(The FASEB Journal. 1999;13:339-346.)
© 1999 FASEB
Caspase-mediated cleavage of APC results in an amino-terminal fragment with an intact armadillo repeat domain
STEPHANIE JOHNSON WEBB 1 ,
DONALD NICHOLSONa ,
VIVIEN J. BUBB and
ANDREW H. WYLLIEb
Department of Pathology, University Medical School, Edinburgh, EH8 9AG, U.K.;
a Merck Frosst Canada, Inc., Pointe Claire-Dorval, Quebec H9R 4P8, Canada; and
b Department of Pathology, Cambridge University, Cambridge CB2 1QP, U.K.
 |
ABSTRACT
|
|---|
During the effector phase of apoptosis, caspase activation appears to be
responsible for the distinctive structural changes of apoptosis and
perhaps for some of the changes in function of the doomed cells. There
is therefore interest in identifying caspase substrates and the details
of the cleavage events. Here we define precisely the event responsible
for generation of a stable 90 kDa fragment from the oncosuppressor
protein adenomatous polyposis coli (APC). Using synthetic radiolabeled
APC peptides as substrate, we demonstrate cleavage by cytosolic
extracts from preapoptotic cells. This cleavage was reproduced by
recombinant caspase-3 and blocked by a tetrapeptide inhibitor
Ac-DEVD-CHO, which is specific for caspase-3 family members. Inhibitors
specific for caspase-1 and -8 however, were less effective in blocking
APC cleavage. Mutation of a candidate DNID caspase-3 target site
completely abolished cleavage. This cleavage may be of biological
importance since the 90 kDa fragment consists of a sequence that is
highly conserved in the human, rat, mouse, Xenopus, and
Drosophila APC, although wide sequence divergence is
observed in Drosophila immediately carboxy-terminal to the
DNID site. Furthermore, cleavage at this site separates two significant
functional domains: an amino-terminal armadillo repeat and an adjacent
series of ß-catenin binding sites. Further circumstantial evidence
for the significance of APC-related pathways in apoptosis is provided
by the observation that apoptosis also induces cleavage of ß-catenin
itself, a protein known to accumulate in cells depleted in functional
APC and that appears to link cell–cell signaling to changes in
transcription and cell movement.—Webb, S. J., Nicholson, D.,
Bubb, V. J., Wyllie, A. H. Caspase-mediated cleavage of APC
results in an amino-terminal fragment with an intact armadillo repeat
domain.
Key Words: apoptosis • adenomatous polyposis coli • PARP • protein kinase • ß-catenin
|
INTRODUCTION
|
|---|
THE FINAL EFFECTOR PHASE of apoptosis is initiated by
the activation of members of a recently characterized family of
cysteinyl aspartate-specific proteases, named caspases. The
identification of several caspase substrates that are important in
vital cellular processes such as genome maintenance, macromolecular
processing, cytoskeletal organization, cell cycle progression and cell
signaling is revealing an unexpected subtlety in the strategy of the
effector phase of apoptosis. Some of the caspase substrates are or are
associated with structural proteins (e.g., lamin, fodrin, actin,
keratin 18, Gas2) whose disassembly may account directly for some of
the characteristic structural features of apoptosis (1)
.
The cleavage of other substrates, such as poly-adenosylribose
polymerase (PARP)2 and DNA-dependent protein kinase
(DNA-PK), results in the immediate inactivation of their enzymatic
activity (2
, 3
). Caspase cleavage releases
apoptosis regulatory functions in other substrates. A notable example
is the recently identified caspase-activated DNAse (CAD), the
apoptosis-specific endonuclease responsible for the characteristic
oligonucleosomal ladder that occurs during apoptosis. Cleavage of the
inhibitor of CAD (ICAD) releases and activates CAD (4)
.
Other death inhibitory proteins (e.g., Bcl-2) are cleaved by caspases,
suggesting a mechanism for positive feedback of signaling in the moment
of commitment to death (5)
. These examples suggest that
study of additional caspase substrates cleaved during apoptosis may
provide information on new critical events in the effector phase of
cell death and may even help to elucidate some of the functions of
these proteins during cell life.
