining
with Ponceau S, the nitrocellulose sheets were saturated for 2 h in Blotto-Tween 20 (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 5% nonfat dry milk,
and 0.1% Tween 20; Sigma Chemical Co.) and incubated overnight at
room temperature with a specific antibody: anti-Gas2, anti-actin, anti- Expression of Caspase-3 (CPP32) in Bacteria and In
Vitro Protease Assay
Caspase-3 cDNA was subcloned in-frame into the BamH1 site of the bacterial expression vector pQE-12 (QIAGEN Inc., Santa Clarita, CA). Cells
were grown to an A600 of 0.6 and expression of caspase-3 was induced by
adding isopropyl- Caspase-3 and Immunoprecipitation
Analysis of Immunocomplexes were released by boiling 5 min in SDS sample
buffer, separated in a 10% SDS-PAGE, and Western blots were performed as described.
Microfilament Reorganization during Apoptosis in
NIH3T3 Cells
NIH3T3 fibroblasts efficiently organize stress fibers and
adhesion plaques and form cell-cell junctional complexes
and membrane ruffles. They represent an ideal system to
study microfilament organization in response to different
environmental conditions. Furthermore, growth-arrested
NIH3T3 cells readily respond to complete absence of serum in the culture medium by undergoing apoptosis.
To study the changes in microfilament organization in
cells undergoing apoptosis, immunofluorescence analysis
was performed on growth-arrested NIH3T3 cells cultured
for a further 3 h in serum-free medium.
As shown in Fig. 1 a, after 3 h of incubation in serum-free medium, changes in the microfilament organization
become clearly detectable in some cells as shown by phalloidin staining (arrows). As a response to the absence of
survival factors, cells apparently sever contacts with neighboring cells, retract from the adhesion substrate, and dismantle stress fibers, extensively reorganizing their actin
cytoskeleton.
Apoptosis is always accompanied by alterations in the
nuclear morphology, finally leading to nuclear fragmentation and formation of the apoptotic bodies. We, therefore,
decided to follow such nuclear alterations in relation to
the changes in actin organization during apoptosis.
When an initial alteration in nuclear architecture was
first detected (arrow, Fig. 1 A) changes in the actin cytoskeleton were also evident (Fig. 1 B). As described above, the
peculiar features at this stage included the severing of contacts with neighboring cells, retraction of fibers, accumulation
of actin in the perinuclear region and loss of stress fibers.
At further stages, nuclear alterations become more evident leading to nuclear fragmentation (Fig. 1 C). At this
stage, the actin cytoskeleton is clearly organized in a ring-like structure (Fig. 1 D, arrow) with membrane blebbing
also clearly evident (data not shown). It is interesting to
note that nuclear fragments are external with respect to
the observed actin ring. This observation suggests that
contraction of actin filaments present in the perinuclear region might be important in the sprouting of the nuclear
bodies. This hypothesis was supported by the most altered
phenotypes, showing the actin reorganization as summarized in Fig. 1, E-H. At these stages the actin ring appeared collapsed, with the nuclear fragments tending to
drop away from the actin filaments. This detachment
probably leads to the appearance of the phenotype represented in Fig. 1, G and H, where only remnants of cytoplasm containing a collapsed actin ring structure were detected. Such a phenotype could represent a final stage of
the microfilament changes taking place during apoptosis.
From this preliminary analysis, it could be appreciated
that apoptosis, as triggered by serum deprivation in NIH3T3
fibroblasts, is coupled to well-defined and ordered changes
of the microfilament system. Such changes are concomitant with membrane blebbing and alteration of the nuclear
architecture.
Analysis of Actin Cleavage during Apoptosis in NIH3T3
Since it has been reported that actin can be cleaved in an
in vitro apoptotic system using purified caspase-1 (Mashima et al., 1995 A time course analysis was performed on growth-arrested
NIH3T3 cells incubated in serum-free medium. Cell survival
was markedly decreased after few hours in serum-free medium (Fig. 2 a) and apoptotic cells could be easily observed
floating in the medium (data not shown).
To study actin cleavage during apoptosis, both adherent
as well as nonadherent (floating dead cells) were combined and Western analysis was performed.
As shown in Fig. 2 b, Gas2 was proteolytically processed
during apoptosis as previously reported (Brancolini et al.,
1995 Under identical culture conditions, actin failed to show
any appreciable variation in size or expression levels as
observed in the different lanes (Fig. 2 b). As a further control, we analyzed the proteolytic cleavage of PKC- The behavior of actins during apoptosis in vivo was also
analyzed by two-dimensional electrophoresis. NIH3T3
cells grown for 36 h in 0.5% FCS were labeled for 12 h
with [35S]methionine, and then simultaneously chased with
cold methionine and cultured in serum-free medium. After 6 h, adherent and nonadherent cells were harvested
separately and two-dimensional gel electrophoresis was
performed as described in Materials and Methods. The
two-dimensional electrophoretic pattern of the different
actin isoforms (Garrels and Franza, 1989 Interestingly, a new band, which might represent a posttranslational modification of an unknown product, became
detectable in the lysates from apoptotic cells (Fig. 3 C, arrowhead). These results clearly demonstrate that during
apoptosis actin fails to be proteolytically processed, as triggered by survival signal deprivation in NIH3T3 cells. Indeed, resistance of actin to cleavage during apoptosis has
been recently reported (Song et al., 1997
Modulation of Cell-Cell Contacts during Apoptosis in
NIH3T3 Fibroblasts
Having demonstrated that actin is not cleaved in vivo during apoptosis in NIH3T3, we decided to search for other
regulators of the microfilament system that could be processed during apoptosis and possibly involved in the dramatic reorganization occurring during cell death.
The previously reported analysis of microfilament changes
during apoptosis clearly suggested that a first hallmark of cell death is the severing of contacts among neighboring cells.
