| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 275, Issue 28, 21402-21408, July 14, 2000
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Received for publication, December 3, 1999, and in revised form, March 9, 2000
DNA fragmentation, a hallmark of apoptosis, is
regulated by a specific nuclease called caspase-activated DNase (CAD)
and its inhibitor (ICAD). When cell lysates from Drosophila S2
cells were chemically denatured and the denatured proteins were removed
after dialysis, the supernatant inhibited Drosophila CAD
(dCAD). To identify the inhibitor, we tested recombinant DREP-1, which
was previously identified using the Drosophila EST data
base and found it also inhibited dCAD DNase. An antibody against DREP-1
inhibited the ICAD activity in the S2 cell extracts, confirming the
identification of DREP-1 as a Drosophila homolog of ICAD
(dICAD). The recombinant DREP-1/dICAD was cleaved at a specific site by
human caspase 3 as well as by extracts prepared from S2 cells
undergoing apoptosis. Biochemical fractionation and immunoprecipitation
of dICAD from S2 cell extracts indicated that dICAD is complexed with
dCAD in proliferating cells. The expression of the caspase-resistant
form of dICAD/DREP-1 in a Drosophila neuronal cell line
prevented the apoptotic DNA fragmentation. Northern hybridization and
the immunohistochemical analyses revealed that the expression of the
dICAD gene is developmentally regulated.
During animal development, many harmful or useless cells are
generated, which are removed by apoptosis, or programmed cell death, to
maintain homeostasis (1). Apoptosis is accompanied by morphological
changes in the cells such as condensation and fragmentation of nuclei
and cells and loss of microvilli from plasma membranes (2). Another
hallmark of apoptosis is the extensive degradation of chromosomal DNA
into nucleosomal units, giving rise to a ladder with multimers of about
180 base pairs (3, 4). Recent genetic and biochemical characterization of the apoptotic signal transduction pathway indicates that apoptosis is mediated by a family of proteases called caspases (cysteinyl aspartate-specific protease) (5). Caspases exist as inactive precursor
forms (zymogens) in proliferating cells and are activated by
proteolytic processing during apoptosis. The caspases involved in the
apoptotic pathway can be grouped into two subfamilies, initiators and
executors. The initiator caspases such as caspases 8 and 9, are
activated at the plasma membrane by death factors, or at mitochondria
by various apoptosis-inducing agents, whereas the executor caspases,
such as caspases 3, 6, and 7, are activated by the initiator caspases.
We and others have recently identified in a mammalian system a DNase
(CAD,1 also called DFF-40 or
CPAN) that is activated by a caspase (6-10) and is responsible for the
characteristic degradation of chromosomal DNA into nucleosomal units.
CAD is complexed with its inhibitor (ICAD, also called DFF-45) in
proliferating cells. When apoptotic stimuli activate a caspase cascade,
caspase 3, which acts downstream in the cascade, cleaves ICAD, which
releases CAD to cleave the chromosomal DNA (11, 12).
The apoptotic signal transduction pathway appears to be well conserved
among metazoans. Genetic analysis of programmed cell death in
Caenorhabditis elegans demonstrated that it is regulated by
a small set of gene products (13): Ced-3 (cell death abnormal) and
Ced-4 function as executors of the process, whereas Ced-9 blocks it.
Mammalian counterparts of these gene products have been identified as
the caspase, Apaf-1 (apoptosis-activating factor), and Bcl-2 families,
respectively (14). Each family in the mammalian system is composed of
several members, thus apoptotic signal transduction seems to be more
elaborate than that in C. elegans.
Development of Drosophila melanogaster is also regulated by
programmed cell death (15-17). Dying cells appear at stage 11 of embryogenesis, which continue throughout embryogenesis. The cell death
is also observed during metamorphosis. As in mammals and C. elegans, the apoptosis in Drosophila is accompanied by
morphological changes in the cells, including DNA fragmentation. This
process is also regulated by Ced-3/caspase- and Ced-4/Apaf-1-like
molecules (16). At least five caspase members have been identified in Drosophila. Of these, DREDD and DRONC resemble the mammalian
initiator caspases 8 and 9. The other three members, Dcp-1, drICE, and
DECAY resemble effector or executioner caspases such as caspases 3 and 7 (18-22).
Despite the apparently well conserved apoptotic system in mammals,
Drosophila, and C. elegans, the molecular
mechanism of the apoptotic DNA fragmentation in Drosophila
has not been well studied. We recently identified a DNase in
Drosophila Schneider line 2 (S2) cells that can be activated
by caspase, and showed it to be the Drosophila counterpart
of CAD (dCAD) (23). In this report, we found that Drosophila
cells also carry an inhibitor of CAD (dICAD). dICAD was complexed with
dCAD and could be cleaved by caspase. Analysis of the expression of the
dICAD gene indicated that it is maternally regulated and
highly expressed during early embryogenesis and metamorphosis. This
expression pattern suggests that dICAD plays an important role in
programmed cell death in Drosophila.
Cell Lines, Antibodies, and Materials--
S2 cells from
D. melanogaster (ATCC CRL1963) and its transformed lines
bearing pMT-reaper (24) were maintained at 25 °C in
Schneider's Drosophila medium (Sigma) supplemented with
10% fetal calf serum (Equitech-Biological Inc.). To induce the
expression of reaper, the transformants were incubated at
25 °C for 12 h in the presence of 0.5 mM
CuSO4, and the cell lysates were prepared as described
(24). The Drosophila neuronal cell line, ML-DmBG2-c2 (BG2-c2) (25) was maintained at 25 °C in Shields and Sang M3 medium
(Sigma) containing 10% fetal calf serum and 10 µg/ml insulin. A
cDNA for the caspase-resistant form of dICAD/DREP-1 was tagged with
FLAG epitope, placed under the Drosophila actin promoter, and introduced into BG2-c2 cells together with the hygromycin-resistant gene. The hygromycin-resistant clones (over 200 clones) were pooled and
used as stable transformed cells.
