Diva, a Bcl-2 Homologue that Binds Directly to Apaf-1 and Induces BH3-independent Cell Death*

We have identified and characterized Diva, which is a novel regulator of apoptosis. Sequence analysis revealed that Diva is a member of the Bcl-2 family of proteins containing Bcl-2 homology domain 1, 2, 3, and 4 (BH1, BH2, BH3, and BH4) regions and a carboxyl-ter-minal hydrophobic domain. The expression of Diva mRNA was detected in multiple embryonic tissues but was restricted to the ovary and testis in adult mice. The expression of Diva promoted the death of 293T, Ramsey, and T47D cells as well as that of primary sensory neurons, indicating that Diva is a proapoptotic protein. Significantly, Diva lacks critical residues in the conserved BH3 region that mediate the interaction between BH3-containing proapoptotic Bcl-2 homologues and their prosurvival binding partners. Consistent with this, Diva did not bind to cellular Bcl-2 family members including Bcl-2, Bcl-X L , Bcl-w, Mcl-1, and A1/Bfl-1. Furthermore, mutants of Diva lacking the BH3 region fully retained their proapoptotic activity, confirming that Diva promotes apoptosis in a BH3-independent manner. Significantly, PCR dideoxy

grammed cell death, is critical during development and tissue homeostasis and plays a role in the pathogenesis of a variety of diseases (1). Several regulatory components of the apoptotic pathway have been identified in various living organisms including man (1,2). A family of proteins that includes Bcl-2, Bcl-X L , Bcl-w, Mcl-1, and A1/Bfl-1 shares conserved regions termed Bcl-2 homology domains 1, 2, 3, and 4 (BH1, BH2, BH3, and BH4) 1 and function by repressing apoptosis (3). The biochemical process by which Bcl-2 family members regulate cell death is poorly understood. Analyses of the nematode cell death regulators CED-3, CED-4, and CED-9 have provided important insight into the biochemical mechanism that regulates apoptosis. CED-9, the nematode homologue of Bcl-2 and Bcl-X L , binds to CED-4 and represses cell death by interacting and inhibiting the killing activity of CED-3 through CED-4 (4 -7). Recently, it was shown that Bcl-X L associates with Apaf-1, a mammalian homologue of CED-4, and inhibits the activation of procaspase-9 (8 -11).

Identification of the Diva cDNA and Preparation of Expression
In Situ Hybridization, Northern Blot, and Reverse Transcription-PCR Analyses-Slides containing Swiss white mouse embryo tissues were prepared as described previously (22). Each specimen was hybridized with a digoxigenin-labeled antisense RNA probe synthesized from a mouse full-length Diva cDNA using an in vitro transcription kit (Promega). As a control, a sense Diva RNA-labeled probe was synthesized and used for hybridization as described above. The hybridization, development, and mounting of slides were performed as described previously (22). Diva cDNA was synthesized from the total RNA of Swiss white mice by reverse transcription and amplified by 30 thermal cycles (95°C for 30 s, 65°C for 30 s, and 72°C for 1 min), using ACTGCAT-GAACGCACTAGACG and TTGGAGAGCAACTTATCTGCC as primers. ␤-Actin cDNA was amplified as described previously (36).
Cell Death Assay-293T cells were transfected with 0.2 g of pcDNA3-␤-gal plus 1 g of pcDNA3-HA-Diva plus 1 g of either pcDNA3-HA-vBcl-2, pcDNA3-HA-Bcl-X L , pcDNA3-p35, or pcDNA3caspase-9 C287S-HA. 24 h after transfection, cells were fixed and stained for ␤-galactosidase as described previously (31). The percentage of apoptotic cells in triplicate cultures was determined by calculating the fraction of membrane-blebbed blue cells from the total population of blue cells. Ramsey and T47D cells were co-transfected with 1 g of pcDNA3, pcDNA3-Myc-Diva, pcDNA3-Flag-Hrk, or pcDNA3-FADD-Myc and 0.5 g of pcDNA3-␤-gal using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instructions. In some experiments, cell death was calculated as a reduction in the number of cells expressing ␤-galactosidase relative to that obtained by transfection with the control plasmid. The staining of nuclei with acridine orange and ethidium bromide was performed as described previously (19).
Microinjection of DNA into Neurons-Primary cultures of rat sympathetic neurons were prepared from the superior cervical ganglia of newborn rats and prepared for microinjection as described previously (37). Individual neurons were injected in the nucleus (DNA solution at 100 ng/l in 0.1 mM Tris-HCl, pH 7.2) as described previously (37). The percentage of microinjected neurons expressing HA-Diva was determined at 20 h postinjection in neurons kept in medium with or without nerve growth factor. Cells were fixed and stained as described previously (36).