In this report, we demonstrate caspase-mediated cleavage of the
oncosuppressor protein adenomatous polyposis coli (APC). APC is widely
expressed in epithelia and some mesenchymal cells, and is essential for
the normal homeostasis of the secretory epithelium of the
gastrointestinal tract. Mutations in the APC gene that result in a
carboxy-truncated version of its 310 kDa protein product have been
identified in the great majority of colorectal tumors, both benign and
malignant. Several domains of APC are now known, including those
responsible for oligomerization, and interaction with ß-catenin,
human discs large protein (hDLG), microtubules, and other proteins.
Although the precise functions of this large protein are still poorly
defined, it clearly plays roles in the regulation of cell adhesion and
migration, cytoskeletal organization, and cell signaling. A 90 kDa
fragment of APC has been observed in apoptotic cells (6)
.
Here we show that this fragment results from cleavage after
Asp777 by a group II caspase, similar or identical to
caspase-3. Caspase-3 is the mammalian homologue of the
Caenorhabditis elegans CED-3, the prototype of all mammalian
caspases 7-9)
. That caspase-mediated cleavage of APC may
be of biological significance is suggested by remarkable conservation
of the tetrapeptide cleavage site and by the similarity of the stable,
cleaved peptide to the truncated peptides engendered in colorectal
tumors.
|
MATERIALS AND METHODS
|
|---|
Cell culture
The human T-leukemic Jurkat cell line and chicken hepatoma DU249
cells were maintained in RPMI 1640 supplemented with 10% fetal calf
serum in 5% carbon dioxide at 37°C. The human colon carcinoma cell
line HCT116 was maintained in BHK-21 medium (Glasgow MEM) supplemented
with 10% heat-inactivated newborn calf serum in 5% carbon dioxide at
37°C.
Preparation of cell-free apoptotic extract
Cytoplasmic extract (S/M extract) was prepared from chicken
hepatoma cells that had been dual-synchronized in S- and M-phases with
aphidicolin and nocodazole, respectively, according to the method of
Lazebnik et al. (10)
. Briefly, DU249 cells (80%
confluency) were arrested in S-phase with 2 µg/ml of aphidicolin
(Sigma, St. Louis, Mo.) for 12 h. The cells were released from the
block and allowed to proceed through the cycle for 6 h, after
which they were blocked in M-phase with 50 ng/ml of nocodazole (Sigma)
for 3 h. Mitotic cells were harvested by mitotic shake-off and
washed in extract preparation buffer consisting of 50 mM Pipes-KOH, pH
7.0, 50 mM KCl, 10 mM EGTA, 2 mM MgCl2, 1 mM DTT, 10 µM
cytochalasin B, 1 mM PMSF, and 1 µg/ml each of chymostatin,
leupeptin, antipain, and pepstatin A. The cells were collected by
centrifugation with complete aspiration of residual supernatant, snap
frozen in a -80°C isopropanol slush, and stored at -80°C. The
pelleted cells were lysed by successive freeze/thaw/homogenization
(dounce) cycles and the homogenate was centrifuged at 150,000 x
g for 3 h in a Beckman SWTi60 rotor. The concentration
of extract in a typical preparation was 15–30 mg/ml. The extract was
aliquoted and stored at -80°C. Each batch of S/M extract prepared
was tested for its ability to induce apoptotic morphological changes to
HeLa nuclei (data not shown).
In vitro transcription/translation of APC peptides
An in vitro translated fragment corresponding to the
amino-terminal third of APC was synthesized using a rabbit reticulocyte
lysate and was used for analysis of proteolytic cleavage during
apoptosis. The human APC cDNA (gift from J. Groden, Cincinnati, Ohio)
was used as a template for synthesis of a fragment of APC that
corresponded to codons 1-1194 (APC1-1194) by polymerase
chain reaction (PCR) using the Boehringer Mannheim Long Template PCR
kit. The forward primer was engineered such that the T7 promoter and
Kozak consensus sequence were immediately upstream of the initiating
methionine and the first 15 bases of the APC sequence; the reverse
primer contained three in-frame stop codons, followed by 15 bases of
the APC coding sequence. The corresponding PCR product was sequence
verified and then used as a template for in vitro
transcription and translation in the presence of
35S-methionine (Amersham, Arlington Heights, Ill.), using
the Promega TNT T7 reticulocyte lysate kit according to the
manufacturer's protocol. A single, major translation product of the
predicted size, 136 kDa, was produced. Human PARP and human caspase-3
cDNAs were used as templates for in vitro
transcription/translation of their respective proteins.