Human caspase 3 was produced in Escherichia coli and
purified as described (26). Biotinylated
Asp-Glu-Val-Asp-chloromethylketone was purchased from the Peptide
Institute (Osaka, Japan). 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Anti-dICAD antiserum was custom-prepared at the Peptide
Institute by immunizing rabbits with GST-DREP-1. A DREP-1-specific antibody was purified from the serum using a dICAD-affinity column, which was prepared by conjugating histidine-tagged DREP-1 to a CNBr-activated Sepharose column (Amersham Pharmacia Biotech). A
monoclonal antibody (clone M2) against the FLAG peptide was obtained
from Sigma.
Isolation of dICAD cDNA and Recombinant dICAD--
Poly(A)
RNA isolated from S2 cells was reverse-transcribed with random hexamer
primers. The coding sequence of the DREP-1 cDNA was amplified by
polymerase chain reaction (PCR) using the following primers:
sense primer, S-1: 5'-GCCTGGAAATAAAGTGCATAGTG-3'; antisense primer,
AS-1: 5'-AAGAATTCTTGGCTCTTGATTTTGATTGGCG-3'. The PCR product of 0.9 kilobase (kb) was inserted into the pGEM-T vector (Promega), and the
nucleotide sequence was determined using a DNA sequencer (PRIZM310,
Applied Systems). DNA encoding a caspase 3-noncleavable DREP-1 mutant
was prepared by recombinant PCR. In brief, an N-terminal portion of
DREP-1 cDNA was amplified with a sense primer (S-2:
5'-CCGGAATTCGAGACTGCAGCGAACTCG-3') and an antisense primer carrying the
mutation (AS-2: 5'-GTTGTTGGCTTCGGTGGT-3'), while a C-terminal
portion of DREP-1 cDNA was amplified with a sense primer
complementary to AS-2 and an antisense primer of AS-1. The products
from the first PCR were isolated by agarose gel electrophoresis, mixed
1:1, and the secondary PCR was carried out using primers S-2 and AS-1.
The coding sequence of DREP-1 cDNA was tagged with a FLAG epitope
and ligated to the E. coli glutathione
S-transferase (GST) gene using pGEX-2T (128/129). The
GST-DREP-1 fusion proteins were expressed in E. coli AD202, and purified by glutathione-Sepharose 4B (Amersham Pharmacia Biotech). To produce N-terminal six-histidine-tagged DREP-1, the DREP-1 coding
sequence was inserted into the pQE-10 vector (Qiagen). The protein was
expressed in E. coli M15 (pREP4) by treating the cells with
2 mM isopropyl-1-thio- Assay for dCAD and dICAD--
To determine the dCAD activity,
plasmid DNA (1 µg) was incubated at 4 °C for 12 h and at
30 °C for 2 h with samples in 20 µl of buffer A (10 mM Hepes-KOH (pH 7.2), 50 mM NaCl, 5 mM MgCl2, 5 mM EGTA, 10% glycerol,
and 5 mM dithiothreitol (DTT)) containing 1 mg/ml bovine
serum albumin and 500 ng of caspase 3. DNA was extracted and analyzed
by electrophoresis on 1.5% agarose gels. The dICAD activity was
determined by its inhibitory activity against dCAD DNase activity. In
brief, partially purified native dCAD (15.5 µg) or recombinant dCAD
(1 µg) produced in COS cells (23) was treated with 500 or 130 ng of
caspase 3 at 4 °C for 12 h in buffer A. Caspase 3 was then
inactivated with 5 µM biotinylated Asp-Glu-Val-Asp-chloromethylketone at 4 °C for 30 min. The test sample was added to the activated dCAD in a final volume of 20 µl of
buffer A and incubated at 30 °C for 2 h. The remaining dCAD activity was determined with 1 µg of plasmid DNA as described above.
The H-7-induced DNA fragmentation in BG2-c2 cells was assayed as
described (23, 27).
Partial Purification of dICAD from S2 Cells--
S2 cells
(9 × 109 cells) were suspended in buffer B (10 mM Hepes-KOH (pH 7.2), 5 mM MgCl2,
5 mM EGTA, 1 mM DTT, and 1 mM
(p-aminophenyl)methanesulfonyl fluoride (pAPMSF) containing
1 µg/ml pepstatin A, 1 µg/ml leupeptin, and 1 µg/ml aprotinin.
Cells were disrupted by three cycles of freezing and thawing in a
Dounce homogenizer accompanied by grinding with a pestle during each
thawing cycle. The homogenate was spun at 30,000 × g
for 30 min and at 100,000 × g for 2 h. Urea (10 M) was added to the supernatant fraction (about 100 mg of
protein) to a final concentration of 8 M, and incubated at
room temperature for 1 h. After adjusting the urea concentration
to 5 M, the sample was loaded onto a DEAE-Sepharose FF
column (10 ml, Amersham Pharmacia Biotech) that was equilibrated with
buffer B containing 10% glycerol, 5 M urea, and 50 mM NaCl. Proteins were eluted with a 50-350 mM linear NaCl gradient in buffer B containing 5 M urea. The
active fractions were pooled, adjusted to 1 M
(NH4)2SO4, and loaded onto a
butyl-Sepharose column (18 ml) that was equilibrated with buffer B
containing 5 M urea and 1 M
(NH4)2SO4. Proteins were then
eluted with a 1 to 0 M
(NH4)2SO4 descending gradient. The
active fractions were pooled and dialyzed against buffer B containing
50 mM NaCl, and the insoluble materials were removed by
centrifugation and used as partially purified dICAD.
Immunohistochemistry--
Immunohistochemical analysis of
Drosophila embryo was carried out essentially as described
(28). In brief, embryos at the stages of 0-3 h and 3-16 h were
collected, dechorionated by treating for 2.5 min in 2.5% NaClO, and
fixed for 20 min with 4% paraformaldehyde in phosphate-buffered saline
(PBS). After blocking nonspecific sites for 1 h with 5% skim milk
in PBS containing 0.02% Tween 20 (PBST), the embryos were incubated
overnight at 4 °C with 2000-fold-diluted rabbit anti-dICAD antibody
in PBST containing 5% skim milk. After several washes in PBST, the
embryos were incubated at room temperature with 1 µg/ml
biotin-conjugated goat anti-rabbit antibody (Jackson Immunoresearch).
