From the Department of Pathology, University of
Michigan Medical School, the § Department of Human Genetics,
University of Michigan Medical School, Ann Arbor, Michigan 48109 and
¶ Drug Safety Evaluation, Pfizer Global Research and Development,
Ann Arbor, Michigan 48105
Received for publication, October 27, 2000, and in revised form, November 10, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
We report the deduced amino acid
sequences of two alternately spliced isoforms, designated DEFCAP-L and
-S, that differ in 44 amino acids and encode a novel member of the
mammalian Ced-4 family of apoptosis proteins. Similar to the other
mammalian Ced-4 proteins (Apaf-1 and Nod1), DEFCAP contains a caspase
recruitment domain (CARD) and a putative nucleotide binding domain,
signified by a consensus Walker's A box (P-loop) and B box
(Mg2+-binding site). Like Nod1, but different from
Apaf-1, DEFCAP contains a putative regulatory domain containing
multiple leucine-rich repeats (LRR). However, a distinguishing feature
of the primary sequence of DEFCAP is that DEFCAP contains at its
NH2 terminus a pyrin-like motif and a proline-rich
sequence, possibly involved in protein-protein interactions with
Src homology domain 3-containing proteins. By using in
vitro coimmunoprecipitation experiments, both long and short
isoforms were capable of strongly interacting with caspase-2 and
exhibited a weaker interaction with caspase-9. Transient overexpression
of full-length DEFCAP-L, but not DEFCAP-S, in breast adenocarcinoma
cells MCF7 resulted in significant levels of apoptosis. In
vitro death assays with transient overexpression of deletion
constructs of both isoforms using Apoptosis (programmed cell death) is the genetically determined
cell suicide program resulting in distinct biochemical and morphological features. Some of the hallmark characteristics of apoptosis include plasma membrane blebbing, nuclear and cytosolic condensation, and ultimately the formation of membrane-bound apoptotic bodies primed for phagocytosis (1). Alterations in the ability of the
cell to initiate and/or execute the proper apoptotic signaling cascade
have been implicated in many diseases such as cancer, autoimmune
diseases, viral infections, and neurodegenerative disorders (2).
Identifying the key mediators of apoptosis and understanding the
molecular mechanisms of programmed cell death is critical to
understanding the pathogenesis of these diseases.
Genetic studies conducted by Horvitz and co-workers (3, 4) using the
roundworm, Caenorhabditis elegans, identified the genes
ced-3, ced-4, and ced-9 as crucial for
initiating proper apoptosis in the developing nematode. Loss of
function mutations in Ced-3 and Ced-4 or an overexpression of Ced-9
prevented programmed cell death in the developing nematode. The
identification and characterization of these genes led to a wealth of
research into the identification of their mammalian counterparts.
Based on the analysis of Ced-3 homologous sequences, the cysteine
protease interleukin 1 To date, only two mammalian Ced-4 homologues, Apaf-1 (5) and Nod1 (6)
CARD4 (7), and one Drosophila Ced-4 homologue, DAPAF-1
(8)/DARK (9), have been reported. Apaf-1, Nod1, DAPAF-1, and Ced-4 are
all similar in having an NH2-terminal CARD followed
directly by a nucleotide binding domain (NBD), also known as the NB-ARC
or NOD domain. Apaf-1 and Nod1 differ from Ced-4 by having a
COOH-terminal regulatory domain containing multiple WD-40 repeats for
Apaf-1 and multiple leucine-rich repeats (LRR) for Nod1. After the
initiation of an apoptotic stimulus and release of cytochrome
c from the mitochondria, cytochrome c is believed to bind the WD-40 repeats of Apaf-1 causing a conformational change in
Apaf-1. Post-translational modifications (i.e. cytochrome
c binding) of Apaf-1 can lead to Apaf-1 oligomerization and
the subsequent ATP-dependent recruitment, processing, and
activation of caspase-9 (5, 10, 11). Apaf-1, caspase-9, and other core
components of the apoptosis machinery interact to form an ~700-kDa
biologically active and ~1.4-MDa biologically inactive complex known
as the apoptosome (12). Formation of the apoptosome and the subsequent
processing of caspase-9 have been shown to be crucial for the
activation of effector caspases, such as caspase-3, leading to the
demise of the cell. In this model, Apaf-1 serves as an adaptor molecule
linking upstream regulatory caspases with downstream effector caspases.
Nod1, on the other hand, appears to function in activating the NF- Here we report the deduced amino acid sequences and characterization of
DEFCAP-L and DEFCAP-S (Death Effector
Filament-forming Ced-4-like
Apoptosis Protein), two isoforms for a novel
member of the mammalian Ced-4 family of proteins. Named for the ability of CARD to form novel cytoplasmic structures termed death effector filaments (14), DEFCAP-L and DEFCAP-S differ in only 44 amino acids,
containing an extra LRR. The NH2-terminal region of DEFCAP consists of a pyrin-like motif (PLM), a proline-rich sequence (PR), followed by a highly conserved NBD, a multiple LRR containing domain, and a COOH-terminal CARD. Based on the primary sequence of
DEFCAP, which contains a well conserved CARD, NBD, and a putative regulatory domain with multiple LRR motifs, we classify DEFCAP as the
third member of the mammalian Ced-4 family of proteins.
High fidelity polymerase chain reaction (PCR) reagents for
cloning were purchased from Roche Molecular Biochemicals. DNA
sequencing was performed by the University of Michigan Core Facilities,
and oligonucleotide synthesis was performed by Operon Technologies, Inc. Restriction enzymes, linkers, and other modifying enzymes were
purchased from New England Biolabs. Antibodies to c-Myc (9E10) AC,
caspase-8 p20 (C-20), caspase-9 p46 (H-170), and caspase-10 (H-19) were
purchased from Santa Cruz Biochemicals. Caspase-2 monoclonal antibodies
(I75620) were purchased from PharMingen/Transduction Laboratories.
RAIDD monoclonal antibodies (4B12) were purchased from StressGen
Biotechnologies Corp. ZVAD-fmk was purchased from Enzyme Systems Products.
Mammalian expression vector pcDNA3.1 was purchased from Invitrogen
and modified to contain an NH2-terminal Myc epitope
(pcDNA3.1 NmycH) by annealing sense and antisense oligonucleotides
coding for the Myc epitope followed by digestion and ligation into the NheI and HindIII sites. 5'-Sequences flanking the
Myc epitope were converted to Kozak consensus translation initiation sites.