Transfection, Expression, Immunoprecipitation, and Immunodetection of Tagged Proteins-5 ϫ 10 6 293T cells were transfected with expression plasmids by the calcium phosphate method as described previously (31). 3 g of pcDNA3 or pcDNA3-Myc-Diva were co-transfected with 3 g of the various expression plasmids listed above and indicated in the figure legends. The total amount of DNA used was always 9 g. After transfection, 293T cells were harvested at the times shown in figure legends and lysed with 0.2% Nonidet P-40 isotonic lysis buffer (12). 1 mg of soluble protein was incubated with 1 g/ml anti-Myc polyclonal Ab or anti-Flag polyclonal Ab for 2 h at 4°C. Tagged proteins were immunoprecipitated with protein A-Sepharose 4B (Zymed Laboratories Inc., San Francisco, CA) and washed as described previously (19). Immunoprecipitates were subjected to 13.5% SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-HA, anti-Myc, or anti-Flag Ab.

Identification of Diva, a Novel Bcl-2 Family Member-To
identify novel apoptosis-regulatory proteins, we screened the GenBank database for cDNAs encoding proteins with homology to NR-13 (27), a Bcl-2 family member, by using the TBLASTN program. Initially, two ESTs (clones 1108004 and 489531) containing overlapping nucleotide sequences with statistically significant amino acid homology to NR-13 and to all known Bcl-2 family members were identified. Subsequently, 20 additional ESTs encoding the same gene were identified through a homology search using the EST 1108004 and 489531 sequences. The longest cDNA (mouse EST clone 1108004) was 1.3 kilobases, and analysis of its nucleotide sequence revealed a novel open reading frame of 191 amino acids with a predicted relative M r 22,300 (Fig. 1A). We designated this protein as Diva (death inducer binding to vBcl-2 and Apaf-1, see below). Alignment analysis revealed that Diva was a Bcl-2-related protein with predicted ␣1-8 helices and BH1, BH2, BH3, and BH4 regions as well as a carboxyl-terminal hydrophobic tail (Fig. 1B). Diva showed significant structural and amino acid homology with all known Bcl-2 family members including Bcl-2, Bcl-X L , Bcl-w, Bax, Bak, Mtd, and Caenorhabditis elegans CED-9 (Fig. 1C). The similarity of Diva with Bcl-2 and Bcl-X L was 41 and 44%, respectively. Diva was most homologous (46% similar) to quail NR-13, a Bcl-2-related protein that is induced by Src kinases (27). However, additional analyses suggested that that Diva is not the mouse orthologue of NR-13, because Diva and NR-13 differ significantly in critical residues and predicted physiological function (see below).

The Sequence of Diva Lacks Critical Residues that Mediate Heterodimerization between Bcl-2 Family Proteins and Contains an Extended Amino Acid Stretch between the Predicted ␣5
and ␣6 Helices-Diva exhibits significant amino acid and structural homology with members of the Bcl-2 family. Close inspection of the Diva amino acid sequence, however, revealed that it differed from that of known Bcl-2 family members in two distinct features. First, Diva lacked critical residues that mediate the interaction between BH3-containing proapoptotic Bcl-2 homologues and their prosurvival binding partners. In the NMR structure of Bcl-X L /Bak BH3 peptide complex, the charged side chain of D83 in the Bak peptide interacts with oppositely charged R139 of Bcl-X L (26). All reported BH3-containing proapoptotic Bcl-2 homologues contain a conserved counterpart of Bak D83 (Fig. 1C; Refs. 12-23). Significantly, the counterpart of Bak D83 in Diva is R47, an amino acid that cannot form ionic interactions with positively charged residues (Fig. 1C). In addition, the BH3 of proapoptotic family members contains amino acids with charged side chains corresponding to positions 76 and 84 of Bak that, in the case of Bak, interact with oppositely charged residues in the BH1 of Bcl-X L (26). The counterpart residues in Diva are A47 and Q48, respectively (Fig. 1C). Conversely, all known prosurvival Bcl-2 family members contain a conserved R or K residue in BH1. In Bcl-X L , the corresponding R residue is located at position 139 and interacts with residue D83 located in the BH3 peptide of Bak (26). The substitution of R139 for Q in Bcl-X L results in a loss of antiapoptotic function and the ability to associate with Bax (26). Significantly, the R139 counterpart in Diva is Q88, an amino acid that cannot form ionic bonds with negatively charged residues. Thus, Diva lacks conserved residues that are critical for heterodimerization between proapoptotic and prosurvival Bcl-2 family members. In addition, Diva contains an extended stretch of eleven amino acids between ␣5 and ␣6 that is not present in known Bcl-2 family members (Fig. 1C).