The signals were amplified using an ABC Elite kit (Vector Laboratories)
according to the manufacturer's instructions. The embryos were mounted
in 70% glycerol in PBS and observed under a microscope.
ICAD Activity in Drosophila S2 Cells--
To examine whether
Drosophila cells express ICAD, the cytosolic fraction
(S-100) was prepared from S2 cells. The extracts had no effect on the
DNase activity of partially purified Drosophila CAD (dCAD)
or of recombinant mouse CAD (data not shown). We observed previously
that mouse ICAD does not show ICAD activity when it is complexed with
CAD (29). Because mouse ICAD but not CAD is resistant to denaturants
such as 6 M guanidine hydrochloride, 8 M urea,
or 0.1% SDS (6), the cytosolic fraction (S-100) from S2 cells was
treated with 5 M guanidine hydrochloride or 8 M
urea. When the denaturants were removed by dialysis, a large amount of
insoluble material formed, which was removed by centrifugation. The
supernatant fraction was then assayed for ICAD activity. As shown in
Fig. 1, the guanidine hydrochloride- or
urea-treated cytosolic fraction inhibited dCAD DNase in a
dose-dependent manner. When this fraction was treated with
caspase 3, the ICAD activity was destroyed. These properties were
similar to those of mouse and human ICADs, suggesting that
Drosophila cells express ICAD (dICAD).
Identification of DREP-1 as dICAD--
Following the inhibitory
activity against dCAD, we partially purified dICAD by chromatography on
DEAE-Sepharose and butyl-Sepharose. Meanwhile, Inohara et
al. (30) found a cDNA clone (DREP-1) in the
Drosophila EST data base that has weak but significant
homology with human ICAD/DFF-45. To examine whether DREP-1 possessed
ICAD activity, we prepared recombinant DREP-1 in E. coli as
a fusion protein with glutathione S-transferase. As shown in
Fig. 2A, GST-DREP-1 efficiently inhibited the dCAD DNase activity, and this ICAD activity was destroyed by treatment with caspase 3. Furthermore, the ICAD activity of partially purified dICAD from S2 cells was inhibited by a
rabbit antibody raised against recombinant DREP-1 protein (Fig.
2B). These results indicated that the DREP-1 cDNA in
fact codes for dICAD.
Fig. 3A shows the amino acid
sequence of DREP-1/dICAD, aligned with those of mouse and human ICAD.
The sequence differed from that published by Inohara et al.
(30) at two positions, but agreed with the sequence in the
Drosophila Genome data base. Drosophila ICAD
consists of 296 amino acids and has a molecular mass of 31,970 Da. It
is rich in acidic residues, containing 23 aspartate and 27 glutamate
residues, and has an isoelectric point of 4.37. Its overall identities
with mouse ICAD (mICAD) and human ICAD (hICAD) are 17.6% and 17.3%,
respectively. As noted previously (30), the homology between
Drosophila ICAD and the mammalian ICADs is more pronounced
in the N-terminal CAD/CIDE domain (7, 30). In situ
hybridization of polytene chromosome with dICAD cDNA indicated that
the dICAD gene is located at band 48EF to 49AB. In fact, we
found the dICAD/DREP-1 gene in P1 clones derived from this region, and the alignment of the genomic sequence in the data base with
that of the cDNA indicated that the 5.5-kb dICAD/DREP-1 gene is split by three introns and is located 1.5 kb downstream of the
gene for the Cleavage of dICAD by Caspase--
The abolishment of the ICAD
activity by caspase 3 suggested that dICAD is cleaved by a caspase. In
fact, treatment of GST-DREP-1 with caspase 3 produced two fragments of
45 and 25 kDa (Fig. 4A). Western blotting analysis of the native dICAD in the S-100 fraction or
partially purified preparation from S2 cells showed a 46-kDa protein,
which was cleaved by human caspase 3 to generate a band of 25 kDa.
Expression of reaper in S2 cells activates caspase 3-like
proteases and induces apoptosis (18, 24, 31). To confirm that
Drosophila caspases can cleave dICAD, GST-DREP-1 was
incubated with the cytosolic fraction from reaper-activated S2 cells, and bound to glutathione-Sepharose beads. When proteins bound
to the beads were analyzed by Western blotting with anti-FLAG antibody,
they showed a 45-kDa band, which was similar to that produced by
treatment with human caspase 3. These results indicated that dICAD
could be cleaved by Drosophila caspases, most likely DCP-1,
drICE, or DECAY, which have the same substrate specificity as human
caspase 3 (18, 19, 22).
To determine the caspase cleavage site of dICAD, the caspase 3-cleaved
GST-DREP-1 was separated by SDS-polyacrylamide gel electrophoresis,
transferred to a polyvinylidene difluoride membrane, and the N-terminal
sequence of the 25-kDa fragment was determined by Edman degradation
using an automated sequencer. This analysis yielded a single sequence,
Ala-Asn-Asn-Ser-Glu-Ser-Ala-Arg-Ile, which corresponded to the sequence
of DREP-1/dICAD from amino acids 120 through 128. This sequence was
preceded by a tetrapeptide (DTTD) that agrees with the consensus
recognition sequence for the caspase 3 subfamily (5, 32). To confirm
that caspase 3 cleaves dICAD at Asp-119, the aspartate was mutated to
glutamate. As shown in Fig. 5, the
wild-type GST-DREP-1 was cleaved by caspase 3 into two fragments, and
its ICAD activity was abolished. On the other hand, the GST-DREP-1
carrying the D119E mutation was not cleaved by caspase 3 and was still
functional after caspase 3 treatment. These results indicated that
dICAD was cleaved at a single site at Asp-119 by a caspase, which
abolished its activity. The caspase-cleavage site determined
experimentally here differs from those (Asp-108 and Asp-212) predicted
from the amino acid sequence of DREP-1 as caspase recognition sites
(30).