Northern Analysis and Semi-quantitative RT-PCR--
Human
12-lane MTN blot (catalog number 7780-1) and a human cancer cell line
MTN blot (catalog number 7757-1) were obtained from
CLONTECH. Full-length DEFCAP-L cDNA was
radiolabeled with [ Chromosomal Localization--
The Stanford G3 Human/Hamster
Radiation Hybrid Panel (Research Genetics) was screened by PCR
with the oligonucleotides
5'-CAGCGTCTCCAAGCTCAGCCATTGGGACCC-3' (sense) and
5'-CAGGCCATGTATTCCATATGCTTCTAGCGT (antisense) yielding an
amplified product of ~300 bp encoding amino acids 560-665. These
primers were believed to be restricted to a single exon based on the
identification of continuous genomic sequences from PAC pDJ891a18
identified by a nucleotide search of the High Through put
Genomic Sequences database.
Site-directed Mutagenesis--
An A box (K340S) point mutation
for full-length DEFCAP-L and -S was created using a two-step PCR
protocol with the following primer pairs: 5'-CGGGGTACCGCTG
GCGGAGCCTGGGGCCGCCTGGCCTGT (sense)/5'-GGCCAGTGTCGACGACCCA ATTCCAGCAGC
(antisense) and 5'-GCTGCTGGAATTGGGTCGTCGACACTGGCC (sense)/5'-CAGGCCATGTATTCCATATGCTTCTAGCGT (antisense) followed by
PCR amplification with the outside primers, digestion with AhdI/BstXI, and a four-part ligation into
pcDNA3.1 NmycH using KpnI, AhdI,
BstXI, and NotI restriction sites.
Expression Vectors--
NH2-terminal Myc-tagged
DEFCAP expression constructs were made in pcDNA3.1 NmycH using
KpnI/NotI restriction sites for the following:
DEFCAP-L (a.a. 1-1473), DEFCAP-S (a.a. 1-1439), K340S-L (a.a.
1-1473), K340S-S (a.a. 1-1439), Construction of EGFP Constructs and Fluorescence
Microscopy--
The multiple cloning site of pEGFPC1
(CLONTECH) was digested with BamHI,
Klenow filled-in to create blunt ends, and ligated with NotI
linkers (New England Biolabs) to create pEGFPC1-NotI. The
NH2-terminal EGFP DEFCAP-CARD (a.a. 1356-1473) fusion
construct was made in pEGFPC1-NotI by ligating DEFCAP
sequences in frame using 5' KpnI and 3' NotI
restriction sites. Cells were visualized at × 100 magnification
by fluorescence microscopy using a PixCell II microscope (Arcturus).
Cell Culture and Transient Transfection--
Human embryonic
kidney 293 cells were maintained in 10% fetal calf serum with
Dulbecco's modified Eagle's medium supplemented with
L-glutamine, penicillin/streptomycin, and nonessential
amino acids. K562 human erythroleukemia cells and MCF7, a human breast carcinoma cell line, were grown in 10% heat-inactivated fetal calf
serum in RPMI 1640 with L-glutamine,
penicillin/streptomycin, and nonessential amino acids. 293 cells were
transfected using standard CaPO Immunoprecipitation and Western Blotting--
293 cells were
seeded at 10% confluency on 10-cm dishes, grown to 50% confluency,
and prior to transfection were given fresh media. A total of 10 µg of
DNA was transfected into each dish as stated previously. 6 h
post-transfection, the transfected cells were given fresh media, and
cells were harvested 12-24 h post-transfection. Cells were harvested
by collecting and pooling floating cells with adherent cells on ice and
washed once with ice-cold phosphate-buffered saline (PBS). The cells
were resuspended in 1 ml of lysis buffer containing 1% Nonidet P-40,
20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol,
2 mM EDTA, 5 mM sodium vanadate, 0.5 mM phenylmethylsulfonyl fluoride, and 1× protease
inhibitors (complete, Mini, BMB 1 836 153) and incubated on ice for 15 min. The cell debris was spun down at 20,000 × g for
15 min, and the supernatant was transferred to a fresh tube. The pellet
was washed once with ice-cold PBS, mixed with 25 µl of 1.5×
SDS-polyacrylamide gel electrophoresis sample buffer containing
dithiothreitol, boiled at 100 °C for 5 min, and saved at Caspase Interaction Experiments--
Coimmunoprecipitation
experiments with either full-length NH2-terminal Myc-tagged
DEFCAP-L or -S and caspases-2, -3, -8, -9, and -10 were performed in
293 cells. Briefly, 293 cells were cotransfected with 5 µg of
Myc-DEFCAP-L or Myc-DEFCAP-S and with 3 µg of caspase construct.
18-24 h post-transfection, cells were harvested and immunoprecipitated
with Myc-AC and immunoblotted with caspase specific antibodies. The
caspase-2 and caspase-9 coimmunoprecipitation experiments with both
DEFCAP isoforms were reproduced in three independent experiments.
Cell Death Assays--
MCF7 cells were plated on 35-mm 6-well
tissue culture dishes, and each well was cotransfected at ~60%
confluency with 0.25 µg of the reporter plasmid pCMV
Cloning of DEFCAP-L from a K562 cDNA Library and Chromosomal
Assignment--
By homology search using BLASTP for CARD-containing
proteins, we identified clone KIAA0926 (GenBankTM accession
number NP_055737), which was kindly provided by the Kazusa DNA Research
Institute (21). For simplicity, clone KIAA0926 will be referred to as
DEFCAP-S. By using the DEFCAP-S nucleotide sequence, we generated
oligonucleotides that were used to PCR-amplify the DEFCAP open reading
frame from a K562 cDNA library. PCR-amplified products were cloned
and restriction-mapped, and DNA sequencing was performed on both
strands to confirm the sequence accuracy of the clones. DNA sequences
with translation products of perfect identity to DEFCAP-S were obtained
from a K562 human erythroleukemia cancer cell line cDNA library. In
addition, we identified some clones that contained a 132-bp insertion
near the 3' end of the DEFCAP open reading frame. DNA sequencing
followed by a BLAST search of the nonredundant data base identified a
perfect match for human hypothetical protein DKFZp58601822.1
(GenBankTM accession number T17255). Analysis of the 132-bp
insertion revealed that these sequences encode for an extra LRR not
found in DEFCAP-S. We designated the full-length DEFCAP sequence
containing the additional 132-bp sequence as DEFCAP-L.
