From the Center for Apoptosis Research and the Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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Molecules that regulate NF- The transcription factor NF- The best characterized active NF- The proinflammatory cytokines TNF- Signals are relayed from the non-enzymatic components of the TNF-R1 or
IL-1R complexes to the enzymatic effector components via homotypic and
heterotypic interactions between domains present in these components.
The death domain for example, which is present in TNF-R1, TRADD, and
RIP, mediates interactions necessary for the assembly of the
NF- Because of the importance of CARD-containing proteins as regulators of
apoptosis, we decided to search the public data base for novel
sequences that contain homologous CARD domains. Here we report the
identification of a CARD-containing viral protein and its mammalian
homolog, and we provide evidence that these proteins, named CLAPs, are
involved in regulation of NF- cDNA Cloning--
The full-length open reading frame of E10
(vCLAP) within the genome of the equine herpesvirus-2 (EHV-2)
(GenBankTM accession number U20824) was cloned by PCR using
the EHV-2 genomic template (a kind gift from Dr. A. J. Davison,
University of Glasgow, UK) and the following PCR adaptor primers:
E10-start, GAAGATCTATGGCGGAGAAATACCCTC; E10-stop,
CCGCTCGAGTCTCTTAGATGTGTTGCAC. The resulting PCR product was
digested with BglII and XhoI and ligated to a
BamHI/XhoI cut T7-pcDNA-3 vector. By using
the TBLASTN program, several human and mouse partial EST sequences with
homology to the CARD domain of vCLAP were identified. The corresponding EST clones (human clones 703916 and 812172; mouse clone 902716) were
obtained from the IMAGE consortium, and their entire nucleotide sequences were determined by automated sequencing. These clones were
found to encode identical full-length open reading frames as evident
from the presence of stop codons upstream of the initiator methionines.
Northern Blot Analysis--
Tissue distribution analysis of
human CLAP mRNA was performed by Northern blot analysis on normal
and tumor MTN Blots (purchased from CLONTECH). Each
lane contains 2 µg of poly(A)+ RNA from specific tissues
or cell lines. The blots were probed with a radiolabeled riboprobe
prepared from a full-length human CLAP cDNA template and then
subjected to autoradiography.
Mammalian Expression Vectors--
The entire open reading frames
of vCLAP and hCLAP were amplified by PCR using complementary PCR
adaptor primers spanning the initiation and stop codons of these genes.
CLAP deletion mutants were also generated by PCR using modified
complementary PCR adaptor primers. The conserved L41 in the CARD domain
of hCLAP was mutated by site-directed mutagenesis using overlapping PCR
as described before (39). FLAG and T7 epitope tagging was done by
cloning the PCR-generated cDNAs of the respective genes in-frame
into pFLAG CMV-2 and pcDNA-3-T7 vectors, respectively. GFP fusion
constructs were generated by cloning the PCR-generated cDNAs of the
respective genes in frame into the mammalian expression vector pEGFP
(CLONTECH). FKBP12 fusion of CLAP-L41R and CLAP-CTD
was constructed in pFkp3-HA vector (generous gift of Dr. X. Yang,
University of Pennsylvania) which contain three tandem repeats of
FKBP12 with an N-terminally fused c-Src myristoylation signal and a
C-terminally fused HA tag (40). Constructs encoding CRADD, caspase-9DN,
Apaf-1, DR5, X-IAP, Bcl-xL, p35, and kinase-inactive mutants of NIK or
IKK Transfection, Immunoprecipitation, and Immunoblot
Analysis--
293 or 293T cells (5 × 106 cells) in
100-mm dishes were transiently transfected with the expression plasmids
(8 µg/plate) using the LipofectAMINETM (Life
Technologies, Inc.) method. Cells were lysed in a lysis buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Nonidet
P-40) and incubated with anti-FLAG-M5 monoclonal antibody (Eastman
Kodak Co.). The immune complexes were precipitated with protein
G-Sepharose, washed extensively, and then eluted by boiling in SDS
sample buffer. The eluted proteins were resolved by SDS-PAGE and
detected by Western analysis with a horseradish peroxidase-conjugated
T7-antibody (Novagen). The total lysates were also resolved by SDS-PAGE
and detected by Western analysis using the anti-FLAG-M5 and T7 antibody.
Phosphatase Treatment--
The phosphatase treatments of hCLAP
were done using Apoptosis Assays--
MCF-Fas cells (0.5 × 105
cells/well) in 12-well plates were transfected with 0.1 µg of LacZ
reporter plasmid, 0.25 µg of various expression plasmids, and 0.75 µg of various apoptosis inhibitor/empty vector plasmids using the
LipofectAMINETM method. Cells were stained for
Assay of NF- Identification and Cloning of Viral and Cellular CLAPs--
To
identify novel molecules that contain CARD domains, we searched public
data bases for sequences that are homologous to the CARD sequence of
Apaf-1 (residues 1-100). By using this strategy, we found that the
210-amino acid deduced open reading frame of the EHV-2 E10 (ORF-E10)
contains an N-terminal CARD domain (residues 21-107) that is ~27%
identical to the CARD of Apaf-1 (Fig. 1, A and C). Interestingly, the E10 CARD domain
exhibits higher identity with the N-terminal prodomains of caspase-9
(~31%), caspase-2 (~28%), and CED-3 (~29%) and exhibits
significant homology with the CARD domain of the adaptor molecule
CRADD/RAIDD (23% identity).