Complex of dICAD with dCAD and Requirement of dICAD Cleavage in the
Apoptotic DNA Fragmentation in Drosophila Cells--
dICAD activity in
the S2 cell extracts was detected only after treatment with
denaturants, suggesting that dICAD is complexed with dCAD or another
molecule that inhibits the ICAD activity. To examine whether dICAD is
complexed with dCAD, the cytosolic fraction from S2 cells was
fractionated on a DEAE-Sepharose column with a NaCl gradient. Western
blotting analysis with an anti-DREP-1 antibody detected the 46-kDa
dICAD protein in fractions 12-18 eluted with 170-230 mM
NaCl. These fractions showed the caspase 3-dependent DNase
activity coincident with the peak of dICAD protein, suggesting that
dCAD was associated with dICAD (Fig.
6A). To further confirm the
association of dICAD with dCAD, the cytosolic fraction from S2 cells
was immunoprecipitated with rabbit anti-DREP/dICAD antibody. When the
immunoprecipitate was treated with caspase 3, it showed strong DNase
activity. These results confirmed that dICAD was complexed with dCAD in
Drosophila cells (Fig. 6B).
A Drosophila neuronal cell line BG2-c2 undergoes apoptosis
by treatment with a kinase inhibitor, H-7, and this process is accompanied by DNA fragmentation (27). We previously reported that
dICAD is cleaved during the apoptotic process of BG2-c2 cells (23). To
confirm the involvement of the dICAD·dCAD system in the DNA
fragmentation during apoptosis of Drosophila cells, the expression plasmid for the non- cleavable form of dICAD/DREP-1 was
introduced into BG2-c2 cells, and the stable transformants expressing
the mutant dICAD/DREP-1 were established (Fig.
7A). As shown in Fig.
7B, the DNA fragmentation was observed in the parental
BG2-c2 cells after treatment with H-7, whereas no DNA fragmentation was
induced in the BG2-c2 cell transformants expressing the
caspase-resistant form of dICAD/DREP-1.
Expression of dICAD during Early Embryogenesis--
To determine
the expression of dICAD mRNA during Drosophila
development, mRNA was prepared from embryos at various stages, larvae, and pupae. Poly(A) mRNA was also prepared from adult male and female flies as well as from S2 cells. As shown in Fig.
8a, Northern hybridization
indicated that the dICAD mRNA of about 2.2 kb was expressed
abundantly in the early stage of embryogenesis (0-3 h), which is
before the onset of zygotic transcription, indicating that the
expression of the dICAD gene was maternally regulated. The
expression level of the dICAD mRNA rapidly decreased in the later
stages of embryogenesis, and no dICAD mRNA was detected in the 16- to 24-h embryonic and larval stages. When the flies entered the pupal
stage, they started to express dICAD mRNA again, and both male and
female adult flies expressed abundant levels of dICAD mRNA. S2
cells that were established from Drosophila embryos also
expressed dICAD mRNA.
We then examined the distribution of dICAD protein in embryos by
whole-mount immunohistochemical analysis using the DREP-1-specific antibody. As shown in Fig. 8b, the dICAD protein was
detected uniformly in embryos at early and late stages (Fig.
8b, A and B). No specific signal was
detected when the antibody was preadsorbed by the recombinant
dICAD/DREP-1 (Fig. 8b, C and D).
In this report, we identified ICAD in D. melanogaster.
Although the homology between DREP-1/dICAD and mouse or human ICAD is
not very high (about 17% identity), DREP-1/dICAD fulfilled the
criteria to be identified as ICAD. That is, it was complexed with dCAD,
inhibited dCAD DNase activity, and could be cleaved by caspase 3, resulting in the loss of its inhibitory activity. There are two
alternatively spliced forms (ICAD-L and ICAD-S) for human and mouse
ICADs, and these are expressed to a similar extent in a variety of
different cell types (6, 29, 33). In contrast, Drosophila S2
cells expressed only a single form of ICAD corresponding to ICAD-L. The
gene structure of dICAD also ruled out the possibility of generating an
alternatively spliced form corresponding to ICAD-S. We recently showed
that ICAD-L but not ICAD-S has a chaperon-like activity in the
production of functional CAD, although both forms of ICAD can equally
inhibit the CAD DNase activity (29). The lack of ICAD-S in
Drosophila may indicate that there is no specific role for
ICAD-S beyond the regulation of CAD activity.
Human and murine ICADs carry two recognition sites for caspase 3 (6,
8), whereas only a single caspase 3 cleavage site was found in dICAD.
The cleavage site in dICAD is located close to the site corresponding
to the first N-terminal cleavage site in mouse and human ICADs (Fig.
3A). Human ICAD-L cleaved at both sites dramatically loses
affinity for CAD, whereas ICAD-L cleaved singly at either site still
has significant affinity for CAD (34). On the other hand, cleavage of
dICAD by caspase 3 at the single site abolished its ICAD activity,
suggesting that the cleaved dICAD loses its ability to bind dCAD. In
Drosophila, but not in mammalian systems, CAD (dCAD) is also
cleaved by caspase 3 to be activated (23). Thus, when caspase 3 is
activated in Drosophila, the single cleavage of dICAD/DREP-1
may be sufficient to release activated, cleaved dCAD, whereas two
cleavages are required in mammalian systems.
The overall similarity between dICAD and mammalian ICADs (mICAD/hICAD),
is low (32.2% similarity). Accordingly, there is species specificity
in the CAD·ICAD system between mammals and Drosophila. That is, although mouse ICAD inhibited dCAD as efficiently as mCAD,
dICAD could not inhibit mCAD DNase. In addition to the inhibitory activity against CAD DNase, mouse and human ICAD function as specific chaperones to facilitate the correct folding of CAD (6, 29, 35).