Cell-cell contacts have been intensively studied in epithelial cells, where specialized domains of the plasma
membrane and the adherens junctions play a critical role
in cellular adhesiveness by providing a link between cell
surface adhesion molecules and the cytoskeleton (Geiger
and Ayalon, 1992 Fibroblasts in culture express functional cadherins (Itoh
et al., 1991 To analyze the dynamics of cell-cell contacts during apoptosis in NIH3T3, as a first step we analyzed for the presence of cadherin-catenin complexes in this cell line. NIH3T3
cell lysates were immunoprecipitated using anti-pan cadherin antibody. After electrophoretic separation, the immunoblots were revealed with anti- In summary, this analysis suggested that Apoptosis Triggered by Both Survival Signal
Deprivation and Genotoxic Stress Induces Proteolytic
Cleavage of We, therefore, asked whether the cell-cell adhesion dynamic
could be regulated by proteolytic processing of the cytosolic adaptor proteins during apoptosis. To test this hypothesis, Western analysis of Deprivation of survival signals induced proteolytic cleavage of
The temporal pattern of such Since Adherens junctions are found in many cell types, including cardiac myocytes and fibroblasts, but they have been
extensively studied in epithelial cells. Therefore, we similarly investigated proteolytic processing of Proteolytic processing of Since it has been demonstrated that Bcl-2 oncoprotein
prevents/delays apoptosis and inhibits activation of the
caspase proteases (Shimizu et al., 1996 When NIH3T3 overexpressing Bcl-2 were analysed for
proteolytic processing of
We can, therefore, conclude that proteolytic cleavage of
We next asked whether enzymes of the caspase family
could be responsible for the proteolytic cleavage of Full-length Treatment with caspase-3 specifically cleaved
Having demonstrated that caspase-3 cleaves Caspase-3 autoprocessing can be easily monitored by
following the disappearance of the p32 precursor form using an in vitro protease assay (Fernandes-Alnemri et al.,
1995b The same caspase-3 activity that is responsible for full
In summary, these data indicate that Experiments using the caspase-3 inhibitor Ac-DEVD-CHO
suggested that After 2.5 min of incubation, partial proteolytic processing of
As a first step to identify the sites of In vitro translated After 2.5 min of incubation with caspase-3, a prominent
~90-kD form and a minor ~65-kD band were revealed by
antibodies against Since antibodies against VSV failed to detect both the
~90- and ~65-kD cleaved forms of Moreover, comparison between From this analysis we can conclude that To explain the observed processing dynamics, we can
hypothesize an initial caspase-3-dependent cleavage event
that removes a fragment of ~2 kD located at the carboxy-terminal of Cleavage of We next assessed whether The apoptotic cleaved form of
To analyze whether the amino-terminal region of As shown in Fig. 8 b, antibodies specific for the amino-terminal region recognize Since the amino-terminal region of Apoptosis was induced in both NIH3T3 and MDCK cells
by serum deprivation, and nonapoptotic and apoptotic
floating cells were harvested separately. The same amount
of proteins derived from apoptotic and nonapoptotic cell
lysates were immunoprecipitated using anti- Fig. 8 c shows that the same amount of The same blots were also analyzed with antibodies against
These results clearly demonstrate that the proteolytic
processing at the amino-terminal of Caspase-3-Dependent Proteolytic Processing of the
Amino-Terminal Region of To further confirm that the amino-terminal region of
As a first attempt to map the amino-terminal cleavage
sites and to further confirm that caspase-3 cleaves the
amino-terminal domain of As predicted by the existence of multiple cleavage sites,
the different point mutants tested were cleaved by caspase-3, and Ac-DEVD-CHO completely abolished this
processing (Fig. 9 b). The cleaved products were efficiently
immunoprecipitated with the anti-VSV antibody, thus indicating that the multiple cleavage sites of Most interestingly, different electrophoretic shifts were
observed, distinguishing the proteolytic products of the
double mutants 144-145D/A and 162-164D/A from the
one generated from the wt The small electrophoretic shift of the digested A possible explanation for the described proteolytic pattern is that in vitro cleavage at residues 162 and/or 164 requires previous cleavage at position 144 and/or 145. Substitution of aspartic residues 144 and/or 145 should also
interfere with proteolytic processing at position 162-164,
thus leading to an incomplete digestion product with a
more evident reduced electrophoretic mobility.
In summary, these results demonstrate that within the
amino-terminal domain of It is becoming evident that the caspase family of cellular
proteases plays an important role in the execution phase
of apoptosis (Kumar, 1995 In this report we show that when apoptosis was induced
either by complete growth factor removal or genotoxic
stress, The cleavage product generated after in vitro treatment
of translated The caspase family of proteases contains a QACXG
pentapeptide in which the cysteine participates in catalysis
and is characterized by the absolute requirement for an aspartic acid residue in the substrate P1 position (Thornberry et al., 1992 We have demonstrated that aspartic residues 144 and/or
145, 162 and/or 164 are target sites for caspase-3 cleavage
in vitro and that upstream, different aspartic residues
within the amino-terminal region of Therefore, we can hypothesize that Adherens junctions link cells together by organizing actin filaments to the plasmamembrane. The transmembrane receptors are the cadherins, a family of homophilic
Ca2+-dependent cell-cell adhesion molecules (Takeichi,
1995 The central function of The fundamental roles of We have demonstrated in vivo that proteolytic processing of the
It is interesting to note that E-cadherin complexes may
promote cell survival and suppress cell death in epithelial
cells (Hermiston and Gordon, 1995 In conclusion, a complex reorganization of the microfilament system was observed during cell death: cells retract
from adhesion substrate, sever the contacts with neighbors, and actin filaments are concentrated in the perinuclear area in a ring-like organization. Collapsing of the
ring-like structure becomes evident with the proceeding of
the morphological changes accompanied by nuclear fragmentation, membrane blebbing, and formation of the apoptotic bodies.
In this context, To unveil the relationships among the specific roles of
; Wyllie, 1995
). Genetic analysis in Caenorhabditis elegans has identified both positive
and negative regulators of apoptosis (Ellis et al., 1991
).
-convertase enzyme (ICE; Yuan et al., 1993
).
precursor at two aspartic residues and is activated by cleavage
after specific aspartic residues of a 45-kD proenzyme, thus
forming two subunits that together form the active site of
the enzyme (Thornberry et al., 1992
). The regulatory mechanisms of the ICE remain to be clarified (Chinnaiyan et
al., 1997
). In general, it exhibits full activity only after proteolytic processing, which occurs either by self-cleavage or
through cleavage by other proteases (Nicholson, 1996
).