To clone the ORF-E10, we generated PCR primers complementary to the
start and stop codons of E10 based on the GenBankTM genomic
sequence of EHV-2-E10 (accession number U20824). The PCR primers were
then used on an EHV-2 genomic template to amplify the ORF-E10 by PCR.
Interestingly, after sequencing of the cloned PCR products, we found an
extra base (G) at position 428 relative to the ATG start site. This
extra base shifts the reading frame of E10, resulting in a longer open
reading frame which encodes a 311-amino acid-long protein (Fig.
1A). The corrected ORF-E10, which represents the actual ORF
of the EHV-2-E10 gene, differs from the GenBankTM ORF-E10
after amino acid 142.
To identify cellular homologs of EHV-2-E10, we searched the
GenBankTM expressed sequence tags (EST) data base with the
ORF-E10 sequence. Several human and mouse EST clones were identified,
and their 3' and 5' sequences were compiled. Sequence analysis of the
human and mouse cDNAs revealed that they encode proteins of 233 amino acids with an overall ~34% identity to the viral E10 (Fig.
1B). The two proteins share a high sequence identity
(~91%), suggesting that they are the human and mouse orthologs.
Interestingly, the highest sequence identity between the cellular and
viral proteins lies within the N-terminal CARD homology domain
(cellular protein, residues 13-99; viral protein, residues 21-107).
In this region the two proteins share ~47% identity. In this region
also, the cellular protein has 20, 21, 26, and 29% identity to the
CARD domain of Apaf-1, caspase-9, caspase-2, and CRADD, respectively (Fig. 1C). The selective conservation of the CARD homology
domain within the mammalian and viral proteins suggests that they might play a role in regulating apoptosis. Based on their homology to the
CARD domain and their apoptotic activity (see below), we call these
proteins CLAPs (for CARD-like
apoptotic proteins).
Structural Organization of Viral and Cellular CLAPs--
Sequence
analysis of the cellular and viral CLAPs revealed that they have a dual
domain structure similar to several adaptor molecules that regulate
apoptosis, such as FADD and CRADD. As mentioned above, the vCLAP and
cCLAP have N-terminal CARD homology domains that might be necessary for
interaction with proximal signal transducing molecules. However, their
C-terminal domains (CTD, cCLAP residues 100-233; vCLAP residues
108-311) are unique in that they share no significant sequence
homology with each other or any other known proteins. Interestingly,
the CTD domain of cCLAP is rich in Ser/Thr residues, whereas that of
vCLAP is rich in Gly residues. An abundance of the Ser/Thr residues in cCLAP suggests that it might be regulated by phosphorylation.
Hydrophilicity plots revealed that the CARDs of CLAPs contain
characteristic hydrophilic stretches similar to the ones present in the
CARDs of several CARD-containing proteins such as caspase-9, Apaf-1, or
CRADD (Fig. 1D). Surprisingly, the hydrophilicity plots revealed that their CTD domains are also structurally related and
contain similar profiles of hydrophilic and hydrophobic stretches (Fig.
1D). Therefore, the cellular and viral CLAPs may be overall structurally related and may function as adaptor molecules for an as
yet unidentified signaling complex(es).
Tissue Distribution of cCLAP--
To determine the distribution of
cCLAP, various normal human tissues and tumor cell line mRNA
samples were subjected to Northern blot analysis. As shown in Fig.
2A, cCLAP mRNA (~2.5
kilobase pairs) is constitutively expressed in all the normal tissues
examined with particularly high expression in the pancreas and in
lymphoid organs. Smaller transcripts (~2.0, ~1.6, and ~1.3
kilobase pairs), which could represent alternatively spliced cCLAP
mRNAs, were detected in skeletal muscle, placenta, spleen, and
peripheral blood leukocyte. The major cCLAP mRNA transcript and the
smaller isoforms were also detected in all the tumor cell mRNA
samples examined (Fig. 2, lower panel).
Viral and Cellular CLAPs Induce Apoptosis in Human Cells--
Many
regulators of apoptosis such as the death receptors, the adaptor
molecules FADD, CRADD, and RIP, the proapoptotic Bcl-2 family members
and others, induce apoptosis when overexpressed in the absence of
additional apoptotic signals (34, 41, 42, 46, 47). It is possible that
overexpression of these molecules mimics or provides the necessary
signal to initiate the apoptotic cascade. We therefore examined whether
the viral and cellular CLAPs are able to induce apoptosis when
overexpressed in human cells. To this end we transiently transfected
MCF-7 cells with constructs encoding full-length CLAP proteins or the
CARD or CTD domains of cCLAP. The transfected cells were then examined
for morphological features of apoptosis. Both the full-length viral and
cellular CLAP proteins induced apoptosis in the absence of additional
apoptotic signals (Fig. 3A).
However, the CARD or the CTD of cCLAP had marginal apoptotic activity,
suggesting that the intact protein is required for induction of
apoptosis. Interestingly, both intact vCLAP and cCLAPs exhibited higher
apoptotic activity in the presence of cycloheximide, suggesting that
these molecules could also regulate genes that inhibit apoptosis (see
below).
