From Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts 02139
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ABSTRACT |
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The nematode CED-4 protein and its human homolog
Apaf-1 play a central role in apoptosis by functioning as direct
activators of death-inducing caspases. A novel human CED-4/Apaf-1
family member called CARD4 was identified that has a domain structure strikingly similar to the cytoplasmic, receptor-like proteins that
mediate disease resistance in plants. CARD4 interacted with the
serine-threonine kinase RICK and potently induced NF- Apoptosis, or programmed cell death, is an evolutionarily
conserved process of cell suicide critical for normal development and
elimination of pathogen-infected cells (1). Genetic studies in the
nematode Caenorhabditis elegans have identified components of the death pathway that show similarity to large families of mammalian proteins involved in apoptosis (2). Surprisingly, Apaf-1 has
been the only CED-4-like cell death protein identified thus far in
humans (3). Like CED-4, Apaf-1 contains a caspase recruitment domain
(CARD)1 as its N-terminal
effector domain and a centrally located nucleotide-binding site (NBS)
domain. The C-terminal region of Apaf-1 lacks homology with CED-4 and
is composed of a long WD-40 repeat domain that regulates Apaf-1
activation. Binding of Apaf-1 to cytochrome c in the
presence of dATP exposes its N-terminal effector domain, which then
mediates a homophillic CARD-CARD interaction with the prodomain of
caspase-9, resulting in caspase oligomerization and activation (4, 5).
The presence of CARD motifs in a variety of other effector and
signaling molecules, including numerous caspases (6) and a recently
identified NF- Yeast Two-hybrid Assay--
The bait vector was constructed by
cloning the CARD-containing N-terminal region of CARD4 (residues
1-145) into yeast vector pGBT9. The resulting plasmid,
pGBT9-CARD4-CARD, was used to screen a human breast library (Millennium
Pharmaceuticals, Inc.) according to the Matchmaker Two-hybrid System
Protocol (CLONTECH). Clones positive for
his and lacZ expression were isolated and
sequenced to determine identity.
Expression Vectors--
HA-CARD4, HA-CARD4-518 (residues
1-518), HA-CARD4-CARD (residues 1-126), HA-CARD4-NBS (residues
127-518), Myc-TRAF6-DN (residues 289-530), Myc-NIK-DN (residues
623-947), Myc-RICK, Myc-RICK-DN (residues 406-541), Myc-RICK/No CARD
(residues 1-435), and Myc-Bcl-XL were amplified by polymerase chain
reaction using oligonucleotides encoding either HA or Myc epitopes and
cloned into the pCI expression plasmid (Promega). The expression
plasmid encoding Apaf-1 was described previously (3).
Luciferase Reporter Assays--
293T cells were plated in 6-well
plates (35-mm wells) and transfected 2 days later (90% confluency)
with 1 µg of NF- Co-immunoprecipitation Assay--
293T cells were plated in
10-cm dishes and transfected the following day (60% confluency) with 4 µg of each expression plasmid using LipofectAMINE (Life Technologies,
Inc.). Cells were harvested 48 h after transfection, lysed in 0.6 ml of buffer (50 mM Tris, pH 8.0, 5 mM EDTA,
120 mM NaCl, 0.5% Nonidet P-40), and incubated with either
anti-HA or anti-Myc monoclonal antibodies (Berkeley Antibody Co.).
Immune complexes were then precipitated with protein A-Sepharose beads
(Amersham Pharmacia Biotech), subjected to 12% SDS-polyacrylamide gel
electrophoresis and immunoblotted with polyclonal antibodies.
Apoptosis Assay--
293T cells were plated in 6-well plates
(35-mm wells) and transfected 2 days later (90% confluency) with 400 ng of pCMV To identify novel human members of the CED-4/Apaf-1 family, we
searched Millennium Pharmaceuticals' proprietary data base of
expressed sequence tags (EST) for clones having sequence similarity to
CARD motifs (6). A CARD-encoding EST was identified and used to screen
a human umbilical vein endothelial cell cDNA library for
full-length clones. A single cDNA of approximately 3.5 kilobases contained an open reading frame encoding a protein of 953 amino acids
with a predicted molecular mass of 108 kDa (Fig.