Drosophila DREP-1/ICAD also had this chaperone-like function for dCAD in COS cells or in a cell free system. However, it did not
assist in the correct folding of mouse CAD, and mICAD could not support
production of functional dCAD (23), indicating that the species
specificity was more strict for the chaperone-like activity than for
the inhibitory activity against CAD. CAD and ICAD have a conserved
domain at the N terminus (the CAD/CIDE domain) (7, 30). The CAD domains
from CAD and ICAD can interact with each other, and this interaction
seems to be important for the chaperone-like activity of ICAD (35). In
this regard, it is interesting to note that the CAD domains between
dICAD and dCAD, and between mICAD and mCAD, are more conserved (32%
and 37% identity, respectively) than between dCAD and mCAD (Fig.
9).
Expression of the caspase-resistant form of dICAD prevented the
apoptotic DNA fragmentation in a Drosophila cell line of
BG2-c2, indicating that dICAD should be cleaved by a caspase to cause the DNA fragmentation. The finding of dICAD complexed with dCAD indicates that the dICAD·dCAD system is solely responsible for the
cell-autonomous DNA fragmentation in Drosophila cells.
Massive programmed cell death occurs during the embryogenesis and
metamorphosis of Drosophila (15, 16). Like several other
apoptosis-related genes (21, 22, 36), the expression of the
dICAD gene seems to be regulated maternally, and its
mRNA and protein could be found during early embryogenesis. The
dICAD mRNA level decreased later in embryogenesis, and the gene was
reactivated when flies entered metamorphosis. The metamorphosis of
flies is regulated by a steroid hormone, ecdysone. In the promoter
region of the dICAD gene (a 1.5-kb DNA fragment between the
RNA polymerase III gene and the first exon of the dICAD gene) and in
the 2.8-kb intron 1, there are several potential binding sites for the
ecdysone-responsive elements, the E74A protein or the Broad complex
(37, 38). It will be interesting to examine whether the ICAD
gene is in fact regulated by ecdysone and whether the elements in the
promoter region are responsible for its expression. We have recently
found, in the mouse system, that the apoptotic DNA fragmentation is
mediated not only cell autonomously by the CAD·ICAD system but also
by phagocytes (39). Mutational analyses of the dICAD and/or dCAD gene
in Drosophila will answer whether a similar auxiliary system for the apoptotic DNA fragmentation also exists in
Drosophila or not and may reveal physiological roles for
apoptotic DNA fragmentation during animal development.
We thank Drs. K. Ui-Tei and Y. Miyata (Nippon
Medical School) for the BG2-c2 cell line. We thank Dr. Y. H. Inoue
(Osaka University of Foreign Studies) and Dr. A. Fukunaga (Osaka City
University) for the in situ hybridization of the polytene
chromosomes, Dr. M. Enari for help in the initial stage of this work,
and S. Kumagai for secretarial assistance.
*
This work was supported in part by grants-in-aid from the
Ministry of Education, Science, Sports, and Culture in Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by research fellowships of the Japan Society for the
Promotion of Science.
Published, JBC Papers in Press, April 25, 2000, DOI 10.1074/jbc.M909611199
The abbreviations used are:
CAD, caspase-activated DNase;
ICAD, inhibitor of CAD;
dCAD, Drosophila CAD;
dICAD, Drosophila ICAD;
hCAD, human CAD;
mICAD, mouse ICAD;
GST, glutathione
S-transferase;
pAPMSF, (p-aminophenyl)methanesulfonyl fluoride;
PCR, polymerase
chain reaction;
DTT, dithiothreitol;
PBS, phosphate-buffered saline;
kb, kilobase(s);
H-7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine;
S2
cells, Schneider line 2 cells;
Apaf-1, apoptosis-activating
factor-1.
Identification and Developmental Expression of Inhibitor of
Caspase-activated DNase (ICAD) in Drosophila melanogaster*
,,§,,§, and¶
Department of Genetics, Osaka University
Medical School and ¶ Core Research for Evolutional Science and
Technology, Japan Science and Technology Corporation, Osaka 565-0871, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside,
and purified using a Ni-chelated Hi-Trap column. The N-terminal
sequence of the caspase-cleaved GST-DREP-1 was determined at the Takara
Shuzo Co. (Kyoto, Japan).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (115K):
[in a new window]
Fig. 1.
ICAD activity in extracts from S2 cells.
The S-100 fraction from S2 cells was treated at room temperature for
1 h with 8 M urea (lanes 2-9) or 5 M guanidine HCl (lanes 10-17), dialyzed against
buffer B containing 50 mM NaCl, and the insoluble material
was removed by centrifugation. The samples were left untreated
(lanes 2-5 and 10-13) or treated with caspase 3 (lanes 6-9 and 14-17). Using 40 µg
(lanes 2, 6, 10, and 14),
20 µg (lanes 3, 7, 11,
15), 10 µg (lanes 4, 8,
12, and 16), or 5 µg (lanes 5,
9, 13, and 17) of protein, the ICAD
activity against the partially purified dCAD (15.5 µg) was
determined. The DNase activity of the partially purified dCAD is shown
in lane 1.
View larger version (71K):
[in a new window]
Fig. 2.
Identification of DREP-1 as dICAD.
A, the ICAD activity of recombinant DREP-1. The recombinant
DREP-1 was produced in E. coli as a fusion protein with GST,
and purified by glutathione-Sepharose as described under
"Experimental Procedures." GST-DREP-1 was left untreated
(lanes 2-4) or treated with caspase 3 (lanes
5-7), and the ICAD activity against the partially purified dCAD
was determined. The amounts of GST-DREP-1 used were 12 ng (lanes
2 and 5), 2.4 ng (lanes 3 and 6),
and 0.5 ng (lanes 4 and 7). The DNase activity of
the partially purified dCAD is shown in lane 1. B,
neutralization of the ICAD activity of S2 cells with anti-DREP-1
antibody. The dICAD activity of the partially purified dICAD (0.7 µg)
against the recombinant dCAD (1.0 µg of the COS cell lysates) was
determined in the presence of various amounts of normal rabbit IgG
(lanes 2-5) or anti-DREP-1 antibody
(lanes 6-9). After incubation at 4 °C
overnight and at 30 °C for 2 h, the substrate DNA (1 µg) was
analyzed by electrophoresis on an agarose gel. The amounts of IgG used
were 6 µg (lanes 2 and 6), 3 µg (lanes
3 and 7), 1.5 µg (lanes 4 and
8), and 0.75 µg (lanes 5 and 9). The
DNase activity of dCAD alone is shown in lane 1. The dICAD
activity of the partially purified dICAD before incubation with IgG is
shown in lane 10.
