mE10, a Novel Caspase Recruitment Domain-containing Proapoptotic
Molecule*
Minhong
Yan
,
James
Lee§,
Sarah
Schilbach§,
Audrey
Goddard§, and
Vishva
Dixit
¶
From the Departments of
Molecular Oncology and
§ Molecular Biology, Genentech, Inc.,
South San Francisco, California 94080
 |
ABSTRACT |
Apoptotic signaling is mediated by homophilic
interactions between conserved domains present in components of the
death pathway. The death domain, death effector domain, and caspase
recruitment domain (CARD) are examples of such interaction motifs. We
have identified a novel mammalian CARD-containing adaptor molecule termed mE10 (mammalian E10). The N-terminal CARD of mE10 exhibits significant homology (47% identity and 64% similarity) to the CARD of
a gene from Equine Herpesvirus type 2. The C-terminal region is unique. Overexpression of mE10 in MCF-7 human breast carcinoma cells induces apoptosis. Mutational analysis indicates that
CARD-mediated mE10 oligomerization is essential for killing activity.
The C terminus of mE10 bound to the zymogen form of caspase-9 and
promoted its processing to the active dimeric species. Taken together,
these data suggest a model where autoproteolytic activation of
pro-caspase-9 is mediated by mE10-induced oligomerization.
 |
INTRODUCTION |
The core components of the death pathway as revealed by genetic
analysis of the nematode Caenorhabditis elegans are
remarkably conserved through evolution (1) although to a much greater degree of complexity in mammals (2). This is reflected in the fact
that, except for CED-4, multiple homologs of each of the core
components are found in mammals. Worm CED-4 and its mammalian homolog
Apaf-1 function to promote the autoproteolytic activation of downstream
effector death proteases termed caspases (3, 4). Caspases are cysteine
proteases related to worm CED-3 that cleave substrates following an Asp
residue (5). They exist as single polypeptide zymogens composed of a
prodomain and a large and a small catalytic subunits. The zymogen is
proteolytically processed to the active dimeric species by either an
upstream caspase or through an autoproteolytic mechanism. This latter
mode of activation is dependent upon the zymogen possessing residual enzymatic activity. Additionally, it is greatly facilitated by adaptor
molecules that bind zymogen, effectively increasing its local
concentration and subsequent rate of proteolytic processing. Caspases
can be classified into upstream initiator caspases and downstream
effector caspases. Effector caspases are cleaved and activated by
upstream initiator caspases. Initiator caspases, on the other hand, are
activated by an autoproteolytic mechanism and dependent upon adaptor
mediated induced proximity. For example, caspase-8, the initiator
caspase for CD-95 receptor induced apoptosis, is complexed by the
adaptor molecule FADD/MORT-1 (6), whereas caspase-9, the initiator
caspase for mitochondrial-induced death, is oligomerized by the adaptor
molecule Apaf-1 in a cytochrome c-dependent
manner (7). Therefore, a recurring theme in the activation of initiator
caspases is adaptor molecule-induced oligomerization (8). This
interaction of caspases with adaptor molecules is mediated by a
homophilic association of structurally related protein modules. Based
on sequence similarity, three such interaction motifs have been
defined: the death domain, the death effector domain (9), and the
caspase recruitment domain (CARD)
(10).1 All contain a
conserved stretch of about 90 amino acids in length. Despite marginal
primary sequence similarities, structural studies suggest that these
domains adopt a similar fold composed of six
-helices (11-13).
The CARD motif is found in many molecules involved in apoptotic
signaling (10). For example, it is present at the N terminus of Apaf-1
and mediates binding to the corresponding domain within caspase-9 (7).
Here we report the molecular cloning and characterization of a novel
CARD-containing molecule designated mE10 that, like Apaf-1, interacts
with caspase-9. Unexpectedly, however, the CARD motif within mE10 was
found not to mediate binding to caspase-9 but rather induced mE10
self-oligomerization. The non-CARD C terminus of mE10 complexed with
caspase-9 and promoted its processing to the active protease.