; Kumar et al., 1994
; Wang et al., 1994
; Faucheau et
al., 1995
; Fernandes-Alnemri et al., 1995a
,b; Boldin et al.,
1996
; Duan et al., 1996a
,b; Fernandes-Alnemri et al., 1996
;
Muzio et al., 1996
). Based on amino acid (aa)1 sequences
and recognized substrates, they have been grouped in different sub-families, and named according to the recently
proposed caspase nomenclature (Alnemri et al., 1996
).
These proteases may overlap in their functions and/or regulate one another's activities.
). Moreover, CD95/FAS/APO-1 receptors use the
adaptor molecule MORT1/FADD to physically engage a
cytosolic ICE-related cystein protease termed FLICE/
MACH (caspase-8) (Boldin et al., 1996
; Muzio et al., 1996
).
Caspase-8 might represent the most upstream protease involved in generating a death signal.
;
Xue and Horvitz, 1995
), or aldehyde and fluormethylketone derivatives of the target cleavage sequences, suppress
mammalian apoptosis as triggered by different stimuli
(Miura et al., 1993
; Gagliardini et al., 1994
; Beidler et al.,
1995
; Nicholson et al., 1995
).
; Song et al., 1996
). Cleavage of the
nuclear lamins is dependent on Mch2 (caspase-6) and
seems to be essential for disassembling the nuclear structure (Lazebnik et al., 1995
; Takahashi et al., 1996
). Other
substrates, whose function is still unclear during apoptosis,
are the 70-kD protein of the U1 small nuclear ribonucleoprotein (Casciola-Rosen et al., 1994
), the sterol regulatory
element-binding proteins SREBP-1 and SRBP-2 (Wang et
al., 1996
), the huntingtin protein (Goldberg et al., 1996
),
D4 GDP dissociation inhibitor (Na et al., 1996
), and pRb
tumor suppressor (Janicke et al., 1996
).
and Gas2, the last being a component of the microfilament system (Brancolini et al., 1992
). In both cases,
proteolytic processing results in a gain of function which
relates to increased kinase activity in the case of PKC
(Emoto et al., 1995
), or to an activity on the microfilament
system and on cell morphology in the case of Gas2 (Brancolini et al., 1995
). Recently, gain of function after caspase-3
processing has also been demonstrated for the p21-activated kinase 2 (Rudel and Bokoch, 1997
), and the DNA
fragmentation factor (Liu et al., 1997
).
; Wyllie et al., 1980
).
-catenin
thereby losing the ability to bind
-catenin. Such proteolytic
cleavage in vitro is mediated by the cystein-protease cpp32/
apopain/yama (caspase-3).
Materials and Methods
).
-catenin cDNA (Hoschuetzky et al., 1994
) was subcloned in-frame with a COOH-terminal VSV tag (
-catenin-VSV) in pMT2SM-tag (Bardelli et al., 1996
) and pGDSV7S-tag (Brancolini et al., 1995
). The
-catenin cDNA was amplified by PCR using a sense primer corresponding to
the T7 promoter (5
-AATACGACTCACTATAGGGC-3
) and a reverse primer (VSV1) containing a SmaI site (5
-CCAAACTATGACTGGAGGGCCCATT-3
). The carboxy-terminal deleted derivative of
-catenin
was produced by endonuclease digestion at the EcoRI site (nucleotides
1361/aa 421) and a COOH-terminal VSV tag was introduced by PCR using
a sense primer corresponding to the T7 promoter, and a reverse primer (VSV2) containing a NotI site (5
-GATTGCGGCCGCAAGTGAGG-3
).
) was used to produce the
point mutants D
A at different positions of the amino-terminal region of
-catenin. As external primers, the olT7 and olVSV2 were used. The pairs
of complementary inverse oligos were the following:
-catenin 162-164D/A:
ol162-164u:5
-CTAAACGCTGAGGCCCAGGTGGTA-3
ol162-164d:
5
-TACCACCTGGGCCTCAGCGTTTAG-3
;
-catenin 144-145D/A: ol144-145u:5
-TATCAGGCTGCCGCGGAACTT-3
ol144-145d:5
-AAGTTCCGCGGCAGCCTGAT A-3
.
-catenin monoclonal antibody directed against aa 571-781 (Signal Transduction Laboratories, Lexington, KY) for 1 h in a moist chamber
at 37°C. TRITC anti-mouse (Southern Biotechnologies, Birmingham,
AL) was used as secondary antibody. Actin filaments were detected using
FITC-phalloidin (Sigma Chemical Co., St. Louis, MO) and nuclei were labeled with propidium iodide 5 mM (Sigma Chemical Co.).
argon laser and a 543 I
helium neon laser. The following sets of filters were used: rhodamine
(BP546, FT580, and LP 590); and fluoresceine (450-490, FT 510, and
LP520).
-catenin and anti-
-catenin (Transduction Laboratories, Lexington, KY), anti-
-catenin amino-terminal (McCrea et al., 1993
), anti-PKC-
(Santa Cruz
Biotechnology, Inc., Santa Cruz, CA), and anti-VSV (Sigma Chemical
Co.). Blots were then rinsed three times with Blotto-Tween 20 and reacted with peroxidase-conjugated goat anti-rabbit (Sigma Chemical Co.)
or goat anti-mouse (Sigma Chemical Co.) for 1 h at room temperature.
The blots were then washed four times in Blotto-Tween 20, rinsed in PBS,
and developed with an enhanced chemiluminescent kit, as recommended
by the vendor (Amersham Int'l.).
-D-thiogalactopyranoside to a final concentration of 1 mM. After 2 h, cells were collected by centrifugation at 3,000 g for 5 min,
and then resuspended in 5 vol of caspase-3 buffer (Goldberg et al., 1996
)
with protease inhibitors (1 mM PMSF and 10 µg/ml each aprotinin, leupeptin, and pepstatin). Cells were lysed by sonication and debris was sedimented by centrifugation at 14,000 g for 20 min. The resulting supernatants were used for in vitro protease assays.
-catenin were labeled with 35S using the TNT-coupled
reticulocyte lysate system (Promega Corp., Madison, WI). 1 µl of translated reticulocyte lysate was incubated with the appropiate dilution of the
bacterial lysates in caspase-3 buffer (final volume of 10 µl) for 1 h at 37°C.
Reactions were terminated by adding 1 vol of SDS gel loading buffer and
boiling for 3 min. The specific caspase-3/cpp32/apopain inhibitor Ac-Asp-Glu-Val-Asp-CHO was obtained from Bachem Bioscience (Bubendorf,
Switzerland).