By using the Stanford G3 radiation hybrid panel, we assigned the DEFCAP
chromosomal localization between sequence-tagged sites (STSs) D17S849
and D17S796 of chromosome 17p13 (LOD score 7.53-9.25). These results
are in agreement with those obtained by Nagase et al.
using the Genebridge4 radiation hybrid panel for clone KIAA0926 (21).
The Amino Acid Sequences of DEFCAP-L and DEFCAP-S Share Homology
with Apaf-1, Nod1, and Ced-4--
The deduced amino acid sequences for
DEFCAP-L and DEFCAP-S encode proteins of 1473 and 1429 amino acids,
respectively. Similar to the other mammalian Ced-4 homologues
identified thus far, DEFCAP contains a CARD domain, a putative
nucleotide binding domain (NBD), and a putative regulatory domain
containing multiple repeat elements (LRRs). However, unlike the two
other mammalian Ced-4 homologues Apaf-1 and Nod1, the positioning of
these protein domains in the primary sequence is not conserved. Both
Apaf-1 and Nod1 contain NH2-terminal CARD domains followed
directly by an NBD. The COOH terminus of Apaf-1 is composed of 12-13
WD-40 repeats due to alternate splicing, whereas that of Nod1 is
composed of 10 LRRs. Without knowing the crystal structure of the
full-length DEFCAP protein or that of any other Ced-4 family member, it
is difficult to comment on the significance of the COOH-terminal CARD
found in DEFCAP versus the NH2-terminal CARD
found in all other Ced-4-like proteins. However, the juxtaposition of
the LRRs and the CARD in DEFCAP may result in an overall structure that
is unique among the Ced-4 family members.
Some features of DEFCAP's primary sequence that distinguish it from
all other Ced-4-like molecules are its NH2-terminal
pyrin-like motif (PLM) (a.a. 1-95), reverse-highlighted in
gray, and its proline-rich sequence (PR) (a.a. 40-257)
containing 9 PXXP motifs, underlined in black (Fig.
1A). DEFCAP's PLM shares 25%
identity to pyrin or marenostrin, a CARD-containing protein originally identified by positional cloning experiments in patients with Familial
Mediterranean Fever disease, an inherited disease characterized by
excessive neutrophil activity resulting in recurrent episodes of
inflammation involving serosal and synovial spaces. The pyrin-like motif is conserved with other mammalian proteins such as ASC
(apoptosis-associated speck-like protein
containing a CARD), a COOH-terminal CARD-containing protein
with an NH2-terminal PLM (22), which shares 28% identity with DEFCAP-PLM. Furthermore, the PLM is evolutionarily conserved as
seen by a protein alignment with Danio rerio ASC1 (Fig.
1B). The recent identification of an emerging number of PLM
and CARD-containing proteins suggests that the PLM may play a role in
regulating the apoptotic machinery.
Directly following the PLM and PR of DEFCAP is a highly conserved Ced-4
homology domain or NBD (a.a. 309-648) containing a consensus A box
(P-loop), B box (Mg2+ binding), and motif III, a conserved
sequence with unknown function (all highlighted in
red, Fig. 1A). Asterisks below
the residues denote the conserved amino acids as determined by Walker
and co-workers (12). A
Amino acids 703-1280 of DEFCAP-L and amino acids 703-1220 of DEFCAP-S
encode a domain containing 9 consensus LRR elements in DEFCAP-L and 8 LRRs in DEFCAP-S with the consensus sequence XAXXAXAXX(N/C/T/Q)+/
The CARD domain of DEFCAP is located at the most carboxyl end of the
protein and is depicted in Fig. 1A by green
highlighting. A Human DEFCAP mRNA Is Expressed in Multiple Tissues but Has the
Highest Level of Expression in Peripheral Blood Leukocytes and the
Chronic Myelogenous Leukemia Cell Line K562--
Northern blot
analysis revealed DEFCAP to be expressed as at least two transcripts of
~7.0 and ~8.0 kilobase pairs in size (Fig.
2A). Both transcripts were
found in a variety of human adult tissues with the highest levels of
expression in peripheral blood leukocytes, heart, thymus, and spleen.
Low levels of DEFCAP mRNA expression were found in skeletal muscle,
colon (no mucosa), kidney, liver, small intestine, placenta, and lung.
No detectable levels of DEFCAP expression were found in the adult
brain. Equal loading of RNA was determined by probing the same blots
with Both DEFCAP-L and DEFCAP-S Isoforms Are Expressed in mRNAs from
Normal Tissue and Cancer Cell Lines--
Since the long DEFCAP isoform
was cloned from a K562 cancer cell line, we first wanted to determine
whether the long isoform exists in RNAs from normal tissues, and second
to gain an understanding of the relative abundance of the two isoforms
in various RNAs by semiquantitative RT-PCR. Oligonucleotides flanking
the alternately spliced sequences of DEFCAP-L were used to amplify the
long isoform as a 322-bp fragment and the short isoform as a 190-bp
fragment (Fig. 3B). Both 322- and 190-bp RT-PCR products were gel-purified and confirmed to be
specific to DEFCAP by DNA sequencing (data not shown). Both long and
short DEFCAP isoforms were identified in RNAs from K562 cells, Jurkat
cells, normal human liver, spleen, PMNs, and PBMCs. Interestingly,
DEFCAP-L levels were relatively constant in all RNAs with the exception
of the Jurkat and spleen RNAs that were slightly diminished. DEFCAP-S
mRNA expression was weakest in K562, Jurkat, and liver but was
significantly increased in spleen, PMNs, and PBMCs. EGFP-DEFCAP-CARD Fusion Proteins Are Capable of Forming Death
Effector Filament-like Structures in MCF7 Cells--
To gain insight
into the subcellular localization of DEFCAP,
NH2-terminal EGFP constructs containing the full-length
DEFCAP-L, DEFCAP-S, and DEFCAP-CARD were created and transfected into
MCF7 cells. Overexpression of full-length EGFP-DEFCAP-L and
EGFP-DEFCAP-S resulted in a mostly diffuse cytoplasmic subcellular
localization (data not shown). However, an EGFP/DEFCAP-CARD fusion
protein was capable of forming novel cytoplasmic filamentous structures similar to the death-effector-filaments (DEF) formed by FADD, the
death-effector domain (DED-B) of procaspase-8 (14), the prodomain of
caspase-2, and RAIDD (24). The formation of DEF-like structures by
DEFCAP-CARD suggests that DEFCAP may have the ability to dimerize or
oligomerize in a CARD-mediated manner. The fact that both full-length
DEFCAP isoforms cannot form DEFs while the CARD alone can suggests that
the CARD of DEFCAP is normally in a conformation that prevents
CARD-mediated oligomerization.