Therefore, the data support a model where mE10 induces apoptosis by
activation of caspase-9 through an induced proximity mechanism.
 |
MATERIALS AND METHODS |
Molecular Cloning of mE10--
A human EST clone
(GenBankTM accession number AA455396) was found to have
significant sequence homology to the CARD of E10 from equine
Herpesvirus 2 (14). To obtain full-length coding information
beyond the known EST cDNA sequence, polymerase chain reaction-based
plasmid library screening was undertaken. Several clones from
independent polymerase chain reaction reactions employing a
proofreading polymerase were analyzed. Searching the EST data base with
the human mE10 open reading frame, we identified two mouse EST clones
(GenBankTM accession numbers AA119259 and W47752) with high
sequence homology to the N terminus and C terminus of the human gene,
respectively. The mouse cDNA containing the entire open reading
frame was generated by polymerase chain reaction using embryonic mouse
cDNA as template and custom primers.
Cell Lines and Expression Vectors--
Human embryonic kidney
293 cells were maintained in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum, L-glutamine, and
penicillin/streptomycin. Human breast carcinoma MCF7 cells were
cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal
bovine serum, L-glutamine, and penicillin/streptomycin. Mammalian expression vectors including pcDNA3.1 V5HisA
(Invitrogen), pEGFP-N2 (CLONTECH), or pRK5B were
engineered using standard recombinant DNA methodology to express
N-terminal or C-terminal epitope-tagged proteins. Deletion or point
mutations were generated by polymerase chain reaction using
Pfu polymerase (Stratagene) and the resulting constructs
confirmed by sequencing.
Northern Blotting--
Human and mouse poly(A)+ RNA
tissue blots (CLONTECH) were hybridized according
to the manufacturer's instructions. 32P-Labeled probes
were generated using DNA fragments corresponding to the entire open
reading frames of human and mouse mE10 genes.
Cell Death Assay--
MCF7 cells were transiently transfected
with LipofectAMINE reagent (Life Technologies, Inc.). pCMV
-galactosidase was used as the reporter plasmid for transfected
cells. 30-36 h following transfection, cells were fixed with 0.5%
glutaraldehyde and stained with
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside.
Apoptotic cells were distinguished by morphological changes including
becoming rounded, condensed, and detached from the cell culture dish
(15).
 |
RESULTS AND DISCUSSION |
Identification of mE10, a Novel CARD-containing Protein--
Data
base searches revealed a human EST clone (GenBankTM
accession number AA455396) with significant amino acid homology to
known CARDs. Cloning and sequence analysis revealed that both human and
mouse open reading frames (90% identity) encoded a protein of 233 amino acids with a CARD residing at the N terminus. Interestingly, its
CARD exhibited highest homology to the CARD present in E10 (14), an
open reading frame found in equine Herpesvirus 2 (47% identity and 64% similarity). Therefore we designated this novel CARD-containing gene mE10 for mammalian E10. In contrast to their N
termini, mE10 and viral E10 lacked significant homology at the C
terminus (Fig. 1).

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Fig. 1.
Sequence analysis of mE10. A,
schematic structure of mE10. B, comparison of amino acid
sequences of human (accession number AF127386) and mouse (accession
number AF127387) mE10 and equine Herpesvirus 2 E10.
C, alignment of the amino acid sequences of CARDs of mE10,
RAIDD, caspase-2, and caspase-9. The asterisks represent
residues conserved among CARDs and mutated in mutation analysis of
mE10.
|
|
Tissue Distribution of mE10--
Northern blot analysis using
32P-labeled cDNA encoding human and mouse mE10 genes
revealed a transcript of about 2.8 kilobases that was constitutively
expressed in a variety of tissues. A smaller transcript of about 1.2 kilobases was also observed in testis. Interestingly, the expression
level of mE10 appeared to be higher in the mouse embryo (Fig.
2).

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Fig. 2.
Tissue distribution of human and mouse
mE10. Northern blot analysis was performed using
32P-labeled cDNA probes encoding human and mouse mE10
genes.
|
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Overexpression of mE10 Induces Apoptosis--
Most characterized
CARD-containing molecules play a role in apoptosis. To investigate
whether mE10 was proapoptotic, human MCF7 breast carcinoma cells were
transfected with a mE10 mammalian expression construct. Extensive cell
death was observed in cells overexpressing mE10 (Fig.