Diva mRNA Is Expressed in Embryonic Tissues and in Adult Ovary and Testis-We performed Northern blot analysis to assess the expression of diva mRNA in various mouse tissues. Hybridization with a Diva probe revealed undetectable expression in the brain, liver, heart, lung, and spleen from adult mice and humans. 2 Because all 22 EST clones of Diva mRNA were derived from oocytes, one-or two-cell-stage embryos, we evaluated the distribution of Diva mRNA in mouse embryos and the reproductive organs of adult mice by in situ hybridization. At stage E15 of embryonic development, intense Diva mRNA labeling was detected in the brain, liver, and heart ( Fig. 2a-c). In adult mice, the expression of Diva was restricted to the reproductive organs. Diva mRNA was detected in granulosa cells of the ovary (Fig. 2, d and e) and the seminiferous tubules of the testis (Fig. 2, g and h). Within the tubules, Diva was expressed in spermatids but not in spermatogonia, spermatocytes, or mature spermatozoa (Fig. 2h). The focal pattern of Diva labeling in seminiferous tubules is consistent with a stage-specific expression during spermatogenesis, because the plane of the section transects the neighboring tubules in different stages of the spermatogenic cycle.
To verify the results of in situ hybridization, the expression of Diva was evaluated by reverse transcription-PCR. Expression of Diva was detected in all embryonic tissues tested (Fig.  3A). After birth, however, Diva mRNA expression dramatically decreased in the liver, brain, heart, lung, and spleen, but it was retained in the adult ovary and testis (Fig. 3B). These results are consistent with those presented in Fig. 2; taken together, they indicate that the expression of Diva mRNA is highly restricted to reproductive tissues in the adult and differs considerably from that reported for other Bcl-2 family members including Bcl-2, Bcl-X L , A1, Bax, and Mtd (22, 30, 38).

Diva Is a Proapoptotic Bcl-2 Family
Member-To begin to assess the biological function of Diva, we constructed a Myctagged Diva expression vector, pcDNA3-Myc-Diva, and transiently transfected the plasmid into 293T kidney cells, Ramsey melanoma cells, and T47D breast cancer cells. A significant percentage of Diva-transfected cells displayed morphological features of adherent cells undergoing apoptosis such as rounding, membrane blebbing, and detachment from the dish when compared with cells transfected with the control plasmid (see below, Fig. 4A). 24 h after transfection, about 50% of the Divatransfected 293T cells underwent apoptosis compared with less than 1% of cells transfected with the empty plasmid ( Fig. 4A; p Ͻ 0.001). The killing activity of Diva was inhibited by baculoviral caspase inhibitor p35 and by a catalytically inactive mutant of caspase-9 (Fig. 4A), suggesting that caspase-9 is involved in the killing activity mediated by Diva. To date, we have been unable to generate any stable 293 cell lines expressing Diva, which is consistent with the observation that Diva 2 N. Inohara and G. Nú ñ ez, unpublished results. causes apoptosis in 293 cells. 2 In addition to 293T cells, the expression of Diva promoted the killing of Ramsey and T47D cells when compared with that of the control plasmid (Fig. 4, B  and C). Similarly, microinjection of the Diva cDNA into primary sensory neurons promoted their death (18 Ϯ 8% survival) after 40 h of nerve growth factor deprivation when compared with neurons injected with control plasmid (35 Ϯ 3% survival).
Apoptosis Induced by Diva Is Inhibited by vBcl-2-Next we tested the ability of vBcl-2 to antagonize apoptosis mediated by Diva in 293T cells. The morphologic features of apoptosis induced by Diva, including rounding and membrane blebbing (Fig. 5B) as well as nuclear fragmentation (Fig. 5C) were inhibited by vBcl-2. Quantitative analysis confirmed that the apoptotic activity of Diva was significantly inhibited by vBcl-2, but not by Bcl-X L (Fig. 5D).
Mutants of Diva Lacking the Conserved BH3 Region Retain their Proapoptotic Activity and the Ability to Interact with vBcl-2-The lack of a critical residue in the BH3 region of Diva suggested that Diva mediates apoptosis independently of the BH3 domain. We engineered two mutant forms of Diva (Diva ⌬39 -51 and Diva ⌬33-52) in which the BH3 region was deleted by PCR mutagenesis to directly test the hypothesis that Diva kills independently of the BH3 region. Diva ⌬39 -51 was expressed at levels comparable to that of wild-type Diva (Fig. 6B).