CLAPs Activate Apoptosis Downstream of the CrmA Block--
To gain
a better understanding of how vCLAP and cCLAP might engage the death
pathway, we investigated their activities with respect to various
inhibitors of apoptosis. The viral serpin CrmA inhibits the initiator
caspase-8 but has relatively weak inhibitory activity toward the
initiator caspase-9 or the effector caspases (caspase-3, -6, and -7)
(48-50). As a result, CrmA inhibits death receptor-induced apoptosis
but not Apaf-1/caspase-9-mediated apoptosis (41, 42, 51). On the other
hand the caspase inhibitor Z-VAD-fmk and the baculovirus p35 protein
have a broad inhibitory spectrum (50) and can inhibit multiple
apoptotic pathways including the death receptor and Apaf-1/caspase-9
pathways. When MCF-7 cells were co-transfected with constructs encoding
CrmA and DR5, CrmA was able to block DR5-induced apoptosis (Fig.
3B). However, when MCF-7 cells were co-transfected with
constructs encoding CrmA and vCLAP or cCLAP, CrmA failed to block
CLAP-induced apoptosis. As expected, Z-VAD-fmk and p35 were able to
block both DR5- and CLAP-induced apoptosis (Fig. 3B). These
data suggest that CLAPs initiate apoptosis by activating caspases
downstream of caspase-8.
To confirm that CLAPs initiate apoptosis downstream of caspase-8, we
used the apoptotic inhibitors Bcl-xL, X-IAP, and caspase-9DN. Bcl-xL
and caspase-9DN act specifically at the level of the Apaf-1-caspase-9 apoptotic complex. Bcl-xL is believed to block the cytochrome c release from the mitochondria, whereas caspase-9DN
interferes with formation of a functional Apaf-1-caspase-9 complex by a
dominant negative mechanism (38, 52-54). X-IAP directly blocks the
activities of the effector caspase-3 and -7 and may also interfere with
the Apaf-1-caspase-9 complex (55, 56). As shown in Fig. 3B,
CLAPs failed to induce apoptosis in MCF-7 cells cotransfected with
constructs encoding CLAPs and either Bcl-xL, X-IAP, or caspase-9DN.
Taken together, these observations suggest that CLAPs initiate
apoptosis some how by triggering the activation of Apaf-1-caspase-9
pathway via caspase-8-independent mechanism.
Viral and Cellular CLAPs Potentiate Death Receptor-induced
Apoptosis in MCF-7 Cells--
Activation of the cytochrome
c/Apaf-1/caspase-9 pathway by the caspase-8-generated
cleavage product of BID is necessary for induction of apoptosis by
death receptors in some cells such as MCF-7 cells (57, 58). In those
cells death receptor-induced apoptosis is inhibited by Bcl-xL and
caspase-9DN (38) (Fig. 3B). Since the CLAP proteins can
activate the cytochrome c/Apaf-1/caspase-9 pathway, we
reasoned that they should be able to potentiate death receptor-induced
apoptosis in MCF-7 cells. To test this hypothesis we transiently
transfected MCF-7 cells with constructs encoding full-length CLAP
proteins or the CARD or CTD domains of cCLAP. The transfected cells
were stimulated with TNF-
The CARD and CTD domains of cCLAP slightly enhanced the apoptotic
activities of TNF- CLAPs Induce NF-
Interestingly, we observed that cCLAP migrates in SDS gels as a doublet
or triplet ranging in size from 29 to 32 kDa (Fig. 4A). The
slower migrating bands appear to be phosphorylated forms of cCLAP,
since treatment of lysates with CLAP-induced NF- The CARD Domain of the CLAP Proteins Is Required for Homo- and
Heterodimerization--
To determine whether cCLAP interacts with
itself and with other CARD-containing proteins, we transfected 293T
cells with expression constructs encoding wild type or mutant T7-tagged
cCLAP and several FLAG-tagged CARD-containing proteins including vCLAP
and cCLAP. cCLAP was found to bind specifically to itself and to vCLAP
and vice versa (Fig. 6, A and
B, lanes 2 and 6). However, cCLAP and vCLAP did
not bind other CARD-containing proteins such as CRADD, APAF-1, and
caspase-9 (Fig. 6, C and D). The interactions of
cCLAP with itself or vCLAP are mediated by the CARD domain, since
deletion of this domain prevented these interactions (Fig. 6,
A and B, lanes 4). The CARD domain of cCLAP was
also able to interact with full-length cCLAP and vCLAP proteins (Fig.
6, A and B, lanes 3). A variant cCLAP with a
point mutation (L41R) in the CARD domain, however, did not interact
with wild type cCLAP or vCLAP (lanes 5). The Leu-41 residue
is conserved in the CARD domains of several apoptotic proteins (Fig.
1C) and has been shown to be essential for heterodimerization of CED-3
and CED-4 or RAIDD and caspase-2 (35, 62). These observations suggest
that the CARD domain is essential for homo- and heterodimerization of
CLAPs. Mutation of a conserved residue in the CARD domain of cCLAP
abrogates these interactions.