Synthesis of mutant APC1-1194D777A
The aspartic acid residue at position 777 in
APC1-1194 was changed to an alanine residue by a PCR-based
overlapping primer extension method for site-directed mutagenesis. A
pair of complementary inverse oligonucleotides, 5'-CAG AAA CTT TTG ACA
ATA TAG CCA ATT TAA GTC CCA AGG C-3' and 5'-GCC TTG GGA CTT AAA TTG GCT
ATA TTG TCA AAA GTT TCT G-3', containing a G to C mutation at position
2348 that changed the aspartic acid residue at codon 777 to an alanine,
was designed. These were used with the forward and reverse primers used
to synthesize APC1-1194 to generate two primary PCR
products with overlapping sequence homology within the region
containing the mutation. These primary PCR products were purified and
used as templates in a second PCR extension to generate an APC product
identical to APC1-1194 except for the mutation at codon
777. The mutation was sequence verified, and translation of the
template produced a single peptide of the expected size, designated
APC1-1194D777A.
Proteolytic cleavage assays
In vitro translated,
[35S]methionine-labeled APC peptide fragments were
incubated with either DU249 S/M apoptotic extract or purified,
recombinant caspases. In a typical cleavage assay, 3 µl of translated
reticulocyte lysate was incubated with either DU249 S/M extract (final
concentration ranged from 0.2 to 8 mg/ml) or purified recombinant
caspases (final concentration ranged from 8.0 to
0.03x10-9 M) in an enzyme dilution buffer consisting of
50 mM HEPES/KOH, pH 7.0, 10% (w/v) sucrose, 2 mM EDTA, 0.1% (w/v)
CHAPS, and 5 mM DTT for 60 min at 37°C. For the inhibition studies,
DU249 S/M extract was preincubated with varying concentrations of
specific tetrapeptide inhibitors of the caspase family of proteases
(11)
acetyl-DEVD-CHO (caspase-3), acetyl-WEHD-CHO
(caspase-1), and acetyl-IETD-CHO (caspase-8) for 20 min at 37°C prior
to the addition of substrate. Cleavage was monitored by sodium dodecyl
sulfate (SDS) -polyacrylamide gel electrophoresis analysis on 8%
Tris-glycine gels (NOVEX) and fluorography (Amplify, Amersham).
Western analysis
For Western analysis of APC and ß-catenin in Jurkat cells,
whole-cell extracts were prepared from untreated cells and cells
treated with 1 µM staurosporine (Sigma) to induce apoptosis.
Apoptosis was assessed morphologically by acridine orange staining. At
the stated times, cells were harvested by centrifugation, washed once
in ice-cold phosphate-buffered saline (PBS) containing 100 µM PMSF
and 1 mg/ml each of chymostatin, leupeptin, antipain, and pepstatin A,
and then lysed in sample preparation buffer [62.5 mM Tris-HCl, pH 6.8,
2% SDS, 10% (v/v) glycerol, 100 mM DTT, 0.01% bromophenol blue].
For studies in HCT-116 cells, whole-cell extracts were prepared from
adherent and detached fractions (95% apoptosis) of untreated cells and
cells treated with 4 mM sodium butyrate (Calbiochem, San Diego,
Calif.). The adherent cells were washed with ice-cold PBS, scraped from
the dish in a small volume of PBS, and snap frozen on dry ice. The
detached cells, the majority of which were apoptotic, were collected
from the media by centrifugation, washed in ice-cold PBS, and snap
frozen on dry ice. The cells were lysed in sample preparation buffer
and repeatedly passed through a 21, then a 23, gauge needle to shear
genomic DNA, boiled for 5 min, aliquoted, and stored at -20°C. The
denatured cellular extracts (10 µg) were electrophoretically
separated on 4–12% Tris/gycine polyacrylamide gels (NOVEX),
transferred to nitrocellulose membranes in Towbin's transfer buffer
(10% methanol) at 300 mAmps for 3 h, and immunoblotted with
antibodies to APC (amino-specific 3122) (12)
or
ß-catenin (Transduction Laboratories, Lexington, Ky.).