-subunit of RNA polymerase III (Fig.
3B).
View larger version (63K):
[in a new window]
Fig. 3.
The amino acid sequence and gene structure of
dICAD. A, alignment of the amino acid sequences of
Drosophila, murine, and human ICADs. The amino acid sequence
of Drosophila ICAD/DREP-1(dICAD) was aligned with
those of murine ICAD (mICAD-L) (6) and human ICAD/DFF-45
(hDFF-45) (8) to give a maximal homology by introducing
several gaps. The positions of intron in dICAD and murine ICAD (33) are
indicated by downward and upward arrows,
respectively. The amino acid residues identical among the three
proteins are shown in bold, and those regarded as favored
substitutions are indicated by underlines. The tetrapeptide
in DREP-1/dICAD recognized by caspase 3 is boxed by
solid lines, whereas those in mICAD-L and hICAD-L
(hDFF-45) are boxed by dashed lines.
Our sequence of dICAD/DREP-1 differs from that published by Inohara
et al. (30) at two positions. Six nucleotides coding for
Lys-Leu are inserted at amino acid position 195, and a nucleotide
change at position 108 causes an amino acid replacement from Asp to Glu
in Inohara et al (30). B, the chromosomal gene
structure of dICAD. The structure of the dICAD chromosomal
gene is schematically shown with that of the gene for -subunit of
RNA polymerase III. Closed boxes and open
boxes represent coding and noncoding regions, respectively.
View larger version (32K):
[in a new window]
Fig. 4.
Cleavage of DREP-1/dICAD by caspase.
A, cleavage of the recombinant DREP-1 by caspase 3. The
GST-DREP/dICAD (2.4 µg) was incubated at 30 °C with 90 ng of human
caspase 3 for the indicated periods of time. Proteins were analyzed by
electrophoresis on a 4-20% gradient polyacrylamide gel and stained
with Coomassie Brilliant Blue. Positions of molecular mass standard
proteins are indicated on the left in kilodaltons. The
intact GST-DREP/dICAD and its cleaved products are indicated by
arrows on the right. B, cleavage of dICAD of S2
cells by caspase 3. The cytosolic fraction from S2 cells (lanes
1 and 2) and the partially purified dICAD (lanes
3 and 4) were incubated at 30 °C for 12 h
without (lanes 1 and 3) or with 90 ng of caspase
3 (lanes 2 and 4). Proteins were then resolved by
electrophoresis on a 4-20% gradient polyacrylamide gel. The Western
blotting was carried out with 4000-fold-diluted rabbit anti-dICAD
antibody, followed by incubation with 3000-fold-diluted horseradish
peroxidase-conjugated goat anti-rabbit IgG (DAKO). Positions of
molecular mass standard proteins are indicated on the left
in kilodaltons. The intact dICAD and its cleaved products are indicated
by arrows on the right. C, cleavage of dICAD by
the apoptotic extracts from S2 cells. The cytosolic extracts (S-30
fraction) were prepared from transformed S2 cells induced to express
reaper (lanes 1-5) or from the nontransformed
parental S2 cells (lane 6). The GST-DREP-1 (30 ng of
protein) was incubated at 30 °C for 3 h with 8 µg (lane
1), 16.0 (lane 2), 33.0 (lane 3), 65.0 (lane 4), and 130.0 (lane 5) of the lysates in
100 µl of buffer A containing 1 mg/ml bovine serum albumin. The
GST-DREP-1 proteins were bound to glutathione-Sepharose beads (20-µl
bed volume) by incubating at 4 °C for 30 min. The beads were heated
at 95 °C for 5 min in Laemmli sample buffer. Proteins released from
the Sepharose beads were then separated by electrophoresis on a 4-20%
gradient polyacrylamide gel. Western blotting was carried out with
3000-fold-diluted anti-FLAG monoclonal antibody, followed by incubation
with the anti-mouse IgG (Jackson Laboratories). The positions of
molecular mass standard proteins are indicated in kilodaltons on the
left.
View larger version (81K):
[in a new window]
Fig. 5.
Resistance of the dICAD mutant to cleavage by
caspase. Wild-type GST-DREP-1 (lanes 1 and
2) or mutant GST-DREP-1 (D119E) (lanes 3 and
4) (2.3 µg) was incubated at 30 °C for 12 h in
buffer A in the absence (lanes 1 and 3) or
presence of 90 ng of human caspase 3 (lanes 2 and
4). Aliquots (2 µg) were then analyzed by electrophoresis
on a 4-20% gradient polyacrylamide gel and stained by Coomassie
Brilliant Blue (A). The positions of the size marker
proteins are shown on the left in kilodaltons. Using 12-ng
aliquots, the ICAD activity was determined with the partially purified
dCAD (B). The DNase activity of the partially purified dCAD
is shown in lane 5.
View larger version (46K):
[in a new window]
Fig. 6.
Complex of dICAD with dCAD in S2 cells.