The Role of CARD-mediated Oligomerization in NF-
Since a point mutation in the CARD domain that inhibits oligomerization
also abrogates NF-
As with the Fkp3-CTD, AP1510 restored the NF- The CARD of hCLAP Inhibits TNF-
In conclusion we have identified and characterized a novel mammalian
protein cCLAP and its homolog in the virus EHV-2. Both the cellular and
viral CLAP proteins activate NF-
The exact molecular order of the TNF-R1 signaling pathway leading to
NF-
The activity and the dual domain structure of CLAP suggest that it
could be an adaptor molecule. Its major function is to connect the
TNF-R1 complex to the downstream NIK complex. The presence of the CARD
domain suggests that this may be the result of CARD domain-mediated
homotypic and heterotypic protein-protein interactions. Data presented
in this report reveal that hCLAP can form homo-oligomers and
hetero-oligomers with vCLAP through its CARD domain and that
oligomerization of CLAP is required for NF-
The CTD domain of CLAP does not appear to be involved in homotypic
protein-protein interactions, but it is essential for CLAP-induced NF-
CLAP proteins appear to have a molecular organization similar to FADD,
where the CARD domain of CLAP corresponds to the structurally related
death domain of FADD and the CTD domain of CLAP corresponds to the DED
domain of FADD. Death receptor-mediated oligomerization of FADD via its
death domain allows FADD to recruit procaspase-8 through heterotypic
interactions of its DED with the DED of procaspase-8. This causes
oligomerization of the C-terminal caspase domain and autoprocessing of
procaspase-8. Similarly, aggregation of the TNF receptor by its ligand
could induce oligomerization of cCLAP via its CARD domain leading to
induction of NF-B activation play
critical roles in apoptosis and inflammation. We describe the cloning
of the cellular homolog of the equine herpesvirus-2 protein E10 and
show that both proteins regulate apoptosis and NF-
B activation.
These proteins were found to contain N-terminal caspase-recruitment domains (CARDs) and novel C-terminal domains (CTDs) and were therefore named CLAPs (CARD-like apoptotic
proteins). The cellular and viral CLAPs induce apoptosis
downstream of caspase-8 by activating the Apaf-1-caspase-9 pathway and
activate NF-
B by acting upstream of the NF-
B-inducing kinase,
NIK, and the IkB kinase, IKK
. Deletion of either the CARD or the CTD
domain inhibits both activities. The CARD domain was found to be
important for homo- and heterodimerization of CLAPs. Substitution of
the CARD domain with an inducible FKBP12 oligomerization domain
produced a molecule that can induce NF-
B activation, suggesting that
the CARD domain functions as an oligomerization domain, whereas the CTD
domain functions as the effector domain in the NF-
B activation
pathway. Expression of the CARD domain of human CLAP abrogates tumor
necrosis factor-
-induced NF-
B activation, suggesting that
cellular CLAP plays an essential role in this pathway of NF-
B activation.
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ABSTRACT
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B is a critical regulator of
cellular response to infectious agents, stress, injury, and
inflammation (reviewed in Refs. 1 and 2). NF-
B has also been
implicated in the regulation of apoptosis (3-7). The transcriptionally
active NF-
B is a homo- or a heterodimer of two subunits, which
belong to a family of transcriptional regulatory proteins known
as the NF-
B/Rel family (reviewed in Refs. 8 and 9). In mammalian cells five family members are known to date that include RelA/p65, c-Rel, RelB, p50, and p52. Among these, the p50 and p52 are made as
inactive precursor molecules (p105 and p100, respectively), which are
proteolytically processed to the smaller transcriptionally active
forms. All family members contain a conserved N-terminal domain called
the Rel homology domain that contains the necessary residues for
dimerization, nuclear translocation, and DNA binding (8, 9).
B complex is the heterodimer
p50-p65 (10-13). In unstimulated cells, the NF-
B heterodimer is
sequestered in an inactive form in the cytosol through non-covalent interactions with the family of the I
B inhibitory proteins which includes I
B
, I
B
, and I
B
(1, 2, 14). These
interactions mask the nuclear localization signals of the NF-
B
heterodimer, thereby preventing it from translocating to the nucleus.
In response to a variety of signaling events, I
B
is
phosphorylated, which targets the protein to degradation via a
ubiquitination-dependent pathway. This unmasks the NF-
B
nuclear localization signals allowing it to translocate to the nucleus
and induce the expression of a number of target genes (9). Some of the
target gene products, such as cIAP-1, cIAP-2, TRAF1, and TRAF2, have
been shown to protect cells from cell death induced by
TNF-
1 (7).
and interleukin-1 induce NF-
B
activation by binding to their cell-surface receptors, TNF-R1, TNF-R2,
or IL-1R, respectively (8, 15-17). Although these receptors do not
share sequence homology, they all activate the NF-
B-inducing kinase
NIK and NF-
B through distinct signaling pathways (9, 17). For
example, the TNF-R1, like several other members of the TNF-R family,
contains an intracellular region known as the death domain which is
required for both apoptosis induction and NF-
B activation (reviewed
in Refs. 18 and 19). Binding of TNF-
to the extracellular ligand
binding domain of TNF-R1 induces aggregation of its death domain and
assembly of a signaling complex containing TRADD (TNF-R1-associated
death domain protein), TRAF2 (TNF-associated factor 2), and RIP
(receptor interacting protein) (20-24). Similarly, aggregation of the
IL-1R by its ligand results in the assembly of an intracellular
signaling complex containing the IL-1R accessory protein AcP, the
adaptor protein MyD88, the IL-1-activated kinases, IRAK1 and IRAK2, and the TRAF protein, TRAF6 (25-29). These two distinct complexes somehow trigger activation of NIK which in turn phosphorylates the I
B kinases
and
(IKK
and IKK
) that then phosphorylate IkB
at Ser-32 and Ser-36 (reviewed in Refs. 9 and 30). The phosphorylation of I
B
targets it for ubiquitination and proteosomal degradation, allowing the active NF-
B heterodimer to translocate to the nucleus (9, 30).