Protein/antibody complexes were detected by enhanced chemiluminescence
(Amersham).
|
RESULTS
|
|---|
APC is processed to a 90 kDa amino-terminal fragment during
apoptosis
We examined endogenous expression of APC in Jurkat cells, a
T-leukemic cell line, and HCT116 cells, a human colon carcinoma cell
line, upon treatment with apoptosis-inducing agents by Western analysis
using an antibody specific to the amino terminus of APC (Fig. 1
). Both these cell lines express only full-length APC. As reported
for other colon carcinoma cell lines, HCT116 cells underwent apoptosis
and detached from the substrate when treated with 4 mM sodium butyrate
as detected by changes in nuclear morphology on acridine orange
staining. In the detached cells (95% apoptotic), full-length APC was
processed to a fragment of approximately 90 kDa detected by the
amino-terminal-specific antibody 3122. After 24 h of treatment
with sodium butyrate, no full-length APC was detectable in the detached
apoptotic cells, but the amino-terminal 90 kDa fragment was abundant
and apparently stable. A similar, approximately 90 kDa APC fragment,
detectable with the amino-terminal antibody, was observed in Jurkat
cells treated with the protein kinase inhibitor staurosporine at 1 µM
to induce apoptosis. After 4 h of treatment, 68% of the cells
were apoptotic, rising to 88% after 8 h. As the percentage of
apoptotic cells increased, the content of detectable full-length APC
decreased. This loss of APC was not due to a general increase in
protein degradation, as Coomassie staining of gels revealed that the
major protein bands were intact even when the majority of the cells
were apoptotic. The 90 kDa fragment appeared in parallel with the
increasing proportion of apoptotic cells and the loss of full-length
APC, although in this situation the observed levels were not
stoichiometric, perhaps indicating limited stability of the
amino-terminal fragment.
View larger version (39K):
[in this window]
[in a new window]
|
Figure 1. APC is processed to a 90 kDa fragment during apoptosis. Whole-cell
extracts from Jurkat cells treated with 1 µM staurosporine
(A) and HCT116 cells treated with 4 mM sodium butyrate
(B) were analyzed by Western analysis using an antibody
specific to the amino terminus of APC. A) Whole-cell
extracts prepared from untreated Jurkat cells and cells treated with 1
µM staurosporine at the times indicated were separated on a 4–12%
SDS-polyacrylamide gel. B) HCT116 cells were treated with 4
mM sodium butyrate; whole-cell extracts were prepared from attached
(adherent) and detached (floating) cells at the indicated times and
separated on a 4–12% polyacrylamide gel. Open symbol indicates
full-length APC, closed symbol indicates amino-terminal 90 kDa
fragment.
|
|
An in vitro translated APC peptide was processed by a
cell-free apoptotic extract
To further define the nature of the 90 kDa APC fragment, we
incubated a synthetic amino-terminal APC peptide with a cell extract
known to contain components of the terminal effector mechanism of
apoptosis (10)
. As described previously, this extract was
prepared from chicken hepatoma (DU249) cells that had been dual
synchronized in the S- and M-phases and were committed to apoptosis. We
confirmed the activity of the extract by demonstrating its capacity to
induce morphological changes characteristic of apoptosis in nuclei of
HeLa cells and to catalyze proteolysis of radiolabeled in
vitro translated PARP. Such active extracts were incubated with an
in vitro translated 35S-radiolabeled synthetic
peptide corresponding to the first 1194 amino acids of APC
(APC1-1194), 136 kDa (Fig. 2
). The apoptotic extract efficiently cleaved this peptide to a
product of approximately 90 kDa (Fig. 3
). The size of this fragment is consistent with that observed
in vivo in apoptotic cells, suggesting a common mechanism of
processing. Furthermore, the temporal pattern of processing of
APC1-1194 by the active extract is similar to that observed
for PARP cleavage (Fig. 3)
.