DEFCAP-L and DEFCAP-S Bind Caspase-2 and Weakly to Caspase--
To
identify DEFCAP/caspase interactions, 293 cells were transiently
cotransfected with either full-length DEFCAP-L or DEFCAP-S in
combination with either pcDNA3.1, caspase-2, caspase-3, caspase-8, caspase-9, or caspase-10. DEFCAP failed to coimmunoprecipitate caspase-3, -8, and -10 and the adaptor proteins FADD and RAIDD (data
not shown). However, both DEFCAP-L and DEFCAP-S were able to
immunoprecipitate effectively caspase-2 (Fig. 4A). 293 cells transfected with caspase-2 alone and immunoprecipitated with Myc-AC served as a negative control and did not immunoprecipitate caspase-2 (data not shown). A ~48-kDa band representing the zymogen form of
caspase-2 can be seen in all of the supernatant lanes but was significantly increased in samples transfected with caspase-2. The
~48-kDa band found in the supernatant and pellet lanes of the vector
control, DEFCAP-L alone, and DEFCAP-S alone most likely represents
specificity to the endogenous procaspase-2 protein. No
caspase-2-specific bands appeared in the IP lanes of the vector control, DEFCAP-L alone, or DEFCAP-S alone suggesting that we were not
able to detect a DEFCAP/caspase-2 interaction with the endogenous
caspase-2 protein (Fig. 4A). These results are not surprising given the fact that the endogenous caspase-2 is localized primarily to the Golgi complex (25) and the mitochondrial intermembrane space (26). Therefore, the endogenous caspase-2 protein was most
likely unable to interact with overexpressed DEFCAP. However, in
cells overexpressing caspase-2 and DEFCAP-L or -S, caspase-2 was
effectively coimmunoprecipitated as seen by bands at ~48 and ~37
kDa. A band at ~33 kDa most likely representing the cleaved caspase-2
product containing the prodomain and the large catalytic subunit (27)
was also found in the DEFCAP-S/caspase-2 (Fig. 4, IP
lane). Interestingly, this ~33-kDa band was significantly diminished in the supernatant of cells cotransfected with
DEFCAP-L/caspase-2 and barely detectable in the IP lane, suggesting
that DEFCAP-L may be able to partially inhibit caspase-2 processing. A
~70-kDa protein in the IP lane denoted by an
asterisk appears to be a nonspecific band that cross-reacted
with the caspase-2 antibody.
To a lesser extent, DEFCAP-L and DEFCAP-S were able to
coimmunoprecipitate caspase-9 (Fig.
4B). Interestingly, the major
band seen in the IP lanes was the ~35-kDa band representing the
processed caspase-9 enzyme (p35). One possible explanation for these
results may be that, like Apaf-1, coimmunoprecipitation of caspase-9 by DEFCAP may lead to the autoactivation and processing of caspase-9 since
the ~50-kDa proenzyme band is barely detectable in the IP lanes (11).
Although difficult to visualize in the figure, the ~50-kDa proenzyme
band was seen in the IP lane of cells cotransfected with both caspase-9
and DEFCAP-L but not in the IP lanes cotransfected with DEFCAP-S.
Another possible explanation why we were only able to detect the
~35-kDa band may be that the DEFCAP interaction is specific to the
processed caspase-9 molecule. Furthermore, the weak DEFCAP/caspase-9
interaction may be indirect possibly requiring an unknown adaptor
molecule. Another important observation is that DEFCAP expression
levels were noticeably reduced when cotransfected with caspase-9 (Fig.
4B, bottom panels). Examination of the pellet lanes for
caspase-9 alone, DEFCAP-L/caspase-9, and DEFCAP-S/caspase-9 suggests
that coexpression of caspase-9 with either DEFCAP construct leads to a
significant increase in the ~48 and ~35-kDa bands in the
membrane-insoluble fraction. Nonspecific bands seen above the ~48-kDa
procaspase-9 are relatively equal suggesting that protein loading does
not adequately explain these differences. The functional significance
of this observation remains unclear; however, DEFCAP may play a role in
targeting caspase-9 to different subcellular membrane fractions.
The fact that DEFCAP interacts strongest with caspase-2 and only weakly
with caspase-9 raises some interesting questions. First of all, what is
the functional consequence of the ability of DEFCAP-L and -S to bind
caspase-2 and can DEFCAP interact with both long and short isoforms of
caspase-2? Mice deficient of both isoforms of caspase-2 exhibit an
increase in facial motor neuron apoptosis, a partial resistance of B
lymphoblasts to granzyme B apoptosis, and a significant increase in the
number of primordial follicles in the postnatal ovary (28). These
results suggest that caspase-2 can have both a pro- and anti-apoptotic
function depending on the cell type. These results raise the
possibility that a DEFCAP/caspase-2 interaction can lead to either a
pro- or anti-apoptotic outcome. Second, where in the cell do the
DEFCAP/caspase interactions take place? Immunohistochemical and cell
fractionation experiments show that caspase-2 and to a lesser degree
caspase-9 have both a cytosolic and nuclear subcellular localization.
Future studies are needed to investigate whether the
DEFCAP/caspase-2 or DEFCAP/caspase-9 interactions are exclusively
cytosolic or whether DEFCAP is able to bind these caspases in the
nucleus to exert its apoptotic function. Moreover, is DEFCAP capable of
being translocated to the nuclear membranes? This idea seems plausible given the data presented by Chen et al. (29) showing that
Ced-4 translocates to the perinuclear membrane upon induction with a death stimulus.
Overexpression of DEFCAP-L, LRR/CARD-L, and LRR/CARD-S Kills MCF7
Cells--
To determine if the ectopic expression of DEFCAP constructs
alone could kill cells in culture, MCF7 breast carcinoma cells were
transiently transfected with NH2-terminal Myc full-length and mutant DEFCAP constructs (Fig.