B-activating complex and the death-inducing signaling complex
(DISC) (9, 17, 19). This domain is also present in proteins of the
IL-1R complex, such as MyD88, IRAK1, and IRAK2 (25, 27-29). Two other
structurally related domains, known as the death-effector domain (DED)
and the caspase-recruitment domain (CARD), mediate interactions between
the initiator caspases and death-inducing signaling complexes (31-33).
The DISC of TNF-R1 utilizes the adaptor molecule FADD which contains
both death and DED domains to relay its death signal (19). After
stimulation of TNF-R1 by its ligand, TNF-R1 recruits TRADD and FADD
through homologous death domain interactions. The DED domain of FADD
then interacts with the homologous DED domains of procaspase-8 and -10, causing their oligomerization and activation (19). The DISC of TNF-R1
also recruits another adaptor molecule with a death domain called CRADD
or RAIDD to transmit its death signal (34, 35). Similar to FADD, CRADD
uses its CARD domain to interact with the homologous CARD domain of
procaspase-2 to induce its oligomerization and activation (34, 35). The
CARD is also found in the prodomain of other apical caspases such as
procaspase-1, -4, -5, and -9 and the apoptosis regulatory molecule
Apaf-1 (31, 36). In the Apaf-1 DISC, this domain is involved in binding and subsequent oligomerization of procaspase-9 by Apaf-1 (37, 38).
B activation and apoptosis.
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ABSTRACT
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MATERIALS AND METHODS
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have been described before (34, 37, 41-44). The
5×
B-luciferase reporter plasmid was from Stratagene.
phosphatase as described in Bellacosa et
al. (45).
-galactosidase activity 30-36 h after transfection. In some
experiments cells were treated with cycloheximide (1 µg/ml) with or
without hTNF-
(10 ng/ml) or Fas antibody (50 ng/ml) or recombinant
soluble TRAIL (200 ng/ml) for 6 h before staining. Normal and
apoptotic blue cells were counted. The percentage of apoptotic cells in
each experiment was expressed as the mean percentage of stained
apoptotic cells as a fraction of the total number of blue cells. Data
represent the average of at least three individual experiments
(n = 3 and S.D. ±1-4%).
B Activation--
NF-
B activation was done
using a luciferase reporter gene. 2 × 105 cells/well
in a 12-well plate were transfected with 5×
B-luciferase reporter
plasmid and various expression plasmids using the
LipofectAMINETM method as per the manufacturer's
instructions. For Jurkat cells, transfections were done by
electroporation using Bio-Rad Gene Pulser II. 24 h after
transfection, cells were harvested and subjected to luciferase assay as
described in Lin et al. (44). In certain experiments, cells
were treated with hTNF-
(20 ng/ml) for 5 h prior to harvesting.
To normalize for transfection efficiency, all transfections included a
LacZ-expressing plasmid, and the lysates were assayed for
-galactosidase activity. Data represent the average of at least
three individual experiments (n = 3 and S.D.
±0.1-0.4).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Sequence and domain structure of viral and
cellular CLAPs. Predicted amino acid sequences of viral CLAP
(A) and cellular CLAP (human and mouse) (B) and
their domain structures. The sequences of the CARD homology domains in
A and B are highlighted. The domain
structures of vCLAP and cCLAP are represented by bar
diagrams below the sequences. The CARD, Ser/Thr-rich, and Gly-rich
domains are labeled inside the bar diagrams. The numbers
indicate the boundaries of these domains. C, co-linear
alignment of the CARD homology domain of CLAPs with that of several
regulators of apoptosis. Identical residues in at least four of seven
sequences are highlighted. D, hydrophilicity plot analysis
of hCLAP and vCLAP. The hydrophilicities of the entire amino acid
sequence of hCLAP (upper panel) and vCLAP (lower
panel) were calculated and plotted using the MacVector 3.5 program.
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Fig. 2.
Expression of hCLAP in normal tissues and
tumor cell lines. The expression of hCLAP mRNA in normal
tissues (upper panel) and tumor cell lines (lower
panel) was determined by Northern blot analysis using premade
multi-tissue Northern blots from CLONTECH. The cell
lines are as follows: HL-60, promyelocytic leukemia; HeLa cell S3,
K-562, chronic myelogenous leukemia; MOLT-4, lymphoblastic leukemia;
Raji, Burkitt's lymphoma; SW480, colorectal adenocarcinoma; A549, lung
carcinoma; G361, melanoma. Numbers on the right
indicate kilobases. PBL, peripheral blood leukocyte.
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Fig. 3.
CLAPs induce apoptosis and potentiate death
receptor-induced apoptosis in human MCF-7 cells. A,
MCF7-Fas cells were transfected with the indicated mammalian expression
plasmids together with a LacZ reporter plasmid. 24-30 h after
transfection, cells were incubated with (+) or without ( )
cycloheximide (Cyclo) and then stained with
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside and
examined for morphological signs of apoptosis. The graphs
show the percentage of round blue apoptotic cells relative to total
blue cells under each condition after subtracting the percentage of
apoptotic cells in the empty vector-transfected cells
(n
3). B, effect of apoptosis inhibitors
on CLAP- and DR5-induced apoptosis. MCF7-Fas cells were transfected
with hCLAP, vCLAP, or DR5 expression constructs or pcDNA3 in the
presence of Z-VAD-fmk (20 µM) or were co-transfected with
these constructs and a 4-fold excess of an empty vector or constructs
encoding CrmA, Bcl-xL, caspase-9DN, X-IAP, or p35. The data are
represented as in A after subtracting the percentage of
apoptotic cells in the control pcDNA3-transfected cells.