-catenin-
-catenin complexes in apoptotic and nonapoptotic
cells was performed as previously described (Hulsken et al., 1994
) with
some modifications. Cells were extracted with 140 mM NaCl, 4.7 mM KCl,
0.7 mM MgSO4, 1.2 CaCl2, 20 mM Tris-HCl, pH 7.5, 5% glycerol, containing 1% Triton X-100 and 1 mM PMSF, and 10 µg/ml each of aprotinin, leupeptin, antipain, and pepstatin. After centrifugation at 14,000 g for 15 min
monoclonal antibodies, anti-
-catenin (Transduction Laboratories), or
anti-pan cadherin (Sigma Chemical Co.) were added. After 3 h on ice,
protein A-Sepharose (Pharmacia Biotech., Inc., Piscataway, NJ) was added
and incubation was prolonged for 1 h on ice. After a brief centrifugation
in Eppendorf centrifuge, immunoprecipitates were washed with 100 mM
NaCl, 5 mM EDTA, 20 mM Tris-HCl, pH 7.5, 1% Triton X-100 containing 1 mM PMSF, and 10 µg/ml each of aprotinin, leupeptin, antipain, and
pepstatin.
Results
Fig. 1.
Confocal analysis of the organization of actin in
NIH3T3 cells induced to enter apoptosis by deprivation of survival factors. Growth-arrested NIH3T3 cells were cultured for 4 h
in serum-free medium, fixed, and stained for actin filaments using
phalloidin-FITC (a), or double stained with propidium iodide, to
visualize nuclei (A, C, E, and G) and phalloidin-FITC (B, D, F,
and H). Bars: (a) 5 µm; (A-H) 1 µm.
[View Larger Version of this Image (100K GIF file)]
; Kayalar et al., 1996
), we analyzed as if in
vivo, during apoptosis, actin was cleaved in our defined
cellular system. In fact, this could possibly explain the dramatic reorganization of the microfilament system occurring during cell death.
Fig. 2.
(a) NIH3T3 cells grown for 48 h in 0.5% FCS were incubated in serum-free medium for the indicated times (relative
numbers and percentages of viable cells were determined by trypan blue dye exclusion). Data represent arithmetic means ± SD
for three independent experiments. (b) Growth-arrested NIH3T3
cells were incubated in serum-free medium for the indicated
times. Lysates from both adherent viable cells and nonadherent
apoptotic cells were combined and Western blot analysis was performed. (c) Two-dimensional gel electrophoresis analysis showing actin patterns in adherent and nonadherent apoptotic cells.
[View Larger Versions of these Images (50 + 23K GIF file)]
). Appearance of a band with increased mobility was
faintly detectable after 3 h from survival signal deprivation. The intensity of the apoptotic band increased after 6 h,
concomitant with the accumulation of apoptotic cells in the
culture medium.
, another identified substrate of caspase proteases (Emoto et
al., 1995
). PKC-
showed a similar kinetics of proteolysis
during apoptosis as previously described for Gas2 (Fig. 2 b).
) did not show appreciable differences between normal and apoptotic cells.
). Nevertheless,
we can not exclude that a minor fraction of actin is proteolytically processed during apoptosis, or that this processing does not occur in NIH3T3 cells.
Fig. 3.
Confocal analysis of cell-cell contacts changes during
apoptosis in NIH3T3 cells. Apoptosis was induced by culturing
growth-arrested confluent NIH3T3 cells for 4 h in serum-free medium. Confluent growth-arrested NIH3T3 cells (A and B) and
apoptotic NIH3T3 cells (C-F) were fixed and double stained for
actin filaments using phalloidin-FITC (A, C, and E) and for
-catenin distribution (B, D, and E), using anti-mouse TRITC as
secondary antibody. Bar, 1.5 µm.
[View Larger Version of this Image (131K GIF file)]
). Such an adhesive role is dependent on
members of the cadherin superfamily of adhesive receptors and the cytoplasmic adaptor proteins
-,
-, and
-catenin/plakoglobin, which connect the cadherins to the
actin filaments (for review see Gumbiner, 1996
).
), interact with one another, and assemble cadherins-catenin complexes (Knudsen et al., 1995).
-catenin or -
-catenin
antibody.
-Catenin,
-catenin, and
-catenin/plakoglobin
were all detected in complexes with cadherin in NIH3T3
cells as previously demonstrated in other fibroblasts (data
not shown). As a next step we performed double immunofluorescences to compare
-catenin distribution in relation
to actin cytoskeleton in apoptotic and nonapoptotic confluent NIH3T3 cells. Intense staining for
-catenin was observed at the cell-cell contacts in nonapoptotic NIH3T3
cells (Fig. 3, A and B). At an early apoptotic stage (Fig. 3
C, arrowhead), when a retraction response was observed,
-catenin staining was rather diffuse, suggesting that disassembly of cell-cell contacts occurred. However, some localization at the cell-cell junction could still be detected, especially where the apoptotic cell still maintains contacts
with its neighbors (Fig. 3 D, arrows). In the more pronounced apoptotic phenotype observed in Fig. 3 E (arrowhead),
-catenin staining was mainly diffuse in the cytoplasm with no evidence for its presence at the cell border
(Fig. 3 F).
-catenin localization at the cell periphery was temporally and spatially
regulated during apoptosis in NIH3T3 cells.
-Catenin
-catenin on both adherent as well
as nonadherent (floating dead cells) NIH3T3 cells induced
to enter apoptosis following serum-free culture condition
was performed.
-catenin giving rise to an ~65-kD stable form clearly
evident at 6 h after treatment (Fig. 4 a).
Fig. 4.
Analysis of -catenin cleavage during apoptosis in
NIH3T3 and MDCK cells. (a) Growth-arrested NIH3T3 cells
were incubated in serum-free medium for the indicated times.
Lysates from both adherent viable cells and nonadherent apoptotic cells were combined and Western blot analysis was performed. (b) Density-arrested NIH3T3 cells were treated with 20 µg/ml of cisplatin or UV irradiated as described in Materials and
Methods. After 24 h adherent (A) and nonadherent (NA) cells
were processed separately and Western analysis was performed.