5C). The base-line apoptosis level as determined by transfection with pcDNA3.1 was 25%
(n = 8, S.E. = 1.655). Full-length DEFCAP-L alone
exhibited 36% (n = 4, S.E. = 0.958) killing, whereas
full-length DEFCAP-S resulted in a 26% killing activity
(n = 4, S.E. = 3.49), a level comparable to vector
control. A lysine to serine (K340S-L) point mutation in the highly
conserved P-loop of DEFCAP did not decrease the killing activity of
DEFCAP-L that resulted in 38% (n = 6, S.E. = 2.611)
apoptotic cells. These results suggest that full-length NBD of DEFCAP-L
may not be functioning as an ATPase during apoptosis. Likewise, a K340S
mutation in DEFCAP-S had no effect on its killing activity that
remained at levels (24%, n = 4, S.E. = 2.743) similar to wild-type DEFCAP-S and vector control. Deleting amino acids 1-309
of DEFCAP-L (
Deletion constructs eliminating the CARD for both DEFCAP isoforms
(
Furthermore, expression constructs containing the PLM/PR/NBD, PLM/PR,
or DEFCAP-CARD were not able to significantly kill MCF7 cells and
resulted in 30 (n = 3, S.E. = 1.402), 32 (n = 3, S.E. = 1.457), and 22% (n = 3, S.E. = 3.11) apoptosis, respectively. In comparison to the vector
control, p values for PLM/PR/NBD and PLM/PR were calculated
at 0.264 and 0.145, respectively. DR3 transfections served as a
positive control for apoptosis and was capable of killing at levels of
66% (n = 4, S.E. = 6.6617). 293 cells transiently transfected with DEFCAP-L, DEFCAP-S, LRR/CARD-L, and LRR/CARD-S exhibited no significant killing activity (data not shown).
The mutational studies also suggest that DEFCAP sequences including the
NBD may act in the negative regulation of DEFCAP-L since a deletion of
the PLM, PR, and NBD (LRR/CARD-L) resulted in a dramatic increase in
apoptosis levels when compared with the full-length DEFCAP-L construct.
Interestingly, the LRR/CARD for the short isoform (LRR/CARD-S) was also
capable of inducing apoptosis but at much lower levels than LRR/CARD-L.
Both LRR/CARD-L and LRR/CARD-S showed comparable levels of protein
expression in transfected cells (data not shown) suggesting that these
differences in killing activity between LRR/CARD-L and -S were not due
to differences in protein expression. Furthermore, these results suggest that, like Apaf-1, a deletion construct containing the CARD and
the domain juxtaposed next to the CARD can act as a constitutively active inducer of cell death. However, the major difference between the
constitutively active Apaf-1 and that of DEFCAP is that the former is
composed of a CARD/NBD and the latter is composed of an LRR/CARD.
Although at present it is not clear which apoptotic signaling pathway
involves DEFCAP, this study demonstrated that DEFCAP LRR/CARD
constructs when transiently overexpressed were able to kill cells
effectively. Future investigations into the mechanism of this killing
may prove useful to understanding the role of this distinct mammalian
Ced-4 molecule.
-galactosidase as a reporter gene
in MCF7 cells suggest the following: 1) the nucleotide binding domain
may act as a negative regulator of the killing activity of DEFCAP; 2)
the LRR/CARD represents a putative constitutively active inducer of
apoptosis; 3) the killing activity of LRR/CARD is inhibitable by
benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone and to a lesser
extent by Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone; and 4) the CARD is
critical for killing activity of DEFCAP. These results suggest that
DEFCAP is a novel member of the mammalian Ced-4 family of proteins
capable of inducing apoptosis, and understanding its regulation may
elucidate the complex nature of the mammalian apoptosis-promoting machinery.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-converting enzyme was identified as the first
mammalian Ced-3-like protein, which led to the identification of a
family of proteases named caspases (cysteine protease with cleavage
specificity after an aspartate residue). Caspases exist in the cell as
inactive zymogens or procaspase molecules that with the proper stimuli
(e.g. death receptor ligation, DNA damage, and
chemotherapeutic agents) can be recruited via their caspase recruitment
domain (CARD)1 to form homo-
and heterodimers. Following their recruitment, caspases can either
autocatalytically or via another caspase become processed into two
catalytic subunits and subsequently converted into active enzymes that
are capable of initiating the execution arm of the apoptotic signaling
pathway. To date, at least 14 members of the caspase family have been
identified. Proapoptotic caspases can be classified into two classes
based on their primary sequence. Upstream regulatory caspases include
caspases-2, -8, -9, and -10 and contain long NH2-terminal
prodomains that are important in regulating their recruitment and
subcellular localization. Downstream effector caspases such as
caspase-3, caspase-6, and caspase-7 contain short or absent prodomains
and upon activation are responsible for the cleavage of many cellular components.
B
pathway via an interaction with RICK, a CARD-containing kinase.
Furthermore, Inohara et al. (6) demonstrate that Nod1 is
capable of interacting with caspase-9 and enhancing caspase-9-mediated apoptosis. Nod1/RICK-induced NF-
B activation and
Nod1/caspase-9-mediated apoptosis are believed to occur via independent
pathways based on the results that an active site caspase-9 mutant was
not able to inhibit Nod1-mediated NF-
B activation (13).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-32P]dCTP, hybridized, and washed
according to the protocol described previously (15) and exposed to
autoradiography film for 5 days.
-Actin cDNA was used as a
control for equal loading of RNA. Random primed reverse transcription
for semi-quantitative RT-PCR was performed using Superscript II reverse
transcriptase (Life Technologies, Inc.) according to the
manufacturer's protocol. RNAs from normal human liver, spleen,
polymorphonuclear cells (PMNs), peripheral blood mononuclear cells
(PBMCs), K562 and Jurkat cancer cell lines, and a negative control
without template were reversed-transcribed. RNAs from K562 and Jurkat
cancer cell lines were isolated using the Trizol reagent (Life
Technologies, Inc.)and PMN and PBMC RNAs were isolated by a
Ficoll-Paque (Amersham Pharmacia Biotech) gradient, following dextran
sedimentation and hypotonic red blood cell lysis. 1 µl of the RT
reaction was used for PCR with the following oligonucleotides: 5'-CGAGAACAGCTGGTCTTCTCCAGGGCTTCG (antisense) and
5'-TCCCCCTTGGGAGTCCTCCTGAAAATGATC (sense) under the following
conditions: 1 cycle at 94 °C for 5 min, 30 cycles at 94 °C for
30 s, 65 °C for 30 s, 72 °C for 30 s, and 1 cycle
at 72 °C for 5 min.