A, cofractionation of dICAD with dCAD. The cytosolic
fraction from S2 cells (S-100, 11 mg protein) was loaded onto a
DEAE-Sepharose column (1 ml) equilibrated with 10 mM
Hepes-KOH buffer (pH 7.2) containing 50 mM NaCl, 20%
glycerol, 0.1 mM DTT, 0.15%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 0.1 mM pAPMSF. Proteins were eluted from the column
with a 50-350 mM linear NaCl gradient, and 0.5-ml
fractions were collected. Using 10-µl aliquots, the dCAD activity in
the indicated fractions was determined in the presence (top
panel) or absence (middle panel) of 0.5 µg of caspase
3. 10-µl aliquots were also separated by electrophoresis on a 4-20%
polyacrylamide gel and analyzed by Western blotting with anti-DREP-1
antibody (bottom panel). B, coimmunoprecipitation
of dCAD with dICAD. The cytosolic fraction of S2 cells (1.1 mg) in 1 ml
of buffer B containing 150 mM NaCl was incubated at 4 °C
overnight with 5 µg of rabbit normal IgG (lane 2),
anti-DREP antibody (lane 3), or anti-DREP-1 antibody
(lane 4) that had been preincubated at room temperature for
1 h with 3.7 µg of His-tagged DREP-1, and protein A-Sepharose
(10-µl bed volume). The beads were thoroughly washed with buffer B
containing 150 mM NaCl. The DNase activity in the
immunoprecipitates was then determined by incubating the beads at
4 °C for 12 h with 1 µg of plasmid DNA in 20 µl of buffer A
containing 0.5 µg of caspase 3. As a control, plasmid DNA was
incubated at 4 °C for 12 h in buffer A containing caspase 3 (lane 1).
View larger version (56K):
[in a new window]
Fig. 7.
Prevention of the H-7-induced DNA
fragmentation in Drosophila BG2-c2 cells by the
caspase-noncleavable dICAD/DREP-1. A, expression of the
caspase-noncleavable dICAD/DREP-1 in the transformants. The cytosolic
fractions (1.0 µg of protein) from the parental BG2-c2 cell line
(lane 1) and its transformed cells (lane 2) were
separated by electrophoresis on a 4-20% polyacrylamide gel and
analyzed by Western blotting with anti-FLAG monoclonal antibody as
described in Fig. 4C. B, H-7-induced DNA
fragmentation. The parental BG2-c2 cell line (lanes 1 and
2) and its transformant expressing the caspase-noncleavable
dICAD/DREP-1 (lanes 3 and 4) were left untreated
(lanes 1 and 3) or treated at 25 °C for
16 h with 300 µM H-7 (lanes 2 and
4). The fragmented DNA was collected as described (27) and
analyzed by electrophoresis on 1.5% agarose gel.
View larger version (50K):
[in a new window]
Fig. 8.
Expression of dICAD in
Drosophila. a, Northern hybridization with
dICAD cDNA was carried out with 2 µg of poly(A) RNA from the
following developmental stages: 0- to 3-h embryo (lane 1),
3- to 16-h embryo (lane 2), 16- to 24-h embryo (lane
3), first and second instar larva (lane 4), third
instar larva (lane 5), early pupa (lane 6), late
pupa (lane 7), adult male (lane 8), and adult
female (lane 9). Lane 10 contains 2 µg of
poly(A) RNA from S2 cells. The membrane filter used for hybridization
was stained with methylene blue and is shown at the bottom.
b, immunohistochemical analysis. The whole-mount embryos
(A and C, 0- to 3-h embryo; B and
D, 3- to 16-h embryo) were immunohistochemically analyzed
with anti-dICAD antibody (A and B) or the
anti-dICAD antibody (C and D), which had been
preincubated for 1 h at room temperature with the equal amount of
His-tagged DREP-1. A scale bar (100 µm) is shown below
panel D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (24K):
[in a new window]
Fig. 9.
Alignment of the CAD/CIDE domains. The
N-terminal amino acid sequences of Drosophila ICAD (dICAD),
Drosophila CAD (dCAD), mouse ICAD (mICAD), and mouse CAD
(mCAD) are aligned to give maximum homology by introducing several gaps
( 
). The identical residues identical between dICAD and dCAD, and
between mICAD and mCAD are shown in bold, and residues
identical among all four proteins are underlined.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Genetics,
Osaka University Medical School, B-3, 2-2 Yamada-oka, Suita, Osaka
565-0871, Japan. Tel.: 81-6-6879-3310; Fax: 81-6-6879-3319; E-mail:
nagata@genetic.med.osaka-u.ac.jp.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Jacobson, M. D.,
Weil, M.,
and Raff, M. C.
(1997)
Cell
88,
347-354
2.
Wyllie, A. H.,
Kerr, J. F. R.,
and Currie, A. R.
(1980)
Int. Rev. Cytol.
68,
251-306
3.
Wyllie, A. H.
(1980)
Nature
284,
555-556
4.
Earnshaw, W. C.
(1995)
Curr. Biol.
7,
337-343
5.
Thornberry, N. A.,
and Lazebnik, Y.
(1998)
Science
281,
1312-1316
6.
Enari, M.,
Sakahira, H.,
Yokoyama, H.,
Okawa, K.,
Iwamatsu, A.,
and Nagata, S.
(1998)
Nature
391,
43-50
7.
Mukae, N.,
Enari, M.,
Sakahira, H.,
Fukuda, Y.,
Inazawa, J.,
Toh, H.,
and Nagata, S.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9123-9128
8.
Liu, X.,
Zou, H.,
Slaughter, C.,
and Wang, X.
(1997)
Cell
89,
175-184
9.
Liu, X.,
Li, P.,
Widlak, P.,
Zou, H.,
Luo, X.,
Garrard, W. T.,
and Wang, X.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8461-8466
10.
Halenbeck, R.,
MacDonald, H.,
Roulston, A.,
Chen, T. T.,
Conroy, L.,
and Williams, L. T.
(1998)
Curr. Biol.
8,
537-540
11.
McIlroy, D.,
Sakahira, H.,
Talanian, R. V.,
and Nagata, S.
(1999)
Oncogene
18,
4401-4408
12.
Sakahira, H.,
Enari, M.,
and Nagata, S.
(1998)
Nature
391,
96-99
13.
Ellis, R. E.,
Yuan, J.,
and Horvitz, H. R.
(1991)
Annu. Rev. Cell Biol.
7,
663-698
14.