View larger version (15K):
[in this window]
[in a new window]
|
Figure 2. In vitro synthesis of an amino-terminal peptide of APC. A
35S-labeled peptide representing the first 1194 amino acids
of APC was synthesized using an in vitro
transcription/translation reticulocyte lysate method. The peptide
included the oligomerization domain, the armadillo repeat domain, and
some of the ß-catenin binding domains.
|
|
View larger version (51K):
[in this window]
[in a new window]
|
Figure 3. APC1-1194 is cleaved to a 90 kDa fragment by a cell-free
apoptotic extract. In vitro translated
35S-labeled APC1-1194 and PARP were incubated
with a cell-free apoptotic extract prepared from chicken hepatoma DU249
cells for 60 min at 37°C, separated on 8% SDS-polyacrylamide gels,
and visualized by fluorography. APC1-1194 was processed to
fragment with an approximate molecular mass of 90 kDa. PARP was
processed to a fragment of approximately 85 kDa, consistent with
previous reports.
|
|
Cleavage of APC by DU249 S/M extract is inhibited by caspase-3
inhibitors
To determine whether processing of APC during apoptosis was
dependent on a caspase, we treated the apoptotic extract with
tetrapeptide inhibitors specific for the different subgroups of the
caspase family (11)
. APC1-1194 was incubated
with DU249 S/M extract (2 mg/ml) in the presence of increasing
concentration of the acetyl-tetrapeptide aldehyde inhibitors WEHD,
DEVD, and IETD (Fig. 4
). For comparison, inhibition of PARP cleavage by DEVD was also
tested. The most efficient inhibition of processing of
APC1-1194 was observed with DEVD, a preferential inhibitor
of caspase-3, and similar (group II) caspases. The IC50 for
inhibition of processing of APC1-1194 by DEVD was 100 nM,
similar to that for DEVD inhibition of PARP (50 nM). WEHD, a
preferential inhibitor of group I caspases such as ICE, exhibited weak
inhibition of APC processing by the extract, evident only at a
concentration of 10 µM. The tetrapeptide IETD, which is a strong
inhibitor of caspase-8, a group III caspase, inhibited processing of
APC1-1194 at a concentration of 1 µM. These findings thus
confirmed that processing of APC1-1194 by DU249 S/M
extracts is dependent on the activity of members of the caspase family
of proteases, in particular, caspase-3. Furthermore, inhibition of
cleavage of APC1-1194 by DEVD correlated with inhibition of
morphological changes to exogenously added HeLa nuclei in a cell-free
assay of apoptosis (data not shown). Partial inhibition of
APC1-1194 processing by the DU249 S/M extract by the
caspase-8 inhibitor IETD may be due to inhibition of caspase-3
activation in the crude cytoplasmic extract. Caspase-8 is thought to
activate group II caspases at (IVL)ExD sites located between the large
and small subunits (13)
.
View larger version (47K):
[in this window]
[in a new window]
|
Figure 4. Cleavage of APC1-1194 by the cell-free apoptotic
extract is inhibited by the group II caspase inhibitor DEVD. In
vitro translated, 35S-labeled APC1-1194
was incubated with the DU249 apoptotic extract (2 mg/ml) that had been
preeincubated for 30 min with varying concentrations of
acetyl-tetrapeptide aldehydes, which specifically inhibit different
subgroups of the caspase family of proteases. WEHD is a group I
inhibitor, IETD a group III inhibitor, and DEVD a group II inhibitor.
The proteolytic products were separated on an 8% SDS-polyacrylamide
gel and visualized by fluorography. Inhibition of PARP cleavage by DEVD
is shown for comparison.
|
|
Caspase-3 cleaves APC1-1194 after amino acid
Asp777 in a conserved site
We next sought to determine whether APC1-1194 is a
substrate for purified caspase-3 in vitro. Incubation of
APC1-1194 with purified, recombinant caspase-3 for 60 min
at 37°C resulted in a stable proteolytic fragment of approximately 90
kDa (Fig. 5
). This fragment migrated at the same rate as the fragment produced
with the DU249 S/M extract (data not shown). To characterize the
caspase-3 cleavage site within APC1-1194, we performed
site-directed mutagenesis. The optimal recognition motif for caspase-3
is DEVD(P1), with a near absolute requirement for an
aspartate residue at position P1 (the first amino acid
amino-terminal to the cleavage site) (11)
. A potential
motif (DNID) was identified; cleavage at this site would release the
amino-terminal 777 amino acids of APC, a fragment with a predicted
molecular mass of 86 kDa. Mutation of Asp777 to alanine at
the P1 position of this site completely abolished cleavage
by recombinant caspase-3 in the in vitro assay (Fig. 6
). This DNID sequence is conserved in the human, mouse, rat,
Xenopus, and Drosophila and is located
immediately carboxy-terminal to the armadillo repeat domain (Fig. 7
).