-Actin oligonucleotides
5'-CGAGAAGATGACCCAGATCATGTTTGAGAC (sense) and 5'-TGGAAGCAGC
CGTGGCCATCTCTTGCTCGA (antisense) were used as a control for RNA
integrity and RT efficiency. PCR products were separated by agarose gel
electrophoresis, stained with ethidium bromide, and photographed under
UV light.
CARD-L (a.a. 1-1355),
CARD-S (a.a. 1-1311),
PR-L (a.a. 309-1473),
PR-S (a.a. 309-1439),
LRR/CARD-L (a.a. 696-1473), LRR/CARD-S (a.a. 696-1439), LRR-L (a.a.
696-1355), LRR-S (a.a. 696-1311), PR/NBD (a.a. 1-648), PR (a.a.
1-308), and DEFCAP-CARD (a.a. 1356-1473). Caspase-2 (Ich1-L) (16),
caspase-3 (Yama, CPP32) (17), caspase-8 (FLICE) (18), caspase-9
(interleukin 1
-converting enzyme-LAP-6, Apaf-3), caspase-9 (C287A)
(19), and caspase-10 (FLICE2) (20) constructs were obtained or created as described elsewhere.
20 °C
for Western analysis. The supernatant was precleared with protein G
beads (Sigma) overnight at 4 °C with gentle rotation. The following
day, the protein G beads were spun down at 2000 × g,
and the supernatant was transferred to a new tube. 35 µl of
precleared supernatant was analyzed for Western analysis, and the
remaining supernatant was used for immunoprecipitations conducted with
gentle rotation at 4 °C with the appropriate antibody for 3 h.
DEFCAP constructs were immunoprecipitated with 10 µl of Myc-AC.
Following incubation, the immunoprecipitations were washed 4 times with
PBS on ice, resuspended in 1.5× SDS-polyacrylamide gel electrophoresis
sample buffer, boiled, and analyzed by Western blotting.
-galactosidase and 1 µg of either pcDNA3.1 alone or an
NH2-terminal Myc-tagged DEFCAP construct. 24 h
post-transfection, the cells were fixed in 0.5% glutaraldehyde and
stained with 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-gal) for 4 h. The cells
were visualized by phase contrast microscopy, and the percentage of
apoptotic cells was determined by counting at least 600 blue cells
(n
3). Round blue cells and/or blue cells exhibiting
plasma membrane blebbing and cell shrinkage were scored apoptotic. The
data presented were from at least two independent experiments conducted
in duplicate or triplicate. A Student's t test using the
computer program SigmaStat (Jandel) comparing vector control with
various DEFCAP constructs was performed to obtain p values.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
View larger version (87K):
[in a new window]
Fig. 1.
Deduced amino acid sequence of human
DEFCAP-L and amino acid alignment of human DEFCAP and its related
proteins. A, the deduced DEFCAP-L open reading frame
encodes a protein of 1473 amino acids. PLM is indicated with
reverse-highlighting in gray. Nine PR motifs
(PXXP) are underlined in black. The
consensus sequence of the Walker's A box (P-loop), B box, and NBD
conserved sequence III with unassigned function are indicated with
red highlighting. Asterisks below
indicate conserved amino acids. K/D LRRs are reverse-highlighted
in blue and non-K/D LRRs are boxed in blue.
The 44 amino acids unique to the DEFCAP-L isoform are underlined
in blue. The CARD is indicated with green
highlighting. B, DEFCAP PLM sequence alignment with
pyrin, ASC1, and ASC. C, a comparison of the putative NBD of
DEFCAP with the NBD of Nod1, Apaf-1, DARK, and CED4. A box, B box, and
motif III are boxed in red. Asterisks denote
highly conserved residues. D, DEFCAP-L LRR (LRRs 1-12)
amino acid alignment. The putative -sheet,
-turn, and
-helix
are labeled on top according to the three-dimensional
structure of the porcine ribonuclease inhibitor. E,
alignment of CARDs for DEFCAP, ASC, Nod1, Apaf-1, Ced-3, and caspase-9.
The residues identical and similar to those of DEFCAP are shown by
reverse and dark highlighting, respectively. The
putative
-helices, H1-H6, are shown according to the
three-dimensional structure of the CARD of Apaf-1 and caspase-9.
F, the domain structures of DEFCAP-L, Nod1, Apaf-1
xL, and Ced-4.
-BLAST search of the nonredundant data
base using amino acids 279-608 of DEFCAP identified the NBD of Nod1
and the mouse gene Mater,
maternal-antigen-that
embryos-require (sequence not shown). A
sequence alignment of the NBDs for all of the Ced-4 family members
suggests that DEFCAP is most homologous to Nod1 with 29% identity. No
significant similarities between the NBD of DEFCAP and Apaf-1, DARK,
and Ced-4 were found when using the NCBI BLAST 2 Sequences tool for
protein sequence alignment.
XA (X
indicates any residue, and A indicates residue with an
aliphatic side chain) according to Kobe and Deisenhofer (K/D LRR)
(reverse-highlighted in blue, Fig. 1A) (23). LRRs 8, 10, and 11 (boxed in blue, Fig. 1A)
represent three non-Kobe and Deisenhofer (non-K/D) LRRs with the
consensus sequence XAXXAXAX(N/C/T/Q)+/
XA. DEFCAP LRRs 2, 4, and 6 are prototypical ribonuclease inhibitor type B
(RI type B) repeats, whereas LRRs 3 and 5 share similarity to the
ribonuclease inhibitor type A (RI type A) repeats. The alternating
nature of the asparagine and cysteine, also known as the
asparagine-cysteine ladder, in residues at position 10 of LRRs 2-6 are
similar to other proteins with multiple internal LRR repeats.
With the exception of leucine at position 20, the putative
-helical sequences of the LRRs do not share significant homology, a
characteristic found in many LRRs. However, the RI type B LRRs 2, 4, and 6 share significant homology among each other as depicted in
purple (Fig. 1D).
-BLAST search of the nonredundant data base
using the CARD of DEFCAP identifies 56% identity with ASC and 29%
identity with Nod1. Less similarity is seen in an alignment comparing
DEFCAP's CARD with the CARD of Apaf-1, Ced-3, and caspase-9.
-actin cDNA. Northern analysis for DEFCAP in cancer cell
lines revealed a high level of expression in the chronic myelogenous
leukemia cell line K562. A weak ~7.0-kilobase pair band was seen in
the Burkitt's lymphoma Raji, colorectal adenocarcinoma SW-480, and
melanoma G-361 cell lines. No significant DEFCAP transcripts were
detected in the promyelocytic leukemia HL-60, cervical carcinoma HeLa
S3, lymphoblastic leukemia MOLT-4, or lung carcinoma A549.