1A). This molecule was designated
CARD4 because it was one of several CARD-containing proteins identified
from the EST search. A BLAST search of protein data bases indicated
that CARD4 was a new protein with at least three putative functional
domains (Fig. 1, A and C). The N-terminal region
of CARD4 (residues 14-104) shares significant sequence similarity with
CARD motifs found in a variety of apoptotic signaling molecules,
including those found in CED-4 and Apaf-1 (Fig. 1B). Contained within the central region of CARD4 (residues 199-398) is a
NBS-like domain that shares approximately 33% sequence identity with
the putative GTP-binding domain (residues 416-618) of CIITA, a
transactivator of major histocompatibility complex class II genes (12).
Within this region of homology are several NBS consensus sequences,
including kinase 1a (P-loop), 2, and 3a motifs that are also found
within the NBS of CED-4 and Apaf-1 (13). The C-terminal region of CARD4
(residues 674-950) encodes at least 10 tandem leucine-rich repeats
(LRR), a protein interaction motif found in a variety of proteins with
diverse functions, including signal transduction (14). The 28-amino
acid consensus sequence derived from the aligned LRRs of CARD4 belongs
to the ribonuclease inhibitor-like subfamily of LRRs that are found
exclusively in animal intracellular proteins. The CARD/NBS/LRR domain
structure of CARD4 justifies its inclusion as a new member of the
CED-4/Apaf-1 family of proteins (Fig. 1C). CARD4 is also
structurally similar to members of the NBS/LRR class of plant disease
resistance gene products whose N-terminal effector domains contain
either a leucine zipper motif or a Toll/interleukin-1 receptor homology
domain (15). Northern blot analysis revealed CARD4 to be constitutively expressed as a 4.5-kilobase transcript with abundant expression in
adult heart, spleen, and lung tissues (Fig. 1D). CARD4 was also found to be expressed in a variety of cancer cell lines and fetal
tissues (data not shown).
B activity through TRAF-6 and NIK signaling molecules. In addition, coexpression of CARD4 augmented caspase-9-induced apoptosis. Thus, CARD4 coordinates downstream NF-
B and apoptotic signaling pathways and may be a component of the host innate immune response.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B-activating kinase (7-9), suggests that other
CED-4/Apaf-1 family members likely exist in humans to coordinate
downstream stress responses. We report here the identity and
characterization of a novel human CED-4/Apaf-1 family member that
activates NF-
B and apoptotic signaling pathways.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B luciferase reporter plasmid (pNF
B-Luc,
Stratagene), 200 ng of pCMV
-gal, 600 ng of pCI vector, and 200 ng
of indicated expression plasmids using SuperFect transfection reagent
(Qiagen). For dominant-negative experiments, 2 ng of CARD4 expressing
plasmid and 800 ng of dominant-negative plasmid were used. Cells were
harvested 48 h after transfection, and luciferase activity in
1000-fold diluted cell extracts was determined using the Luciferase
Assay System (Promega). In addition,
-galactosidase activities were
determined and used to normalize transfection efficiency.
-gal vector (Stratagene) and 800 ng of each expression
plasmid using SuperFect transfection reagent (Qiagen). At 48 h
cells were fixed and stained for
-galactosidase expression. Cell
viability was determined by counting the number of flat, blue-staining cells.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Sequence and expression of CARD4.
A, amino acid sequence of CARD4. N-terminal CARD motif is
indicated by bold lettering. NBS kinase 1a, 2, and 3a
domains and the C-terminal LRRs (LRR1-LRR10) are underlined.
B, amino acid sequence alignment of the CARD of CARD4 with
CARDs found in RICK, C. elegans CED-4 and Apaf-1.