View larger version (76K):
[in this window]
[in a new window]
|
Figure 5. APC1-1194 is cleaved by caspase-3 in vitro.
In vitro translated, 35S-labeled
APC1-1194 and PARP were incubated with varying
concentrations of purified caspase-3 for 60 min at 37°C. The
proteolytic product was separated on 8% SDS-polyacrylamide gels and
visualized by fluorography.
|
|
View larger version (34K):
[in this window]
[in a new window]
|
Figure 6. Mutation of Asp777 in APC1-1194 to an alanine
completely abolishes cleavage by caspase-3. An in vitro
synthesized peptide corresponding to amino acids 1-1194 of APC that had
an alanine residue in place of an aspartic acid at position 777,
APC1-1194D777A, APC1-1194, and PARP were
incubated with varying concentrations of purified caspase-3 for 60 min
at 37°C. The proteolytic products were separated on 8%
SDS-polyacrylamide gels and visualized by fluorography.
|
|
View larger version (19K):
[in this window]
[in a new window]
|
Figure 7. The caspase-3 cleavage site in APC is conserved. The
recognition/cleavage tetrapeptide sequence DNID located at the carboxy
terminus of the armadillo repeat domain of APC is conserved in the
human, mouse, rat, and Xenopus and has a conservative
isoleucine to leucine change in Drosophila.
|
|
ß-Catenin is processed to a lower molecular weight form during
apoptosis
To begin to address the functional significance of APC cleavage
during apoptosis, we looked at expression levels of ß-catenin. APC
has been shown to bind to ß-catenin and regulate its cytoplasmic
levels and subcellular distribution 14-16)
. Western
analysis of whole-cell extracts prepared from Jurkat cells induced to
undergo apoptosis by treatment with staurosporine (1 µM) was
performed using an antibody that recognizes the carboxy-terminal end of
ß-catenin (Fig. 8
). With an increasing percent of apoptosis there is a decrease in
the level of full-length ß-catenin concomitant with an increase in an
immunoreactive protein of approximately 65 kDa. At 4 h after
treatment with staurosporine, when 60% of the cells were apoptotic,
approximately 60% of ß-catenin had been processed to the lower
molecular weight form. After 6 h of treatment (78% apoptosis),
nearly all full-length ß-catenin had been processed to the 65 kDa
form.
View larger version (52K):
[in this window]
[in a new window]
|
Figure 8. ß-Catenin is cleaved during apoptosis. Whole-cell extracts from
Jurkat cells either untreated or treated with 1 µM staurosporine were
analyzed by Western analysis using an anti-ß-catenin antibody.
Whole-cell extracts were prepared from untreated and treated cells at
the indicated times, separated on an 8% SDS-polyacrylamide gel, and
immunoblotted with an antibody specific for ß-catenin.
|
|
|
DISCUSSION
|
|---|
Cleavage of the oncosuppressor protein APC in apoptosis, which
results in the generation of a large, apparently stable amino-terminal
fragment, has been demonstrated in cells of hemopoietic and epithelial
lineages by ourselves and others (6)
. Here we have shown
that the cleavage event occurs at a conserved DNID tetrapeptide in a
highly conserved region carboxy-terminal to the armadillo repeat region
and is mediated by an enzyme with many properties of caspase-3. APC
thus joins the list of protein substrates cleaved during apoptosis at
provocative intramolecular sites. The question arises as to whether
this cleavage of APC plays any functional role in the process of
apoptosis.
In colorectal adenocarcinomas, the majority of mutations in APC are
chain-terminating, resulting in the expression of a truncated protein.
These truncated proteins have lost the functional domains of APC
responsible for interactions with microtubules, EB1, and hDLG, GSK-3ß
phosphorylation sites, and, usually, the ß-catenin binding sites.