3). Cell death was effectively blocked by
a broad spectrum of cell death inhibitory genes including CrmA, p35,
cIAP1, cIAP2, and caspase-9 dominant negative mutant (Fig. 3C). To identify the domains that mediate mE10-induced cell
death, truncated and single amino acid mutant versions were generated. Partial deletion (amino acids 1-55) of the CARD abolished the pro-apoptotic activity of mE10. Furthermore, point mutations of several
conserved residues (m1, L28A; m2, L47A; m3, I55A; m4, I46A; m5, E53A;
m6, L41A) also eliminated the killing ability of mE10. Surprisingly,
deletion of 71 amino acids at the C terminus (
C71) also impaired the
cell death-inducing capability of mE10 (Fig. 3A), suggesting
that both the N-terminal CARD and an intact C terminus are required for
inducing apoptosis.

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Fig. 3.
Overexpression of mE10 induces apoptosis in
mammalian cells. A, MCF7 cells were transiently
transfected with 0.15 µg of reporter gene -galactosidase plasmid
together with 0.75 µg of indicated expression constructs
(pcDNA3.1-V5 His). Transfected cells were visualized with
5-bromo-4-chloro-3-indolyl- -D-galactopyranoside
staining and scored for morphological feature of apoptosis.
B, diagrammatic representation of mE10 deletion and point
mutants. C, MCF7 cells were transiently transfected with
0.25 µg of pcDNA3.1 mE10-V5His and 3-fold excess of the indicated
apoptosis inhibitors. Data (means ± S.D.) were from three
experiments, and in each transfection more than 500 blue cells were
counted.
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CARD-mediated Oligomerization of mE10--
Because CARD-CARD
interactions play an important role in signaling apoptosis, we asked
whether the CARD of mE10 could interact with other known
CARD-containing proteins. To directly assess the binding activity of
the N-terminal CARD of mE10, we used
C71 mE10 fused to GST at its C
terminus. In cotransfection experiments performed in 293 cells,
C71-GST failed to bind known CARD-containing molecules including
caspase-1, caspase-2, caspase-9, Apaf-1, and RAIDD (data not shown).
However, mE10 itself was found to oligomerize in a
CARD-dependent manner. When we examined the cellular
localization of mE10 fused to EGFP at its C terminus (mE10-EGFP), a
perinuclear, compact, and filamentous pattern of expression was
observed in MCF7 cells (Fig.
4A). The localization was not
due to cell death perturbing cellular architecture because a similar
pattern was observed when mE10-EGFP was coexpressed with dominant
negative caspase-9 that effectively blocked apoptosis (Fig.
4A). Mutation of the CARD either by deletion (
N55) or
point mutation (L47A) resulted in a diffuse distribution of mE10-EGFP,
suggesting that the CARD was essential for mE10 aggregation.
Furthermore, it appeared that the CARD was not only required but also
sufficient to promote oligomerization because C-terminal truncated
versions of mE10 that retained an intact CARD (
C71,
C99, and
C103) still aggregated. This finding was consistent with the result
of coprecipitation assays performed in transiently transfected 293 cells expressing GST fusion and Flag epitope-tagged mE10 proteins.
Intact mE10-Flag coprecipitated with full-length mE10-GST. However,
CARD deletion but not the C-terminal truncation abolished this
interaction between the differentially tagged molecules (Fig.
4B). The importance of the CARD in mediating homophilic
interaction was further supported by the finding that mutation of
conserved CARD residues resulted in attenuation of oligomerization
(Fig. 4C).

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Fig. 4.
Oligomerization of mE10 mediated by its
CARD. A, cellular localization of mE10-EGFP in MCF7
cells. MCF7 cells were transfected with wild type, point mutant L47A,
and deletion mutants N55, C71, C99, and C103 fused at the C
terminus with EGFP. In some transfections, caspase-9-DN was
cotransfected to inhibit cell death. 24 h following transfection,
cells were examined by confocal microscopy. The nucleus
(blue) was counterstained with Hoechst 33342 (Molecular
Probes). B and C, self-association of mE10 as
assessed by in vivo coprecipitation assays. B,
293 cells were transiently transfected with wild type mE10-Flag
together with mE10-GST fusion constructs of full-length
(FL), C-terminal deletion mutants ( C71 and
C99) or N-terminal deletion mutants ( N55
and N127). 24 h after transfection, GST fusion
proteins were precipitated with glutathione-agarose beads, and the
binding of mE10-Flag was detected by anti-Flag Western blotting.