Raff, M.
(1998)
Nature
396,
119-122
15.
Abrams, J. M.,
White, K.,
Fessler, L. I.,
and Steller, H.
(1993)
Development
117,
29-43
16.
Abrams, J. M.
(1999)
Trends Cell Biol.
9,
435-440
17.
Bergmann, A.,
Agapite, J.,
and Steller, H.
(1998)
Oncogene
17,
3215-3223
18.
Song, Z.,
McCall, K.,
and Steller, H.
(1997)
Science
275,
536-540
19.
Fraser, A. G.,
McCarthy, N. J.,
and Evan, G. I.
(1997)
EMBO J.
16,
6192-6199
20.
Chen, P.,
Rodriguez, A.,
Erskine, R.,
Thach, T.,
and Abrams, J. M.
(1998)
Dev. Biol.
201,
202-216
21.
Dorstyn, L.,
Colussi, P. A.,
Quinn, L. M.,
Richardson, H.,
and Kumar, S.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
4307-4312
22.
Dorstyn, L.,
Read, S. H.,
Quinn, L. M.,
Richardson, H.,
and Kumar, S.
(1999)
J. Biol. Chem.
274,
30778-30783
23.
Yokoyama, H.,
Mukae, N.,
Sakahira, H.,
Okawa, K.,
Iwamatsu, A.,
and Nagata, S.
(2000)
J. Biol. Chem.
275,
12978-12986
24.
Kondo, T.,
Yokokura, T.,
and Nagata, S.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
11951-11956
25.
Ui, K.,
Nishihara, S.,
Sakuma, M.,
Togashi, S.,
Ueda, R.,
Miyata, Y.,
and Miyake, T.
(1994)
In Vitro Cell Dev. Biol. Anim.
30A,
209-216
26.
Kamens, J.,
Paskind, M.,
Hugunin, M.,
Talanian, R.,
Allen, H.,
Banach, D.,
Bump, N.,
Hackett, M.,
Johnston, C.,
Li, P.,
Mankovich, J.,
Terranova, M.,
and Ghayur, T.
(1995)
J. Biol. Chem.
270,
15250-15256
27.
Nagano, M.,
Suzuki, H.,
Ui-Tei, K.,
Sato, S.,
Miyake, T.,
and Miyata, Y.
(1998)
Neurosci. Res.
31,
113-121
28.
Nakagoshi, H.,
Hoshi, M.,
Nabeshima, Y.,
and Matsuzaki, F.
(1998)
Genes Dev.
12,
2724-2734
29.
Sakahira, H.,
Enari, M.,
and Nagata, S.
(1999)
J. Biol. Chem.
274,
15740-15744
30.
Inohara, N.,
Koseki, T.,
Chen, S.,
Wu, X.,
and Nunez, G.
(1998)
EMBO J.
17,
2526-2533
31.
Pronk, G.,
Ramer, K.,
Amiri, P.,
and Williams, L.
(1996)
Science
271,
808-810
32.
Nicholson, D. W.,
and Thornberry, N. A.
(1997)
Trends Biochem. Sci.
22,
299-306
33.
Kawane, K.,
Fukuyama, H.,
Adachi, M.,
Sakahira, H.,
Copeland, N. G.,
Gilbert, D. J.,
Jenkins, N. A.,
and Nagata, S.
(1999)
Cell Death Differ.
6,
745-752
34.
McCarty, J. S.,
Toh, S. Y.,
and Li, P.
(1999)
Biochem. Biophys. Res. Commun.
264,
181-185
35.
McCarty, J. S.,
Toh, S. Y.,
and Li, P.
(1999)
Biochem. Biophys. Res. Commun.
264,
176-180
36.
Rodriguez, A.,
Oliver, H.,
Zou, H.,
Chen, P.,
Wang, X.,
and Abrams, J.
(1999)
Nat. Cell Biol.
1,
272-279
37.
Urness, L. D.,
and Thummel, C. S.
(1990)
Cell
63,
47-61
38.
von Kalm, L.,
Crossgrove, K.,
Von Seggern, D.,
Guild, G. M.,
and Beckendorf, S. K.
(1994)
EMBO J.
13,
3505-3516
39.
McIlroy, D.,
Tanaka, M.,
Sakahira, H.,
Fukuyama, H.,
Suzuki, M.,
Yamamura, K.-i.,
Ohsawa, Y.,
Uchiyama, Y.,
and Nagata, S.
(2000)
Genes Dev.
14,
549-558
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  D. Lechardeur, S. Dougaparsad, C. Nemes, and G. L. Lukacs Oligomerization State of the DNA Fragmentation Factor in Normal and Apoptotic Cells J. Biol. Chem., December 2, 2005; 280(48): 40216 - 40225. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Lechardeur, M. Xu, and G. L. Lukacs Contrasting nuclear dynamics of the caspase-activated DNase (CAD) in dividing and apoptotic cells J. Cell Biol., December 6, 2004; 167(5): 851 - 862. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Mukae, H. Yokoyama, T. Yokokura, Y. Sakoyama, and S. Nagata Activation of the innate immunity in Drosophila by endogenous chromosomal DNA that escaped apoptotic degradation Genes & Dev., October 15, 2002; 16(20): 2662 - 2671. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Fukumoto, R. Watanabe-Fukunaga, K. Fujisawa, S. Nagata, and R. Fukunaga The Fused Protein Kinase Regulates Hedgehog-stimulated Transcriptional Activation in Drosophila Schneider 2 Cells J. Biol. Chem., October 12, 2001; 276(42): 38441 - 38448. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Cao, W. Pei, J. Lan, R. A. Stetler, Y. Luo, T. Nagayama, S. H. Graham, X.-M. Yin, R. P. Simon, and J. Chen Caspase-Activated DNase/DNA Fragmentation Factor 40 Mediates Apoptotic DNA Fragmentation in Transient Cerebral Ischemia and in Neuronal Cultures J. Neurosci., July 1, 2001; 21(13): 4678 - 4690. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