However, the majority of the APC mutants retain a highly conserved
armadillo repeat domain located in the amino-terminal third of the
protein. The precise function of the armadillo repeat domain of APC has
not been elucidated. However, it is likely to be functionally essential
as it is the most highly conserved region of the APC molecule, 60%
identical with the Drosophila APC as compared to an overall
identity of less than 30% (15)
. Armadillo repeat domains
have been found in a number of other proteins, including ß-catenin,
and appear to mediate protein/protein interactions (17)
.
The APC cleavage site we have described here, responsible for its
proteolysis in apoptosis by a member of the caspase family, occurs
after Asp777 at the carboxy-terminal end of the armadillo
repeat domain, thus resulting in preservation of an apparently stable,
90 kDa amino-terminal fragment with an intact armadillo repeat domain.
Whether the preservation of this domain is functionally relevant to
apoptosis remains to be determined. It is of considerable interest,
however, that cleavage of ß-catenin by caspase-3 during apoptosis
also results in the preservation of its armadillo repeat domain
(18)
. We speculate that preservation of the armadillo
repeat domains of both APC and ß-catenin may be pivotal in the
effector process of commitment to apoptosis.
The release of apoptotic regulatory functions by caspase-mediated
cleavage during apoptosis is not without precedence, as has been
observed with the recently identified apoptosis-specific DNase complex
(DFF and CAD/ICAD) 4, 19)
. Thus, cleavage of APC may
unmask a latent, as yet uncharacterized, function of the armadillo
repeat domain. Further characterization of the subcellular localization
of this APC peptide and identification of associating proteins will
provide a clearer understanding of its role in apoptosis.
Cleavage of APC will undoubtedly have significant effects on other
functions of APC downstream of the cleavage site such as cell signaling
through its interaction with ß-catenin, and cytoskeletal structure
and cell migration through its interactions with microtubules.
Elimination of the microtubule binding domain in the carboxy terminus
of APC may facilitate microtubule disassembly. Cleavage of ß-catenin
by caspase-3 during apoptosis eliminates its actin binding domain,
resulting in the dismantling of cell–cell contacts and rearrangement
of the actin cytoskeleton during apoptosis (18)
. Thus, the
role of APC and ß-catenin in apoptosis may be related to their
maintenance of microtubule and microfilament structural arrays, with
proteolytic cleavage being necessary for the physical dismantling of
these cytoskeletal networks and the gross structural changes that occur
in a cell dying by apoptosis.
|
ACKNOWLEDGMENTS
|
|---|
S.J.W. is a Fellow of the Jane Coffin Childs Memorial Fund for
Medical Research. A.H.W. and V.J.B. are supported by the Cancer
Research Campaign. This work was also supported by the Scottish
Hospital Endowments Research Trust. The authors would like to thank
Yuri Lazebnik and William Earnshaw for their invaluable insight and
Vicky Houtzager for technical assistance.
|
FOOTNOTES
|
|---|
1 Correspondence: Department of Pharmacology and Toxicology, School of Medicine, University of Louisville, Louisville, KY 40292, USA. E-mail: sjwebb01{at}homer.louisville.edu 
2 Abbreviations: APC, adenomatous polyposis coli; CAD,
caspase-activated DNAse; hDLG, human discs large protein; ICAD,
inhibitor of CAD; PARP, poly-adenosylribose polymerase; PBS,
phosphate-buffered saline; PCR, polymerase chain reaction; PK, protein
kinase; SDS, sodium dodecyl sulfate.
Received for publication July 8, 1998.
Revision received October 27, 1998.