View larger version (45K):
[in a new window]
Fig. 2.
Human DEFCAP mRNA expression by Northern
blot analysis and semi-quantitative RT-PCR. A, Northern
blot analysis of DEFCAP expression in human adult tissues and in cancer
cell lines with -actin mRNA levels shown below.
B, semi-quantitative RT-PCR of DEFCAP-L and DEFCAP-S
mRNA expression in cancer cell lines K562 and Jurkat and in normal
liver, spleen, PMNs, PBMCs, and negative control with
-actin levels
shown below.
-Actin mRNA
levels served as a control for RNA integrity and RT-PCR efficiency and
were relatively constant with the exception of the K562 RNA which is
slightly diminished. The weak band at ~590 bp seen in the K562,
spleen, PMN, and PBMC lanes may represent a PCR artifact. This band was
gel-purified from spleen, PMN, and PBMC RT-PCR samples, subjected to
PCR with the same oligonucleotides used in the RT-PCR, and did not
yield a ~590-bp PCR product.
View larger version (88K):
[in a new window]
Fig. 3.
EGFP-DEFCAP-CARD forms death-effector
filament-like structures when overexpressed in MCF7 cells. Phase
contrast microscopy of MCF7 cells transiently transfected with pEGFP
vector control and GFP-DEFCAP-CARD are shown in A and
C, respectively, with red arrowheads pointing to
transfected cells. The same fields for pEGFP vector control and
GFP-DEFCAP-CARD viewed by fluorescence microscopy are shown in
B and D. A black bar represents a
15-µm scale.
View larger version (52K):
[in a new window]
Fig. 4.
DEFCAP interacts with caspase-2 and weakly
with caspase-9. 293 cells were transiently transfected as
described under "Materials and Methods." Supernatants
(S), pellets (P), and coimmunoprecipitated
proteins (IP) were analyzed by Western analysis with
caspase-2- (A) or caspase-9 (B)-specific
antibodies. The pellet lane was included to show that caspase-2,
caspase-9, and both DEFCAP-L and -S are found in the membrane-insoluble
fraction. A, arrows at ~48, ~37, and ~33
kDa represent the caspase-2 proenzyme and two processed forms of the
enzyme, respectively. IgG heavy and light chains are depicted with
arrows at 55 and 18 kDa, respectively. B, a weak
DEFCAP interaction with caspase-9 is seen by a band at ~35 kDa
representing the processed caspase-9 protein. A and
B, DEFCAP expression was determined by Western analysis with
Myc-horseradish peroxidase antibodies shown at the
bottom of each panel.
PLM/PR-L) resulted in 44% (n = 3, S.E. = 1.649) apoptosis, a slight increase in apoptosis versus
the wild-type full-length DEFCAP-L construct. A
PLM/PR-S construct
showed no significant killing activity, 26% (n = 3, S.E. = 3.936) versus vector control or full-length DEFCAP-S.
A deletion construct containing only the leucine-rich repeats and the
CARD (LRR/CARD-L and-S) exhibited a significant level of killing
activity at 63% (n = 3, S.E. = 0.627) for LRR/CARD-L
and 41% (n = 4, S.E. = 1.54) for LRR/CARD-S. These
results suggest that the LRR/CARD for both long and short isoforms may
act as a constitutively active proapoptotic form of DEFCAP. In the
presence of 25 µM ZVAD-fmk, the pan-caspase inhibitor,
the killing activity of LRR/CARD-L and LRR/CARD-S were dramatically
reduced to 21 (n = 4, S.E. = 1.319) and 18%
(n = 4, S.E. = 1.624), respectively (data not shown),
suggesting that the mechanism of killing is most likely
caspase-dependent. Base-line apoptosis with pcDNA3.1
alone in the presence of ZVAD-fmk was 11% (n = 3, S.E. = 0.284) (data not shown). In the presence of DEVD-fmk, the killing
activity of LRR/CARD-L and -S was slightly reduced to 40 and 34%,
respectively (data not shown). These results suggest that the
activation of both a caspase with DEVD specificity and one with a
non-DEVD specificity are required for maximal apoptotic activity by the
LRR/CARD of DEFCAP.
View larger version (29K):
[in a new window]
Fig. 5.
Mutational analysis of DEFCAP-L and
DEFCAP-S in -galactosidase
death assays in MCF7 cells. A,
NH2-terminal Myc-tagged full-length and mutant expression
constructs. PLM, PR, NBD, LRRs, and CARD are indicated by gray,
black, red, blue, and green boxes, respectively. The
additional LRR (LRR12) found in DEFCAP-L is shown within a yellow
box representing the 44-a.a. insertion. B, phase
contrast microscopy of MCF7 cells transiently transfected with
CMV-
-galactosidase and pcDNA3.1 (a) or LRR/CARD-L
(b). Black arrowheads depict live transfected
cells, and red arrowheads depict apoptotic cells.
C, killing activity of the various DEFCAP deletion
constructs. A dashed line represents the basal level of
killing as determined by transfection with pcDNA3.1 alone.
DR3-transfected cells represent a positive control for apoptosis.
CARD-L and
CARD-S) were unable to induce apoptosis and exhibited killing activities of 25 (n = 3, S.E. = 1.737) and 23% (n = 3, S.E. = 1.085), respectively.
These results suggest that the CARD is critical for the ability of
DEFCAP-L to kill. This conclusion is further supported by a comparison
of the LRR/CARD-L and -S constructs with the LRR-L and-S only
constructs, which gave 22 (n = 3, S.E. = 0.778) and
17% (n = 3, S.E. = 3.118), respectively.
![]() |
FOOTNOTES |
---|
* 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) NP_055737.
To whom correspondence should be addressed: Dept. of
Pathology, University of Michigan Medical School, 1301 Catherine Rd., Ann Arbor, MI 48109-0602. Tel.: 734-763-6384; Fax: 734-763-4782; E-mail: pward@umich.edu.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M009853200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CARD, caspase recruitment domain; NBD, nucleotide binding domain; LRR, leucine-rich repeat; PR, proline-rich sequences, PLM; pyrin-like motif, EGFP, enhanced green fluorescent protein; DR3, Death Receptor 3; ZVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone; DEVD-fmk, Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone; RT-PCR, reverse transcriptase-polymerase chain reaction; bp, base pair; a.a., amino acids; PBS, phosphate-buffered saline; PMN, polymorphonuclear; PBMCs, peripheral blood mononuclear cells; IP, immunoprecipitation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Kerr, J. F., Wyllie, A. H., and Currie, A. R. (1972) Br. J. Cancer 26, 239-257[Medline] [Order article via Infotrieve] |
2. | Thompson, C. B. (1995) Science 267, 1456-1462[Medline] [Order article via Infotrieve] |
3. | Hengartner, M. O., and Horvitz, H. R. (1994) Philos. Trans. R. Soc. Lond-B Biol. Sci. 345, 243-246[Medline] [Order article via Infotrieve] |
4. |
Yuan, J.,
and Horvitz, H. R.