C, MCF7-Fas cells were transfected with the indicated
mammalian expression plasmids together with a LacZ reporter plasmid.
24 h after transfection, cells were incubated with TNF-
,
anti-Fas antibody, or TRAIL for 6 h and then stained with
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside and
examined for morphological signs of apoptosis. Similarly, the
vector-transfected cells were also incubated with TNF-
, anti-Fas
antibody, or TRAIL together with cycloheximide (Vector + Cyclo) for 6 h and then examined for apoptosis as described
above. The data are represented as in A and B.
CARD, residues 1-104; CTD, residues 97-233.
, Fas agonist antibody, or TRAIL and then
examined for morphological features of apoptosis. As expected, we
observed that both full-length vCLAP and hCLAP significantly enhanced
the apoptotic activities of TNF-
, Fas agonist antibody, or TRAIL,
compared with the vector controls (Fig. 3C). These findings
suggest that activation of the cytochrome c/Apaf-1/caspase-9
pathway by the CLAP proteins enhances apoptosis induction by death
receptors in some cells.
, Fas agonist antibody, or TRAIL, compared with
the vector controls. Cycloheximide also produced a similar effect on
MCF-7 cells comparable to that observed with the CARD and CTD (Fig.
3C). The slight enhancement effect produced by the CARD and
CTD could be due to their marginal proapoptotic activity (see Fig.
3A). It is also possible that the dominant negative effect
of the CARD on NF-
B activation (see below) may contribute to its
marginal apoptosis enhancement activity.
B Activation--
The ability of cycloheximide
to potentiate the apoptotic activity of CLAPs (Fig. 3A)
suggests that these molecules may have an intrinsic anti-apoptotic
signaling activity that is inhibited by protein synthesis inhibitors.
Cycloheximide has also been shown to potentiate apoptosis induced by
stimulation of the TNF-R1 (59), possibly by inhibiting the synthesis of
NF-
B-regulated anti-apoptotic proteins. Since TNF-R1 can induce both
apoptosis through activation of caspase-8 and proliferation through
induction of NF-
B activation, we reasoned that CLAPs could also have
a dual signaling role that leads either to apoptosis or to NF-
B
activation. To test this possibility, we transiently transfected 293T
cells with an NF-
B reporter construct and increasing amounts of
expression constructs encoding these proteins. The transfected cells
were lysed 16-20 h later and assayed for NF-
B activity and protein
expression. As shown in Fig. 4,
A and B, both human and viral CLAPs were capable of inducing NF-
B in a dose-dependent manner. At
comparable expression levels, vCLAP was ~40-50-fold more potent than
cCLAP in inducing NF-
B activation. Similar results were also
obtained with the human MCF-7 and Jurkat cell lines (data not shown).
Furthermore, both human and viral CLAPs failed to induce AP1 or NFAT
reporter activity (Fig. 4C), suggesting that the activation
is NF-
B-specific.
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Fig. 4.
CLAPs induce NF- B
activation. 293T cells were transfected with an empty vector
(V) or increasing amounts of FLAG-tagged hCLAP
(A) or vCLAP (B) constructs together with an
NF-
B luciferase reporter construct. The cells were harvested 24 h after transfection and lysed in a luciferase assay buffer. The
lysates were assayed for luciferase activity as described under
"Materials and Methods" and subjected to Western blot analysis. The
relative amounts of hCLAP and vCLAP in the lysates were determined by
densitometric scanning of the immunoreactive bands on the Western
blots. The intensity of the vCLAP band at 0.2 µg of
plasmid/transfection was ~0.5 the intensity of the hCLAP band at the
same plasmid concentration. C, 293T cells were transfected
with FLAG-tagged hCLAP, vCLAP, or v-Src constructs together with an
NF-
B, AP1, or NFAT luciferase reporter construct. The cells were
harvested 24 h after transfection and assayed for luciferase
activity as described above. The v-Src oncoprotein can activate both
AP1 and NFAT and was used as a control. D, lysate from 293T
cells expressing FLAG-hCLAP were incubated with (+) or without (
)
phosphatase for 1 h at 37 °C and then subjected to Western blot
analysis with an anti-FLAG antibody.
phosphatase reduced their intensity
and increased the intensity of the faster migrating band (Fig.
4D). These observations suggest that cCLAP might be regulated by phosphorylation. It is likely that phosphorylation of
hCLAP occurs in the Ser/Thr-rich CTD domain, since the CTD domain
(residues 97-233) migrates as a doublet in a high percentage of SDS
gels, whereas the CARD domain (residues 1-104) migrates as a single
band (data not shown).
B Activation Is Inhibited by Dominant Negative
NIK and IKK
--
To determine how CLAP proteins activate NF-
B,
we transiently transfected 293T cells with the NF-
B controlled
luciferase reporter plasmid in combination with cCLAP or vCLAP
expression constructs and kinase-inactive NIK or IKK
(mNIK, mIKK
)
expression constructs. NIK and IKK
transduce the signals that
activate NF-
B. Their kinase-inactive mutants block TNF-
-induced
NF-
B activation (60, 61). As shown in Fig.
5, A and B, the two
kinase mutants completely blocked NF-
B activation by CLAP proteins.
These results demonstrate that the kinase activities of NIK and IKK
are essential for CLAP-induced NF-
B activation and suggest that
cCLAP may contribute to the transduction of the NF-
B activation
signals from the TNF receptor.