Expression of Diva ⌬39 -51 induced the apoptosis of 293T as well as wild-type Diva (Fig. 6A). Identical results were obtained with Diva ⌬33-52. 2 Immunoprecipitation analysis revealed that Diva ⌬39 -51 and Diva ⌬33-52 interact with vBcl-2 ( Fig.  6B; data not shown), indicating that the BH3 region is dispensable for the association of Diva with vBcl-2. These results indicate that Diva induces apoptosis and heterodimerizes with vBcl-2 in a BH3-independent manner.
Diva Interacts with Apaf-1 and Functions as a Competitor of Bcl-X L for Apaf-1 Binding-We reasoned that Diva might interact with a cellular target other than Bcl-2 family members to induce apoptosis. Because apoptosis induced by Diva was inhibited by a catalytically inactive mutant of caspase-9, we tested the ability of Diva to associate with Apaf-1, an upstream regulator of caspase-9 (9 -11). In the two-hybrid assay, Diva interacted with Apaf-1, but not with Bcl-X L , Bcl-2, Bcl-w, Bfl-1/A1, Mcl-1, or Bax (Fig. 7). In addition, Diva was unable to interact with adenovirus E1B 19K, Hrk, Bak, or Mtd in the two-hybrid assay. 2 We verified the interaction between Diva and Apaf-1 in mammalian cells and tested whether Diva might block the binding of Bcl-X L to Apaf-1, because Bcl-X L can also interact with Apaf-1 (10,11). 293T cells were co-transfected with plasmids producing HA-tagged Diva, HA-tagged Bcl-X L , and Myc-tagged Apaf-1. Immunoprecipitation of Apaf-1-Myc co-immunoprecipitated Diva (Fig. 8A), in agreement with the two-hybrid results. We also detected an interaction between Diva and CED-4, the nematode Apaf-1 homologue, 2 which is consistent with the observation that Diva interacts with the amino-terminal half of Apaf-1, the region that is homologous to CED-4 (9). As reported previously (10 -11), Bcl-X L was coimmunoprecipitated with Apaf-1 (Fig. 8A). Importantly, the association of Bcl-X L and Apaf-1 was inhibited by the expression of Diva, as determined by immunoblotting with an anti-HA antibody (Fig. 8A). In contrast, the binding of Diva to Apaf-1 was not significantly affected by Bcl-X L (Fig. 8A). Immunoblotting of the lysates showed comparable levels of Bcl-X L , Apaf-1, and Diva (Fig. 8A, bottom panel), indicating that the results were not due to the differential expression of these proteins. To verify the results, we performed reciprocal experiments in which Bcl-X L was immunoprecipitated with anti-Flag antibody. As observed in the reciprocal experiment, the association of Bcl-X L and Apaf-1 was inhibited in the presence of Diva (Fig. 8B). In agreement with the two-hybrid assay, Diva did not associate with Bcl-X L (Fig. 8C). We conclude that Diva binds to Apaf-1 and acts as an antagonistic competitor of Bcl-X L for Apaf-1 binding. In this study, we show that Diva is a proapoptotic protein that binds to the caspase-9 regulator Apaf-1. In the adult, the expression of Diva is restricted to the granulosa cells of the ovary and the germ cells of the testis, two cell types in which apoptosis plays a critical role during follicular maturation and spermatogenesis, respectively. The expression of Diva is re-  stricted to spermatids, a cell stage that undergoes a complex differentiation process that includes a loss of the majority of the cytoplasm before being released as mature sperm into the lumen of the seminiferous tubules. These cytoplasmic remnants containing degenerating excess organelles are engulfed by neighboring Sertoli cells. Thus, it is possible that Diva is involved in the dissolution of the cytoplasm, because the formation and engulfment of these cytoplasmic remnants resembles, in part, the apoptotic process. Bax is also expressed in germ cells of the testis but exhibits a cellular pattern that is different from that of Diva (39). Although both Diva and Bax are proapoptotic proteins that inhibit the binding of Bcl-X L to Apaf-1, they appear to act through different mechanisms. Bax has been shown to heterodimerize with Bcl-X L but not with Apaf-1 (10). The interaction between Bax and Bcl-X L prevents the binding of Bcl-X L to Apaf-1 (10). In contrast, Diva associates with Apaf-1 but not with Bcl-X L and acts as a competitor of Bcl-X L for Apaf-1 binding. Therefore, cell death in the ovary and testis can be regulated through proapoptotic Bcl-2 family members by at least two different mechanisms. Bok/Mtd is another cell death agonist of the Bcl-2 family that is expressed in the granulosa cells of the ovary and in the testis (29). Unlike Bax, Bok/Mtd predominantly binds to Mcl-1 and A1, but not to Bcl-2 and Bcl-X L (29). Thus, cells utilize different proapoptotic Bcl-2 homologues and strategies to block the function of prosurvival Bcl-2 family members during oogenesis and spermatogenesis.