Black shading indicates identical residues. C,
domain structure of CARD4 compared with C. elegans CED-4,
human Apaf-1, and plant disease resistance proteins: Tobacco N protein
(10) and Arabidopsis RPS2 protein (11). TIR,
toll/interleukin-1 receptor homology domain; LZ, leucine
zipper motif. D, CARD4 expression in various adult human
tissues (CLONTECH) by Northern blot analysis.
PBL, peripheral blood lymphocytes.
By analogy to CED-4 and Apaf-1 proteins, the N-terminal CARD motif of
CARD4 likely functions as an effector domain that mediates specific
homophilic interactions with downstream CARD-containing signaling
molecules. To gain insight into signaling pathways engaged by CARD4, we
used its N-terminal CARD (residues 1-145) as bait in a yeast
two-hybrid screen of a human breast cDNA library to identify
CARD-containing interactors. From approximately 8 million transformants, 12 positive clones showing activation of his
and lacZ reporter genes were identified. Of these 12 interactors, six were partial-length cDNAs of RICK (RIP2, CARDIAK)
(7-9), a recently identified serine-threonine kinase that contains a C-terminal CARD motif (Fig. 2A).
In similar screens of human prostate and brain cDNA libraries,
approximately 30-40% of all positive clones were found to be RICK
(data not shown). Interestingly, the CARD of CARD4 shows the most
similarity (26% identity) with the corresponding domain in RICK when
compared with the family of known CARD motifs (Fig. 1B).
This interaction was mediated by the CARD of RICK because this domain
alone (residues 435-540) was sufficient for binding to the bait (data
not shown). Furthermore, immunoprecipitation of epitope-tagged RICK
expressed in mammalian cells quantitatively coprecipitated CARD4 (Fig.
2B). This association was mediated specifically by the CARD
motif of RICK, because CARD4 did not coprecipitate with a truncated
form of RICK lacking its CARD (RICK/No CARD). Taken together, these
results suggest that CARD4 selectively interacts with RICK through a
homophilic CARD-CARD interaction.
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Recent studies have shown that RICK activates NF-B signaling
pathways (8, 9). Because the putative N-terminal effector domain of
CARD4 was found to interact with the CARD of RICK, we determined
whether CARD4 can activate NF-
B signaling using a luciferase
reporter gene directed by an NF-
B-responsive promoter. Expression of
CARD4 in 293T cells potently induced NF-
B activity in a
concentration-dependent manner; maximum induction of
luciferase activity was 40-fold compared with control vector (Fig.
3A). Induction of NF-
B activity
was not a general activity of CED-4/Apaf-1 family members because
expression of Apaf-1 failed to activate this signaling pathway (Fig.
3B). Although RICK also activates the Jun N-terminal kinase
signaling pathway (Ref. 9 and data not shown), CARD4 expression in 293T
cells failed to either induce phosphorylation of Jun N-terminal kinase
or activate a luciferase reporter gene with AP-1 promoter elements
(data not shown). We next determined the domains of CARD4 that mediate
the induction of NF-
B activity (Fig. 3B). A C-terminal
truncated mutant (CARD4-518) activated NF-
B at levels comparable
with full-length CARD4, demonstrating that the LRR domain was not
required for induction. The N-terminal CARD (CARD4-CARD), but not the
NBS domain (CARD4-NBS), was found subsequently to be sufficient for
induction, establishing the CARD motif as the NF-
B-activating domain
of CARD4 (Fig. 3B). Induction of NF-
B activity by RICK is
mediated in part by TRAF-6 and NIK kinase (8). Dominant-negative
versions of these molecules (TRAF6-DN and NIK-DN) were also found to
inhibit induction when coexpressed with CARD4, suggesting that TRAF-6
and NIK act downstream of CARD4 to activate NF-
B (Fig.