|
REFERENCES
|
|---|
-
Nicholson D., Thornberry N. A.. Caspaseskiller proteases. Trends Biochem. Sci. 1997;22:299-306.[Medline]
-
Kaufmann S. H., Desnoyers S., Ottaviano Y., Davidson N. E., Poirier G. G.. Specific proteolytic cleavage of poly(ADP-ribose) polymerasean early marker of chemotherapy-induced apoptosis. Cancer Res 1993;53:3976-3985.[Abstract/Free Full Text]
-
Casciola-Rosen L. A., Anhalt G. J., Rosen A.. DNA-dependent protein kinase is one of a subset of autoantigens specifically cleaved early during apoptosis. J. Exp. Med. 1995;182:1625-1634.[Abstract/Free Full Text]
-
Sakahira H., Enari M., Nagata S.. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature (London) 1998;391:96-99.[Medline]
-
Polakis P.. Mutations in the APC gene and their implications for protein structure and function. Curr. Opin. Genet. Dev. 1995;5:66-71.[Medline]
-
Browne S. J., Williams A. C., Hague A., Butt A. J., Paraskeva C.. Loss of APC protein expressed by human colonic epithelial cells and the appearance of a specific low-molecular weight form is associated with apoptosis in vitro. Int. J. Cancer 1994;59:56-64.[Medline]
-
Fernandes-Alnemri T., Litwack G., Alnemri E. S.. CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein ced-3 and mammalian interleukin-I-converting enzyme. J. Biol. Chem. 1994;269:30761-30764.[Abstract/Free Full Text]
-
Tewari M., Quan L. T., O'Rourke K., Desnoyers S., Zeng Z., Beidler D. R., Poirier G. G., Salveson G. S., Dixit V. M.. Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 1995;81:801-809.[Medline]
-
Nicholson D. W., Ali A., Thornberry N. A., Vaillancourt J. P., Ding C. K., Gallant M., Gareau Y., Griffin P. R., Labelle M., Lazebnik Y. A., Munday N. A., Raju S. M., Smulson M. E., Yamin T.-T., Yu V. L., Miller D. K.. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature (London) 1995;376:37-43.[Medline]
-
Lazebnik Y. A., Cole S., Cooke C., . A,Nelson W. G., Earnshaw W. C.. Nuclear events of apoptosis in vitro in cell-free mitotic extractsa model system for analysis of the active phase of apoptosis. J. Cell Biol. 1993;123:7-22.[Abstract/Free Full Text]
-
Thornberry N. A., Rano T. A., Peterson E. P., Rasper D. M., Timkey T., Garcia-Calvo M., Houtzager V. M., Nordstrom P. A., Roy S., Vaillancourt J. P., Chapman K. T., Nicholson D. W.. A combinatorial approach defines specificities of members of the caspase family and granzyme BFunctional relationships established for key mediators of apoptosis. J. Biol. Chem. 1997;272:17907-17911.[Abstract/Free Full Text]
-
Midgley C. A., White S., Howitt R., Save V., Dunlop M. G., Hall P. A., Lane D. P., Wyllie A. H., Bubb V. J.. APC expression in normal human tissues. J. Pathol. 1997;181:426-433.[Medline]
-
Liu X., Kim C. N., Pohl J., Wang X.. Purification and characterization of an interleukin-1-converting enzyme family protease that activates cysteine protease p32 (CPP32). J. Biol. Chem. 1996;271:13371-13376.[Abstract/Free Full Text]
-
Papkoff J., Rubinfeld B., Schryver B., Polakis P.. Wnt-1 regulates free pools of catenins and stabilizes APC-catenin complexes. Mol. Cell. Biol. 1995;16:2128-2134.[Abstract]
-
Hayashi S., Rubinfeld B., Souza B., Polakis P., Wieschaus E., Levine A. J.. A Drosophila homolog of the tumor suppressor gene adenomatous polyposis coli down-regulatesß-catenin but its zygotic expression is not essential for the regulation of armadillo. Proc. Natl. Acad. Sci. USA 1997;94:242-247.[Abstract/Free Full Text]
-
Munemitsu S., Alber I., Souza B., Rubinfeld B., Polakis P.. Regulation of intracellular ß-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein. Proc. Natl. Acad. Sci. USA 1995;92:3046-3050.[Abstract/Free Full Text]
-
Peifer M., Berg S., Reynolds A. B.. A repeating amino acid motif shared by proteins with diverse cellular roles. Cell 1994;76:789-791.[Medline]
-
Brancolini C., Lazarevic D., Rodriguez J., Schnieder C.. Dismantling cell–cell contacts during apoptosis is coupled to a caspase-dependent proteolytic cleavage of ß-catenin. J. Cell Biol. 1997;139:759-771.[Abstract/Free Full Text]
-
Liu X., Zou H., Slaughter C., Wang X.. DFF, a beterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 1997;89:175-184.[Medline]
TOP
ABSTRACT
INTRODUCTION