C, 293 cells were cotransfected with wild type or point
mutant V5 and GST tagged mE10. 24 h after transfection, GST fusion
proteins were precipitated from the cell lysates with
glutathione-agarose beads, and the binding of mE10-V5 was determined by
anti-V5 Western blotting.
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mE10 Enhances Pro-caspase-9 Processing--
Given that
mE10-induced apoptosis was effectively blocked by a dominant negative
version of caspase-9, we reasoned that it might be involved in
mE10-induced cell death. To test this possibility, the processing of
caspase-9 was examined in transiently transfected 293 cells or MCF7
cells in the presence or absence of mE10 (Fig. 5). During activation, caspase-9 is
cleaved at Asp315, generating a signature p12 small
subunit. Activated caspase-9 cleaves and activates the downstream
effector, caspase-3, which in turn cleaves caspase-9 at
Asp330, generating a characteristic p10 fragment. Because a
gene rearrangement has inactivated caspase-3 in MCF7 cells, only the
p12 form is observed (4). In some transfections, plasmid expressing
baculovirus protein p35 was also included. Baculovirus p35 binds and
inhibits activated caspases, allowing for facile detection of processed subunits. In 293 cells, expression of caspase-9-Flag alone led to
auto-processing and apoptosis in a dose-dependent manner.
However, coexpression of mE10 greatly enhanced processing of
caspase-9-Flag as judged by generation of the small catalytic subunit.
Similar experiments using deletion mutants of mE10 demonstrated that
both the N-terminal CARD and the unique C-terminal region of mE10 were required for the enhancement of caspase-9 processing. This is in
agreement with the result that both domains are necessary for mE10-induced cell death (Fig. 3). To determine whether the role of the
CARD motif was simply to provide an oligomerization platform, a
chimeric mE10 was constructed where the first 55 amino acids were
replaced with a 28-amino acid leucine zipper that promotes the
formation of tetrameric oligomers (16). The chimera (Leu-tetra
N55
mE10) did oligomerize when expressed in mammalian cells (data not
shown) but failed to induce cell death (data not shown) or enhance
caspase-9 processing (Fig. 5B). This result suggested that
oligomerization is not the sole function conferred by the CARD motif.
Rather, the oligomerized CARD must in some other way (such as by
influencing conformation or stability) enhance the proapoptotic
activity of mE10.

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Fig. 5.
mE10 enhances caspase-9 processing in
vivo. A, mE10 enhances caspase-9 processing
in 293 cells. Caspase-9-Flag was transfected into 293 cells in
combination with the baculovirus anti-apoptosis protein p35 and mE10-V5
as indicated. Cells were lysed 24 h post-transfection, and cell lysates were subjected to immunoprecipitation with
monoclonal anti-Flag beads (M2 beads from Kodak) followed by Western
blotting using polyclonal anti-Flag antibody (Santa Cruz
Biotechnology). Because caspase-9 was C-terminally tagged, the
processing of caspase-9 was monitored by appearance of the C-terminal
small subunit. The bottom panel is a longer exposure of the
top panel. B, both the N-terminal CARD and the
C-terminal region of mE10 are required for the enhancement of caspase-9
processing. Caspase-9-flag was transfected with full-length
(FL) or deletion mutants ( N55 and
C71) of mE10. Apaf-1 (1-412), a truncated version of
Apaf-1 consisting of only the CED-3 and CED-4 homology domains, was
used as a positive control, because it has previously been shown to
enhance caspase-9 processing (4). Leu-tetra N55 mE10 was constructed
by an N terminus of N55 a leucine zipper sequence that promoted the
formation of a tetramer. The processing of caspase-9-Flag was assessed
as described for A. C, caspase-9 processing in
MCF7 cells. Various amounts of caspase-9-Flag were transfected with or
without mE10 into MCF7 cells. The processing of caspase-9-Flag was
carried out as described for A.