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Fig. 5.
CLAP-induced NF- B
activation is inhibited by kinase-inactive NIK and
IKK
proteins. 293T cells were transfected
with an empty vector (0.135 µg) or with constructs encoding hCLAP
(A, 0.135 µg) or vCLAP (B, 0. 02 µg) together
with 0.135 µg of kinase-inactive mutants of NIK (mNIK) or
IKK
(mIKK
), or empty vector (V) and an
NF-
B luciferase reporter construct. Cells were then harvested and
assayed for NF-
B activity by the luciferase assay as described under
"Materials and Methods."
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Fig. 6.
cCLAP and vCLAP can self-associate and
interact with each other through the CARD domain. 293T cells were
transfected with expression plasmids encoding T7 epitope-tagged hCLAP
or vCLAP and FLAG epitope-tagged hCLAP, hCLAP-CARD (CARD, residues
1-104), hCLAP-CTD (CTD, residues 97-233), hCLAP-L41R (L41R), vCLAP,
Apaf-1, or procaspase-9. 293T cells were also transfected with T7-CRADD
and FLAG epitope-tagged hCLAP (C, lane 3) or vCLAP (D,
lane 3). After 36 h, extracts were prepared and
immunoprecipitated (IP) with a monoclonal antibody to the
FLAG epitope. The immunoprecipitates (upper panel) and the
corresponding cellular extracts (middle panel) were analyzed
by SDS-PAGE and immunoblotted with a horseradish peroxidase-conjugated
T7-antibody. The cellular extracts were also Western blotted
(WB) with anti-FLAG antibody (lower panel).
B
Activation--
To define the role of the CARD and CTD domains of the
CLAP proteins in NF-
B activation, we transfected 293T cells with
expression constructs encoding the CARD and CTD domains of cCLAP. In
addition we transfected cells with a construct encoding the cCLAP-L41R mutant. As shown in Fig. 7B,
unlike the full-length cCLAP protein, both the individual CARD and CTD
domains failed to induce NF-
B activation. Similar results were
obtained with vCLAP (data not shown). The cCLAP-L41R mutant also failed
to activate NF-
B (Fig. 7B). These data suggest that
NF-
B activation by CLAP requires both the CARD and the C-terminal
domains.
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Fig. 7.
NF- B-inducing
activity of the truncated and chimeric cCLAP proteins.
A, schematic diagram of the FLAG-tagged full-length hCLAP,
hCLAP-CARD, and hCLAP-CTD and the FKBP fusions of hCLAP-L41R and
hCLAP-CTD. FKBP, FK506-binding protein FKBP12. The
asterisk indicates the L41R point mutation. The c-Src
myristoylation signal (M) and HA and FLAG tags are
indicated. B, 293T cells were transfected with an empty
vector (V, 0.135 µg) or with 0.135 µg of constructs
encoding hCLAP, hCLAP-CARD (residues 1-104), hCLAP-CTD (residues
97-233), or hCLAP-L41R mutant and an NF-
B luciferase reporter
construct and then assayed for NF-
B activity by the luciferase assay
as in Fig. 4 legend. C and D, 293T cells were
transfected with the NF-
B luciferase reporter construct and 0.2 µg
of Fkp3, Fkp3-CLAP-CTD, or Fkp3-CLAP-L41R alone or together with 0.2 µg of Fkp3 or 0.2 µg mNIK as indicated. The total amount of
transfected DNA in all the samples was maintained constant by inclusion
of empty vector. Cells were then treated with different amounts of
AP1510 for 10 h and then assayed for NF-
B activity.
B activation (Figs. 6, A and
B, and 7B), we concluded that the CARD domain
functions by promoting oligomerization. If this is the case the CTD,
which is also required for NF-
B activation, is likely to function as
the NF-
B activation domain. To test this hypothesis, we fused the
CTD of cCLAP and vCLAP and the L41R mutant to an inducible FKBP12
oligomerization cassette (Fig. 7A). We then transfected the
fusion proteins into 293T cells and treated the cells with the
oligomerization drug AP1510 (63). Treatment of the Fkp3-CTD-transfected
cells with increasing amounts of AP1510 resulted in a
dose-dependent increase in NF-
B activity (Fig.
7C). At an AP1510 concentration of 500 nM, more
than 16-fold induction of NF-
B activity was observed, a level
comparable to that observed with cells expressing full-length hCLAP.
This activity was inhibited by co-expression of the nonfused FKBP12
cassette (Fkp3) and by the kinase-inactive NIK mutant (mNIK). NF-
B
was not activated in cells expressing Fkp3 in the presence of the
highest concentration of AP1510. These results show that NF-
B
activation is due to the oligomerization of the cCLAP CTD domain.
B-inducing activity of
Fkp3-CLAP-L41R (Fig. 7D). The CTD alone or the L41R mutant of cCLAP failed to activate NF-
B both in the presence or absence of
the drug. Similar data were obtained with vCLAP-CTD (data not shown).
These data support the hypothesis that the CARD domain functions as an
oligomerization domain, whereas the CTD functions as the effector
domain in the NF-
B activation pathway.
-induced NF-
B
Activation--
To determine the role of cCLAP in the transduction of
the TNF-
signals that activate NF-
B, we transfected 293T cells
with constructs encoding FLAG- or GFP-tagged wild type or mutant cCLAP proteins and then stimulated the cells with TNF-
. The ability of
TNF-
to induce NF-
B activation was slightly enhanced by
overexpression of FLAG-cCLAP or cCLAP-GFP (Fig.