(1992)
Development
116,
309-320 |
5. | Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405-413[Medline] [Order article via Infotrieve] |
6. |
Inohara, N.,
Koseki, T.,
del Peso, L.,
Hu, Y.,
Yee, C.,
Chen, S.,
Carrio, R.,
Merino, J.,
Liu, D.,
Ni, J.,
and Nunez, G.
(1999)
J. Biol. Chem.
274,
14560-14567 |
7. |
Bertin, J.,
Nir, W. J.,
Fischer, C. M.,
Tayber, O. V.,
Errada, P. R.,
Grant, J. R.,
Keilty, J. J.,
Gosselin, M. L.,
Robison, K. E.,
Wong, G. H.,
Glucksmann, M. A.,
and DiStefano, P. S.
(1999)
J. Biol. Chem.
274,
12955-12958 |
8. | Kanuka, H., Sawamoto, K., Inohara, N., Matsuno, K., Okano, H., and Miura, M. (1999) Mol. Cell 4, 757-769[Medline] [Order article via Infotrieve] |
9. | Rodriguez, A., Oliver, H., Zou, H., Chen, P., Wang, X., and Abrams, J. M. (1999) Nat. Cell Biol. 1, 272-279[CrossRef][Medline] [Order article via Infotrieve] |
10. | Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997) Cell 91, 479-489[Medline] [Order article via Infotrieve] |
11. |
Zou, H.,
Li, Y.,
Liu, X.,
and Wang, X.
(1999)
J. Biol. Chem.
274,
11549-11556 |
12. |
Cain, K.,
Bratton, S. B.,
Langlais, C.,
Walker, G.,
Brown, D. G.,
Sun, X. M.,
and Cohen, G. M.
(2000)
J. Biol. Chem.
275,
6067-6070 |
13. |
Inohara, N.,
Koseki, T.,
Lin, J.,
del Peso, L.,
Lucas, P. C.,
Chen, F. F.,
Ogura, Y.,
and Nunez, G.
(2000)
J. Biol. Chem.
275,
27823-27831 |
14. |
Siegel, R. M.,
Martin, D. A.,
Zheng, L.,
Ng, S. Y.,
Bertin, J.,
Cohen, J.,
and Lenardo, M. J.
(1998)
J. Cell Biol.
141,
1243-1253 |
15. | Church, G. M., and Gilbert, W. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1991-1995[Abstract] |
16. |
Duan, H.,
Chinnaiyan, A. M.,
Hudson, P. L.,
Wing, J. P.,
He, W. W.,
and Dixit, V. M.
(1996)
J. Biol. Chem.
271,
1621-1625 |
17. | Tewari, M., Quan, L. T., O'Rourke, K., Desnoyers, S., Zeng, Z., Beidler, D. R., Poirier, G. G., Salvesen, G. S., and Dixit, V. M. (1995) Cell 81, 801-809[Medline] [Order article via Infotrieve] |
18. | Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996) Cell 85, 817-827[Medline] [Order article via Infotrieve] |
19. |
Duan, H.,
Orth, K.,
Chinnaiyan, A. M.,
Poirier, G. G.,
Froelich, C. J.,
He, W. W.,
and Dixit, V. M.
(1996)
J. Biol. Chem.
271,
16720-16724 |
20. |
Vincenz, C.,
and Dixit, V. M.
(1997)
J. Biol. Chem.
272,
6578-6583 |
21. | Nagase, T., Ishikawa, K., Suyama, M., Kikuno, R., Hirosawa, M., Miyajima, N., Tanaka, A., Kotani, H., Nomura, N., and Ohara, O. (1999) DNA Res. 6, 63-70[Medline] [Order article via Infotrieve] |
22. |
Masumoto, J.,
Taniguchi, S.,
Ayukawa, K.,
Sarvotham, H.,
Kishino, T.,
Niikawa, N.,
Hidaka, E.,
Katsuyama, T.,
Higuchi, T.,
and Sagara, J.
(1999)
J. Biol. Chem.
274,
33835-33838 |
23. | Kobe, B., and Deisenhofer, J. (1994) Trends Biochem. Sci. 19, 415-421[CrossRef][Medline] [Order article via Infotrieve] |
24. | Shearwin-Whyatt, L. M., Harvey, N. L., and Kumar, S. (2000) Cell Death Differ. 7, 155-165[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Mancini, M.,
Machamer, C. E.,
Roy, S.,
Nicholson, D. W.,
Thornberry, N. A.,
Casciola-Rosen, L. A.,
and Rosen, A.
(2000)
J. Cell Biol.
149,
603-612 |
26. |
Susin, S. A.,
Lorenzo, H. K.,
Zamzami, N.,
Marzo, I.,
Brenner, C.,
Larochette, N.,
Prevost, M. C.,
Alzari, P. M.,
and Kroemer, G.
(1999)
J. Exp. Med.
189,
381-394 |
27. |
Li, H.,
Bergeron, L.,
Cryns, V.,
Pasternack, M. S.,
Zhu, H.,
Shi, L.,
Greenberg, A.,
and Yuan, J.
(1997)
J. Biol. Chem.
272,
21010-21017 |
28. |
Bergeron, L.,
Perez, G. I.,
Macdonald, G.,
Shi, L.,
Sun, Y.,
Jurisicova, A.,
Varmuza, S.,
Latham, K. E.,
Flaws, J. A.,
Salter, J. C.,
Hara, H.,
Moskowitz, M. A.,
Li, E.,
Greenberg, A.,
Tilly, J. L.,
and Yuan, J.
(1998)
Genes Dev.
12,
1304-1314 |
29. |
Chen, F.,
Hersh, B. M.,
Conradt, B.,
Zhou, Z.,
Riemer, D.,
Gruenbaum, Y.,
and Horvitz, H. R.
(2000)
Science
287,
1485-1489 |