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C Terminus of mE10 Binds Caspase-9--
Recent studies support an
induced proximity or oligomerization model for the activation of
initiator caspases (8). For example, the N-terminal CARD of Apaf-1
interacts with the corresponding CARD within the prodomain of
Caspase-9. When Apaf-1 oligomerizes by self-associating through its
CED-4 homology domain, pro-caspase-9 molecules are brought into close
proximity, facilitating their autoproteolytic activation (4). Recently
a report by Yang et al. (3) showed that CED-4 itself also
takes advantage of this induced aggregation strategy to promote
autoactivation of CED-3. Given that mE10 forms oligomers through
self-association of its CARD and enhances pro-caspase-9 processing
in vivo, we hypothesized that mE10 may facilitate the
processing of pro-caspase-9 in a manner analogous to that employed by
Apaf-1 and CED-4. If so, mE10 should complex with pro-caspase-9
in vivo. Because the CARD of mE10 failed to interact with
other CARD-containing molecules including caspase-9, we reasoned that
the interaction between mE10 and pro-caspase-9 must be mediated by the
C-terminal non-CARD containing region. To test this, 293 cells were
transiently transfected with caspase-9-DN-Flag and various GST-mE10
fusion constructs (Fig. 6).
Caspase-9-DN-Flag coprecipitated with GST fused with full-length mE10
and the CARD deletion mutant (
N55) but not with the C-terminal
truncation (
C71), confirming that the C-terminal non-CARD containing
region of mE10 is essential for binding caspase-9. Experiments using
various caspase-9 deletion mutants suggested that both the CARD and the
catalytic domain of caspase-9 contributed to the interaction with mE10
(data not shown). This is different from the interaction between Apaf-1
and caspase-9 that involves only the CARDs. In similar experiments,
caspase-3 failed to coprecipitate with mE10 (Fig. 6B), as
did the CARD-containing caspase-2 (data not shown), suggesting that
mE10 interacts specifically with caspase-9 (Fig. 6B). Taken
together, the results supported an oligomerization model of caspase-9
activation by mE10 and was consistent with prior death assay results,
which indicated that both CARD and C-terminal regions were
indispensable for mE10 function.

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Fig. 6.
mE10 Binds caspase-9 through the
non-CARD. A, 293 cells were cotransfected with
caspase-9-DN-Flag and indicated GST-mE10 fusion constructs. GST fusion
proteins were precipitated with glutathione-agarose beads and
immunoblotted with Flag antibody. B, the C-terminal region
of mE10 specifically binds caspase-9 but not caspase-3. 293 cells were
transfected with the indicated expression constructs. GST- N55 fusion
protein was precipitated with glutathione-agarose beads and
immunoblotted with epitope-tag antibody to detect complex formation
with caspase-9-Flag and caspase-3-GD, respectively. Lanes 1 and 4 represent the amount of caspase-9-Flag and
caspase-3-GD in the total cell lysates (Tol). WB,
Western blot.
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|
In summary, we have identified a novel CARD-containing molecule that
promotes the activation of caspase-9 and is proapoptotic. Although the
CARD of mE10 is required for the activation of caspase-9 in
vivo, it does not directly interact with caspase-9. Rather, this
interaction is mediated by the C-terminal region of mE10. Therefore,
these data suggest a mode of caspase-9 activation that is independent
of Apaf-1. It is important to note, however, that an inherent
limitation of our conclusions is that they are based on the study of
overexpressed proteins. The physiological mechanism by which mE10
induces apoptosis awaits determination.
 |
ACKNOWLEDGEMENTS |
We thank Jeffrey Hooley and Steve Sherwood
for help with confocal microscopy, the Genentech Biorganic Chemistry
Department for oligonucleotide synthesis, and colleagues in the Dixit
laboratory for reagents and valuable discussions.
 |
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.
¶
To whom correspondence should be addressed: Dept. of Molecular
Oncology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. Tel.: 650-225-1312; Fax: 650-225-6127; E-mail: dixit{at}gene.com.
 |
ABBREVIATIONS |
The abbreviations used are:
CARD, caspase
recruitment domain;
EGFP, enhanced green fluorescent protein;
GST, glutathione S transferase;
EST, expressed sequence tag.
 |
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