(c) MDCK cells grown for 4 d in 10% FCS were incubated in serum-free medium for 12 h. Adherent (A) and nonadherent (NA)
cells were processed separately and Western analysis was performed. (d) MDCK cells grown for 4 d in 10% FCS were treated for 4 h with the indicated amount of MMS. After 24 h, lysates from both adherent viable cells and nonadherent apoptotic cells were combined and Western blot analysis was performed.
[View Larger Version of this Image (38K GIF file)]
-catenin processing was
closely related to the above described Gas2 and PKC-
apoptotic processing. In fact, processing of
-catenin paralleled
the appearance of apoptotic cells in the culture medium
(data not shown).
-catenin is known to associate with
-catenin, to
mediate the formation of cell-cell adhesion we also followed
-catenin behavior during apoptosis. In this case
there was no evidence for proteolytic degradation of
-catenin in NIH3T3 cells induced to enter apoptosis under the
same conditions (Fig. 4 a).
-Catenin proteolytic processing was next analyzed using different apoptotic stimuli, such as genotoxic stress as
mediated by DNA-damaging agents. NIH3T3 cells were
treated with the chemotherapeutic agent cisplatin or irradiated with UV, and after 24 h adherent and nonadherent
(floating dead cells) were separately harvested. Western
blot analysis revealed that
-catenin was cleaved to a ~65-kD
form exclusively in the apoptotic cells and, on the contrary,
-catenin failed to be processed under the same conditions (Fig. 4 b).
-catenin during apoptosis in MDCK cells. Apoptosis was induced by
both serum deprivation and DNA damage using increasing amounts of the alkylating agent methylmethanesulfonate (MMS). Western analysis was performed on adherent
and nonadherent cells harvested separately, in the case of
serum starvation, or jointly, in the case of MMS treatment.
-catenin in MDCK cells was
detected when apoptosis was induced either by serum starvation (Fig. 4 c) or by DNA damage (Fig. 4 d). In the last
instance, the relative amount of the processed form paralleled the increased level of genotoxic insult and the appearance of apoptotic cells in the culture medium (data
not shown). Here again, both
-catenin and actin failed to
be processed.
; Chinnaiyan et al.,
1997
), we investigated the fate of
-catenin during apoptotic stimuli in the presence of increased Bcl-2 expression.
-catenin under serum-free conditions, apoptosis and proteolytic cleavage of Gas2 and
-catenin were dramatically suppressed (Fig. 5).
Fig. 5.
(A) NIH3T3-NEO or NIH3T3 cell lines transfected
with bcl-2 were grown for 48 h in 0.5% FCS and incubated in serum-free medium (relative numbers and percentages of viable
cells were determined by trypan blue dye exclusion). Data represent arithmetic means ± SD for three independent experiments.
(B) Growth-arrested NIH3T3-NEO and NIH3T3-bcl-2 cells
were incubated in serum-free medium for the indicated times.
Lysates from both adherent viable cells and nonadherent apoptotic cells were combined and Western blot analysis was performed.
[View Larger Version of this Image (28K GIF file)]
-Catenin cleavage during apoptosis was also observed
in the presence of the proteosome inhibitor N-acetyl-leucinyl-leucinyl-norleucinal-H, an inhibitor of the chymotryptic site on the proteosome (Pagano et al., 1995
), thus suggesting that it is not mediated by the ubiquitin/proteosome
system (data not shown).
-catenin correlates with activation of a cell death program in both fibroblast and epithelial cells, all the
-catenin pool is cleaved during apoptosis and this processing is
suppressed by increased expression of Bcl-2.
-Catenin Is Cleaved by Caspase-3 In Vitro
-catenin during apoptosis. We analyzed whether caspase-3, one
of the late-activated proteases in the apoptotic proteolytic
cascade, was able to cleave
-catenin in vitro.
-catenin cDNA was translated in vitro and
treated with caspase-3 from a bacterial lysate (Fernandes-Alnemri et al., 1995b
; Song et al., 1996
; Xue et al., 1996
).
-catenin,
producing a fragment of ~65 kD, whereas lysates from
bacteria not expressing caspase-3, grown and extracted under identical conditions, failed to cleave
-catenin (Fig. 6
a). Moreover, caspase-3-dependent proteolytic cleavage
of
-catenin could be prevented by addition of the specific
caspase inhibitor, the acidic tetrapeptide aldehyde Ac-DEVD-CHO (Fig. 6 b). It is worth noting that when Ac-DEVD-CHO was used at a final concentration of 20 nM,
only a partial inhibition of the
-catenin cleavage occurred, and in parallel an intermediate proteolytic product
was detected.
Fig. 6.
In vitro protease assays. (a) 35S labeled in vitro translated -catenin was incubated for 1 h at 37°C with caspase-3
buffer alone or with the indicated amounts of caspase-3-expressing bacteria lysates or control bacteria lysates. (b) 35S labeled in
vitro translated
-catenin was incubated for 1 h at 37°C with
caspase-3 buffer alone, with control bacteria lysates, with
caspase-3-expressing bacteria lysates, or in the presence of the indicated amount of caspase-3-specific inhibitor Ac-DEVD-CHO. (c) 35S labeled in vitro translated caspase-3 precursor was incubated for 1 h at 37°C with caspase-3 buffer alone, or with the indicated amount of caspase-3-expressing bacterial lysates or control
bacterial lysates.
[View Larger Version of this Image (35K GIF file)]
-catenin,
we tried to define whether
-catenin is a relevant substrate
for this protease. Caspase-3, like other proteases of this
family, can undergo autocatalytic cleavage, thus producing
an intermediate form (p21) and two active forms, p17 and
p12 (Fernandes-Alnemri et al., 1995b
; Nicholson et al.,
1995
; Martin et al., 1996
; Wang et al., 1996
; Xue et al., 1996
).
; Martin et al., 1996
; Wang et al., 1996
; Xue et al.,
1996
). In vitro translated caspase-3 was incubated with increasing amounts of bacterial lysates expressing active
caspase-3 and compared to the efficiency of
-catenin
cleavage over the same time course.
-catenin processing could only partially self-cleave the in
vitro translated caspase-3 precursor form (Fig. 6 c). In fact,
12 µg, the equivalent of total proteins from bacterial extracts expressing caspase-3, were required for full processing of caspase-3 precursor with respect to the 2 µg sufficient for full
-catenin processing. Caspase-3-dependent
processing specificity of
-catenin is also strengthened by
the observation that an amount of caspase-2 (ICH-1), enough for full autoprocessing, was unable to cleave
-catenin in vitro (data not shown).