3B). Conversely, coexpression of the antiapoptotic protein
Bcl-XL had no effect on NF-
B induction. Coexpression of the CARD of
RICK (RICK-DN) also functioned as a dominant-negative mutant,
consistent with this molecule being a potential downstream mediator of
CARD4 function.
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Besides having a role in NF-B signaling, RICK also affects apoptotic
signaling pathways by an unknown mechanism, because it increases
apoptotic death induced by caspase-8 and caspase-10 and augments the
processing of pro-caspase-1 (7, 9). Confirming the role of RICK as a
general activator of caspases, we found that RICK coexpression also
increases caspase-9 apoptotic activity (see below), even though RICK
does not interact with caspase-9 (7, 9). We therefore determined
whether CARD4 coexpression increases apoptotic death induced by
caspase-9. 293T cells expressing caspase-9 underwent apoptosis with
rounding-up, membrane blebbing, and lifting off from the plate, thereby
reducing the number of viable cells (Fig.
4A). In contrast, expression of
CARD4, RICK, or Apaf-1 had no effect on cell viability when expressed
alone. However, CARD4 coexpression increased the apoptotic death
induced by caspase-9 at levels comparable with those observed with
either RICK or Apaf-1 (Fig. 4B). This activity was mapped to
the N-terminal CARD motif of CARD4 because coexpression of this domain
alone was sufficient to increase apoptotic death induced by caspase-9 (Fig. 4B). Interestingly, CARD4 coexpression failed to
increase apoptotic death induced by caspase-8, suggesting that the
enhancing activity was specific for caspase-9-mediated apoptosis (data
not shown).
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We have shown that CARD4 is structurally related to the CED-4 and
Apaf-1 cell death proteins and activates NF-B and apoptotic activity
through interactions with its N-terminal CARD. Our finding that CARD4
interacts with RICK through a homophilic CARD-CARD interaction
implicates the serine-threonine kinase as a potential downstream
mediator of CARD4 signaling. It is possible, however, that other
CARD-containing molecules are responsible for mediating CARD4 function
in 293T cells. Although a role for the C-terminal LRRs remains
uncertain, it is likely that this domain functions in a manner
analogous to the Apaf-1 WD-40 domain to mediate CARD4 activation by
upstream signaling molecules. Our finding that CARD4 also displays
structural similarity to the NBS/LRR class of plant disease resistance
gene products is intriguing. NBS/LRR-containing plant proteins initiate
a complex defense response to pathogen infection, including changes in
gene expression and localized cell death (15). Interestingly, tomato
resistance response to bacterial speck disease involves both an NBS/LRR
protein and a serine-threonine kinase. Recent studies suggest that
plant defense mechanisms activated in response to pathogen infection
are analogous to the innate immune response of vertebrates and insects
(16). CARD4 may therefore be a component of the innate immune response that transduces upstream stress or pathogen signals to the activation of NF-
B and apoptotic signaling pathways.
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ACKNOWLEDGEMENTS |
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We thank E. Alnemri for caspase-9 vector, X. Wang for Apaf-1 vector, T. Libermann and Y. Akbarali for luminometer help, and M. Jacobson, N. Roy, L. Chiang, R. Curtis, and R. Breitbart for comments and discussion.
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FOOTNOTES |
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* 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) AF126484.
To whom correspondence should be addressed: Millennium
Pharmaceuticals, Inc., 640 Memorial Dr., Cambridge, MA 02139. Tel.: 617-679-7215; Fax: 617-374-9379; E-mail: bertin{at}mpi.com.
§ These authors contributed equally to this work.
¶ Present address: Astra Arcus USA, Inc., Three Biotech, One Innovation Dr., Worcester, MA 01605.
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ABBREVIATIONS |
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The abbreviations used are:
CARD, caspase
recruitment domain;
NBS, nucleotide-binding site;
EST, expressed
sequence tag;
LRR, leucine-rich repeat;
HA, hemagglutinin;
-gal,
-galactosidase.
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