mE10, a Novel Caspase Recruitment Domain-containing Proapoptotic Molecule*

Minhong YanDagger , James Lee§, Sarah Schilbach§, Audrey Goddard§, and Vishva DixitDagger

From the Departments of Dagger  Molecular Oncology and § Molecular Biology, Genentech, Inc., South San Francisco, California 94080

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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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 alpha -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
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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 beta -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-beta -D-galactopyranoside. Apoptotic cells were distinguished by morphological changes including becoming rounded, condensed, and detached from the cell culture dish (15).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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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.

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 (Delta 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 beta -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-beta -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.

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 Delta C71 mE10 fused to GST at its C terminus. In cotransfection experiments performed in 293 cells, Delta 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 (Delta 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 (Delta C71, Delta C99, and Delta 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 Delta N55, Delta C71, Delta C99, and Delta 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 (Delta C71 and Delta C99) or N-terminal deletion mutants (Delta N55 and Delta 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.

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 Delta 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 (Delta N55 and Delta 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 Delta N55 mE10 was constructed by an N terminus of Delta 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.

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 (Delta N55) but not with the C-terminal truncation (Delta 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-Delta 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.

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.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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