-catenin is a better substrate for caspase-3 than itself, thus strongly suggesting that
-catenin is a relevant caspase-3 substrate.
-Catenin Is Cleaved at Different Sites by Caspase-3
-catenin could be cleaved at different sites.
To study the overall pattern of
-catenin processing, we
performed a time course analysis of
-catenin processing
in vitro using bacterial lysates expressing caspase-3. In vitro
translated
-catenin was incubated for the indicated times
at 37°C with caspase-3-expressing or control bacterial lysates.
-catenin could be detected (Fig. 7 a). A major
cleavage product of ~90 kD, in addition to the common
but minor ~65-kD form, was, in fact, revealed at this time.
After a 10-min incubation, only the ~65-kD form was
present, thus suggesting that the ~90-kD form could represent an intermediate degradation product of
-catenin. This result, however, suggested that caspase-3 has greater
affinity for full-length
-catenin to generate the ~90 kD
than for the full-length or the ~90 kD to generate the
~65-kD form. Incubation with control bacterial lysates did
not induce such proteolytic processing.
Fig. 7.
(a) 35S labeled in vitro translated -catenin was incubated for 1 h at 37°C with caspase-3 buffer (lane B), or with 2 µg
of caspase-3-expressing bacterial lysates or control bacterial lysates for the indicated times. (b) Western immunoblots were performed on in vitro translated
-catenin and cellular lysates from
(A) adherent nonapoptotic, and (NA) nonadherent apoptotic
MDCK cells using antibodies against
-catenin or against VSV-tag. In vitro translated
-catenin was incubated for 1 h at 37°C
with caspase-3 buffer (lane B) or with 2 µg of caspase-3-expressing bacterial lysates or control bacterial lysates.
[View Larger Version of this Image (58K GIF file)]
-catenin that are
cleaved by caspase-3, we constructed a tagged
-catenin
with the in-frame VSV epitope at its carboxy terminus.
-catenin-VSV was incubated for 2.5 or 60 min at 37°C with caspase-3-expressing or control
bacterial lysates and then Western blot was performed using antibodies against
-catenin or VSV.
-catenin. After 60 min of incubation,
only the ~65-kD form was detectable (Fig. 7 b).
-catenin, we can suggest that the carboxy tail is the first one to be proteolytically attacked by caspase-3.
-catenin in extracts
from MDCK cells undergoing apoptosis revealed a similar
electrophoretic mobility with respect to the in vitro,
caspase-3 processed, ~65-kD form of
-catenin.
-catenin, as
synthesized in vitro, is cleaved by caspase-3 at at least two
different sites generating the final ~65-kD form that
closely resembles the processed form previously identified
in vivo during apoptosis.
-catenin.
-Catenin during Apoptosis Abolishes Its
Ability to Bind
-Catenin
-catenin was cleaved at the
carboxy-terminal domain during apoptosis in vivo. NIH3T3
were transfected with the
-catenin-VSV construct, apoptosis was induced by serum deprivation, and adherent and
nonadherent cells were harvested separately for Western
analysis.
-catenin (65 kD) was
detected in apoptotic cells (nonadherent) using the antibody specific for aa 571-781, but it was undetectable using
antibodies against the VSV tag (Fig. 8 a). To exclude the
possibility that during apoptosis the VSV tag was cleaved
instead of the
-catenin carboxy-terminal region, we transfected the cytoplasmic adenylate kinase containing the
VSV tag at its carboxy-terminus as a control. Apoptosis
was similarly induced by serum deprivation, and nonadherent cells were harvested separately for Western analysis. Adenylate kinase VSV was similarly detected in both
apoptotic and nonapoptotic cells (Fig. 8 a). These results
indicate that the carboxy terminal domain of
-catenin is
specifically processed during apoptosis at a site further upstream of the tag in vivo.
Fig. 8.
(a) NIH3T3 cells transfected
with -catenin-VSV or with adenylate kinase were incubated in serum-free medium for 12 h. Adherent (A) and nonadherent (NA) cells were processed
separately and Western analysis was performed using anti-
-catenin (aa 571-781)
or anti-VSV-tag antibodies. (b) Growth-arrested NIH3T3 cells were incubated in serum-free medium for 12 h. Adherent
(A) and nonadherent (NA) cells were processed separately and Western analysis
was performed using anti-
-catenin (aa
571-781), or anti-amino-terminal
-catenin (aa 6-138) (McCrea et al., 1993
). (c)
NIH3T3 and MDCK cells were incubated
for 12 h in serum-free medium. Adherent
(A) and nonadherent (NA) cells were processed separately. Immunoprecipitation
using anti-
-catenin was performed as described in Materials and Methods. The immunocomplexes were resolved in SDS-PAGE and processed for Western blot
analysis with anti-
-catenin or anti-
-catenin as indicated.
[View Larger Version of this Image (38K GIF file)]
-catenin was also cleaved during apoptosis in vivo, we used an
antibody against the amino-terminal region of
-catenin
(aa 6-138) (McCrea et al., 1993
). Apoptosis was induced in
NIH3T3 cells by serum deprivation and in MDCK by
MMS treatment, and adherent and nonadherent cells were
harvested separately for Western analysis.
-catenin only in nonapoptotic
cells, thus indicating that the amino-terminal region of
-catenin is also proteolytically cleaved both in MDCK
and NIH3T3 apoptotic cells.
-catenin is critically
involved in binding
-catenin, we decided to analyze whether,
during apoptosis,
-catenin was able to maintain the complex with
-catenin or, due to proteolytic cleavage at the
amino-terminal, such a complex was compromised.
-catenin antibody. After electrophoretic separation, the immunoblots
were revealed with anti-
-catenin antibody.
-catenin could
be detected in the lysates of apoptotic and nonapoptotic
cells used for immunoprecipitation. However,
-catenin
was not found in complexes with the apoptotic form of
-catenin, whereas it was detected in complexes with
-catenin in the viable cells.
-catenin to demonstrate that the two different forms of
-catenin were similarly immunoprecipitated under the
different experimental conditions (Fig. 8 c).
-catenin during apoptosis is the major determinant for the loss of interaction
with
-catenin, which should, therefore, contribute to dismantling actin anchorage from the cell adhesion receptors.