8). Interestingly, the ability of TNF-
to induce NF-
B activation was blocked in a
dose-dependent manner by overexpression of increasing
amounts of the FLAG-tagged CARD domain of cCLAP but not by the
FLAG-tagged CTD domain or the L41R mutant. A dose-dependent
inhibition of TNF-
-induced NF-
B activation was also observed when
cells were transfected with increasing amounts of GFP-tagged CARD
domain of cCLAP. These data demonstrate that the CARD domain of cCLAP
is a dominant negative regulator of TNF-
-induced NF-
B activation
and suggests that cCLAP might be a downstream component of the
TNF-
-induced NF-
B activation pathway.
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Fig. 8.
TNF- -induced
NF-
B activation is inhibited by the CARD
domain of hCLAP. 293T cells were transfected with an empty vector
(1.2 µg) or with increasing amounts (0.2-1.2 µg) of constructs
encoding FLAG-tagged hCLAP-CARD, hCLAP-CTD, hCLAP-L41R, or
hCLAP-CARD-GFP and an NF-
B luciferase reporter construct and then
treated with TNF-
. Cells were also transfected with constructs
encoding FLAG-hCLAP (1.2 µg) or hCLAP-GFP (1.2 µg) and an NF-
B
luciferase reporter construct and then treated with TNF-
. Cells were
harvested 5 h after treatment and assayed for NF-
B activity by
the luciferase assay as described above. The lysates were also
subjected to Western blot analysis with anti-FLAG antibody (FLAG-tagged
proteins) or anti-GFP antibody (GFP-tagged proteins) (upper
panels). The FLAG-tags are on the N termini, whereas the GFP-tags
are on the C termini of the tagged proteins.
B and induce apoptosis. Although
vCLAP and cCLAP induce apoptosis in transfected cells, their ability to
induce NF-
B activation suggests that they may also promote cell
survival. During viral infection, it is possible that inhibition of
apoptosis may be temporarily advantageous for the enhancement of viral
replication (64). EHV-2 has developed an anti-apoptotic strategy by
acquiring genes that encode anti-apoptotic proteins such as FLIP (E8)
which suppresses death receptor-induced apoptosis. Moreover, it has
acquired an open reading frame (E4) that has homology to the
anti-apoptotic protein Bcl-2 (64). During viral infection FLIP and the
Bcl-2 homolog(s) are expected to prevent vCLAP (E10)- and death
receptor-induced apoptosis. In this anti-apoptotic environment, vCLAP
is likely to induce massive activation of NF-
B which may further
extend the survival of the infected cells by inducing the expression of
cellular anti-apoptotic proteins. NF-
B activation by vCLAP may also
promote the expression of EHV-2 genes. It is possible that deregulation
of the anti-apoptotic proteins (i.e. E4 and FLIP) during the
lytic phase of viral replication may unmask the proapoptotic properties
of vCLAP which may therefore induce apoptosis.
B activation is not fully understood. Several components of this
pathway remain to be identified as evident from the recent identification of two additional components of the IKK complex, IKAP
and NEMO/IKK-
(65-67). One of the missing links between the upstream TNF-R1-TRADD-RIP complex and the downstream NIK-IKK-IKAP complex could be CLAP. This is consistent with the ability of dominant
negative NIK and IKK
to inhibit NF-
B activation by CLAP and with
the ability of the CARD domain of CLAP to inhibit TNF-
-induced
NF-
B activation.
B activation. Its ability
to act as a dominant negative inhibitor of TNF-
-induced NF-
B
activation indicates that it may interact with the upstream
TNF-R1-TRADD-RIP complex directly or indirectly through its CARD domain.
B activation. Deletion of this domain abrogates the ability of
cCLAP to induce NF-
B activation. However, artificially induced oligomerization of this domain is sufficient to induce NF-
B
activation independent of the CARD domain. This indicates that the CTD
domain is involved in heterotypic interactions with downstream
regulators of NF-
B and that oligomerization of these regulators is
necessary for induction of NF-
B activation. These regulators may
include the NIK-IKK-IKAP complex and other proteins that remain to be identified.
B through the interaction of the CTD effector domain
with downstream regulators of NF-
B activation.
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ACKNOWLEDGEMENTS |
---|
We thank A. J. Davison for the EHV-2 DNA; W. C. Greene and X. Lin for the dominant negative mutants of NIK and IKKa; and X. Yang for pFkp3-HA.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Research Grant AG13487, the Charlotte Geyer Foundation, and IDUN Pharmaceuticals.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) AF134394-AF134396.
These authors contributed equally to this work.
§ To whom correspondence should be addressed: Thomas Jefferson University, Kimmel Cancer Institute, Bluemle Life Sciences Bldg., Rm. 904, 233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-503-4632; Fax: 215-923-1098; E-mail: E_Alnemri{at}lac.jci.tju.edu.
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ABBREVIATIONS |
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The abbreviations used are: TNF, tumor necrosis factor; CARD, caspase-recruitment domains; IL-1R, interleukin-1 receptor; DISC, death-inducing signaling complex; CTD, C-terminal domains; PCR, polymerase chain reaction; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; ORF, open reading frame; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; DED, death-effector domain; EHV, equine herpesvirus; HA, hemagglutinin.
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REFERENCES |
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