-Catenin
-catenin was cleaved during apoptosis by a caspase, a deleted
carboxy-terminal version of
-catenin (
-catenin
CT),
missing aa 422-781 was produced, and a VSV tag was introduced at the carboxy-terminal. Digestion of the in vitro
translated
-catenin
CT with caspase-3 produced a major
band of ~32 kD, which was completely suppressed by addition of the specific inhibitor Ac-DEVD-CHO (Fig. 9 a).
Using decreasing amounts of caspase-3, a complex pattern
of digestion products was observed, probably representing
partial proteolytic products (Fig. 9 a). This evidence suggests the existence of multiple target sites for caspase proteases within the amino-terminal region of
-catenin.
Fig. 9.
(a) 35S labeled in vitro translated -catenin
CT was incubated for 1 h at 37°C with control bacterial lysates or with increasing amount of caspase-3-expressing bacterial lysates. (b) 35S
labeled in vitro translated
-catenin
CTwtVSV,
-catenin
CT162-164D/AVSV, and the
-catenin
CT144-145D/AVSV
were incubated with caspase-3-expressing bacterial lysates or
with caspase-3-expressing bacterial lysates in the presence of 200 nM Ac-DEVD-CHO. Immunoprecipitations were performed using anti-VSV antibody.
[View Larger Version of this Image (32K GIF file)]
-catenin, we decided to perform a mutagenesis analysis. Different mutants of the
-catenin amino-terminal region were constructed by introducing point mutations that substitute the aspartic residue with alanine within the amino-terminal region. More
specifically, the double-point mutants
-catenin
CT144-
145D/A and
-catenin
CT162-164D/A were created, and
the resulting constructs were in vitro transcribed/translated. The in vitro translated products were then incubated
with caspase-3-expressing bacterial lysates and immunoprecipitated using the antibody against the VSV tag.
-catenin
CT
lie in the amino-terminal part of the protein (Fig. 9 b).
-catenin
CT (Fig. 9 b).
-catenin
CT162-164D/A suggests that at least one or both of
the two aspartic residues represent cleavage sites for
caspase-3. Since caspase-3 digestion of
-catenin
CT144-
145D/A double mutants produced a larger electrophoretic
shift, we can infer that the mutated 144-145 aspartic residues could represent caspase-3 cleavage sites.
-catenin, different aspartic
residues are targets for caspase-3 cleavage. More specifically, aspartic residues at position 162 and/or 164 and 144 and/or 145 of
-catenin serve as caspase-3 targets within
the amino-terminal region.
Discussion
; Fraser and Evan, 1996
; Nicholson, 1996
). The activity of a protease results in the cleavage of various substrates. Some specific substrates for
caspase proteases have been identified and in some cases a
relationship with the apoptotic process could be clarified
(Casciola-Rosen et al., 1994
; Lazebnik et al., 1994
; Brancolini et al., 1995
; Emoto et al., 1995
; Lazebnik et al., 1995
;
Goldberg et al., 1996
; Janicke et al., 1996
; Na et al., 1996
;
Song et al., 1996
; Wang et al., 1996
; Liu et al., 1997
; Rutel
and Bokoch, 1997). However, further identification of critical substrates for caspase proteases is fundamental to unveil the execution phase of apoptosis.
-catenin becomes efficiently cleaved to produce a
stable ~65-kD form in both epithelial and fibroblast cells.
-Catenin is a ~92-kD protein component of cell-cell contacts and adherens junctions having both structural and
signaling functions (Miller and Moon, 1996
). The reported
cleavage of
-catenin seems to be (a) specific, since
-catenin, another component of adherens junctions, is not apparently processed during apoptosis, (b) coupled to the
onset of apoptosis, since it was coincident with Gas2 activation, and (c) suppressed by the antiapoptotic protein
Bcl-2 and proportional to the level of apoptotic insult.
-catenin protein with active caspase-3 (cpp32/
apopain/yama) migrates with the same apparent size as
the
-catenin cleavage product of apoptotic MDCK cells,
supporting the idea that in vivo such cleavage is also mediated by a caspase protease. Kinetics studies in vitro and
functional studies in vivo indicate that
-catenin is cleaved
at different sites, thus trimming both the amino- and carboxy-terminal regions of the protein.
). In addition to the requirement for a P1
Asp, caspase-3 shows preference for an anionic Asp residue in the P4 residue (DXXD) (Nicholson, 1996
). A
DXXD consensus site is located in the carboxy-terminal
part of
-catenin (aa 760-764: DLMDG), thus possibly explaining the rapid cleavage of the carboxy-terminal site of
the protein as observed in vitro after incubation with
caspase-3. This sequence is similar to the DQMDG site in
p35, a baculovirus antiapoptotic protein that is efficiently
cleaved by caspase-3 (Xue and Horvitz, 1995
).
-catenin are also
cleaved by caspase-3, none of them showing a canonical
DXXD sequence. These observations raised the question
whether caspase-3 is the protease involved in processing of
the amino-terminal domain in vivo, or other caspases show
higher affinity for such amino-terminal sites. Indeed, the
recent generation of caspase-3 null mice (Kuida et al.,
1996
), clearly demonstrates that caspase-3 play a critical
role during morphogenetic cell death in the mammalian
brain but raises the possibility that other caspase proteases
may have an important role during apoptosis in other tissues or in response to cell death-trigger.
-catenin could also
be a target for other caspases; further studies are required
to understand if other caspases are involved in
-catenin
processing, and to identify the different aspartic residues
cleaved during apoptosis.
). Their crystal structures suggest that cadherin-mediated cell adhesion is not based solely on the stability of association among individual molecules, but rather by the
generation of a cell adhesion zipper which provides a
mechanism to form strong intercellular bonds (Shapiro et
al., 1995
). In vivo strong intercellular bonds are dependent
upon the association of the cadherin carboxy-terminal cytoplasmic domain to the central region of
-catenin and
-catenin/plakoglobin (Nagafuchi and Takeichi, 1988
; Ozawa
et al., 1989
; Hulsken et al., 1995; Sacco et al., 1995
). Deletion of the
-catenin-binding site from cadherin cytoplasmic
domain renders the cadherin nonfunctional in a cell aggregation assay (Nagafuchi and Takeichi, 1988
; Ozawa et al.,
1989
).
-catenin as a regulator of adherens junctions has been further demonstrated in different
organisms using different experimental approaches (Peifer
et al., 1993
; Oyama et al., 1994
; Haegel et al., 1995
). It has
been extensively demonstrated that
-catenin serves as an
adapter to link cadherins to
-catenin and thereby to the
actin cytoskeleton, since
-catenin seems to interact with
both actin and
-actinin (Knudsen et al., 1995; Rimm et
al., 1995
). In this context,
-catenin seems to be the critical
component for cadherin-catenin complexes in the regulation of cellular adhesiveness (Hamaguchi et al., 1993
;
Cowin and Bruke, 1996
).
-catenin in cadherin cytoskeleton interactions have also been clearly established in
various cell types. Nonadhesive epithelial tumor cells lacking
-catenin can be induced to form tightly adherent epithelia after reintroduction of
-catenin (Watabe et al.,
1994
). Moreover, expression of E-cadherin-
-catenin chimeras has been reported to induce a strong and inflexible
adhesive phenotype (Nagafuchi et al., 1994
).
-catenin at its amino-terminal domain impairs
its ability to bind
-catenin. Amino acid residues 120-151
of
-catenin, embedded in the amino-terminal domain and
containing the first ARM repeat, are required for interaction with
-catenin (Aberle et al., 1994
). This is confirmed
in the human gastric cell line HSC-39 where a truncated
-catenin originated from a homozygous in-frame deletion that removes aa 28-134, cannot interact with
-catenin
(Oyama et al., 1994
; Kawanishi et al., 1995
). The observations that: (a) an antibody raised against aa 6-138 (McCrea et al., 1993
) of the amino-terminal region of
-catenin fails to detect the apoptotic form of
-catenin, and (b)
aspartic residues 144 and/or 145, 162 and/or 164 are targets
for caspase-3 cleavage in vitro, further confirming that
proteolytic processing of
-catenin during apoptosis impairs interaction with
-catenin. Removal of the
-catenin domain responsible for interaction with
-catenin should
contribute to disassemble the connections among the adhesion receptors, cadherins, and the actin cytoskeleton,
thereby impairing a sustained cell-cell adhesive interaction (Fig. 10). An early marker of apoptosis in confluent
cells is the retraction from neighboring cells, which is
probably coincident with disassembly of cell-cell contacts. This process seems to be both temporally and spatially
regulated and probably not exclusively dependent on
-catenin cleavage. In fact,
-catenin-related member,
-catenin/plakoglobin, also plays a similar role in regulating cadherin adhesive activity in adherens junctions and in the
specialized desmosomal junctions. The aspartic residues
144 and/or 145 and 162 and/or 164 possible target sites for
caspase activity in
-catenin, are conserved among
-catenin,
-catenin, and armadillo (the Drosophila form of
-catenin). Indeed, we also noticed that
-catenin/plakoglobin was proteolytically processed both in vitro after incubation
with caspase-3, and in vivo during apoptosis in fibroblast
and epithelial cells (Brancolini, C., manuscript in preparation).
Fig. 10.
Schematic representation of -catenin cleavage during
apoptosis. Apoptotic stimuli lead to activation of the caspase proteases, removing both a segment of
-catenin from the carboxy-terminal domain and from the amino-terminal domain including
the first armadillo repeat (black arrows). Cleaved
-catenin is released from
-catenin, thereby losing the connections to the cytoskeleton. Unmapped caspase cleavage sites are indicated by
hatched arrows.
[View Larger Version of this Image (25K GIF file)]
). In this context, cleavage of
-catenin might also suppress transduction of survival signals, consequently contributing to the one-way
proceeding of the apoptotic process. The evidence that
some components of microfilament system are substrates
for the caspase proteases (Brancolini et al., 1995
; Martin et
al., 1996
; Na et al., 1996
), suggests that these proteases coordinate actin architectural changes during apoptosis and
that such changes are important for a normal apoptotic
phenotype (Cotter et al., 1992
).
-catenin and Gas2 processing seem to
have different roles. Cleavage of Gas2 seems to be important to switch on the ability to rearrange the microfilament
system, which may be relevant for the organization of the
ring-like structure (Brancolini et al., 1995
). Cleavage of
-catenin seems to be responsible for switching off its
function as a regulator of adherens junctions, and is possibly
instrumental for severing contacts with neighboring cells.
-Catenin is also found in the nucleus and cytoplasm,
where its function is not restricted to modulation of adherens junctions. Association of
-catenin with the tumor-suppressor gene product APC (adenomatous polyposis colon) (Rubinfeld et al., 1993
) and the transcription factors
LEF-1/XTcf-3 (Behrens et al., 1996
; Molenaar et al., 1996
)
have, in fact, been reported. Moreover,
-catenin, together with the related protein
-catenin/plakoglobin and
Armadillo, is implicated in the Wnt/wingless signaling
pathway, which determines the dorsoventral axis in Xenopus and segment polarity in Drosophila (for review see
Miller and Moon, 1996
).
-catenin in the regulation of cell growth/differentiation
and its proteolytic processing during apoptosis will be an
exciting challenge for the future.
Received for publication 3 April 1997 and in revised form 28 August 1997.
Address all correspondence to Claudio Schneider, Laboratorio Nazionale, Consorzio Interuniversitario Biotechnologie, AREA Science Park, Padriciano 99, 34142 Trieste, Italy. Tel.: 39.40.398.985. Fax: 39.40.398.990. E-mail: sch{at}icgeb.trieste.itWe would like to thank A. Sgorbissa for help in some experiments. We
are grateful to B.M. Gumbiner for providing anti-amino-terminal -catenin
antibody. D. Lazarevic is a European Association Cancer Research fellow.
J. Rodriguez is a Howard Hughes Medical Institute Predoctoral Fellow.
This work was supported by Associazione Italiana Ricerca sul Cancro, Consorzio Nazionale delle Ricerche-Applicazioni Cliniche della Ricerca Oncologica, Ministero dell' "UNIVERSITA" and della Ricerca Scientifica and Tecnologica. 40% to C. Schneider, and CNR Progetto Strategico Ciclo cellulare e Apoptosi to C. Brancolini.
aa, amino acid;
Gas, growth arrest specific protein;
ICE, interleukin-1-convertase enzyme;
MMS, methylmethanesulfonate.
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