Bcl-rambo, a Novel Bcl-2 Homologue That Induces Apoptosis via Its Unique C-terminal Extension*

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 22, Issue of June 1, pp. 19548 19554, 2001 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Bcl-rambo, a Novel Bcl-2 Homologue That Induces Apoptosis via Its Unique C-terminal Extension* Received for publication, November 21, 2000, and in revised form, March 20, 2001 Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M010520200 Takao Kataoka, Nils Holler, Olivier Micheau, Fabio Martinon, Antoine Tinel, Kay Hofmann**, and Jürg Tschopp From the Institute of Biochemistry, University of Lausanne, Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland and **MEMOREC Stoffel GmbH, Stoeckheimer Weg 1, D-50829 Koeln, Germany The Bcl-2 family of proteins plays a central regulatory role in apoptosis. We have identified a novel, widely expressed Bcl-2 member which we have named Bclrambo. Bcl-rambo shows overall structural homology to the anti-apoptotic Bcl-2 members containing conserved Bcl-2 homology (BH) motifs 1, 2, 3, and 4. Unlike Bcl-2, however, the C-terminal membrane anchor region is preceded by a unique 250 amino acid insertion containing two tandem repeats. No interaction of Bcl-rambo with either anti-apoptotic (Bcl-2, Bcl-x L, Bcl-w, A1, MCL-1, E1B-19K, and BHRF1) or pro-apoptotic (Bax, Bak, Bik, Bid, Bim, and Bad) members of the Bcl-2 family was observed. In mammalian cells, Bcl-rambo was localized to mitochondria, and its overexpression induces apoptosis that is specifically blocked by the caspase inhibitors, IAPs, whereas inhibitors controlling upstream events of either the death receptor (FLIP, FADD-DN) or the mitochondrial pro-apoptotic pathway (Bcl-x L ) had no effect. Surprisingly, the Bcl-rambo cell death activity was induced by its membrane-anchored C-terminal domain and not by the Bcl-2 homology region. Thus, Bclrambo constitutes a novel type of pro-apoptotic Bcl-2 member that triggers cell death independently of its BH motifs. mammalian Bad, Bik/Nbk, Blk, Hrk, Bid, Bim/Bod, Noxa, and C. elegans EGL-1 share homology only within the short BH3 motif and are therefore called BH3-only proteins (2, 3). Most members of the wider family possess a hydrophobic C-terminal segment, which facilitates their interaction with the endoplasmic reticulum/nuclear envelope and/or the outer mitochondrial membrane, where the prosurvival members normally reside, and where most others assemble during apoptosis. The ability of the prosurvival and antisurvival family members to form heterodimers raises the possibility that each type might titrate the other, potentially accounting for their opposing actions. The relative concentrations of the opposing subfamily members would then determine whether the cell lives or dies (4, 5). Mutagenesis of Bcl-2 initially suggested that its ability to inhibit cell death required binding to a pro-apoptotic family member (6), but Bcl-x L mutants have been identified that do not bind Bax or Bak yet still block apoptosis (7). Thus, it remains unclear whether the ability to associate with other family members is central to regulating apoptosis. To search for additional regulators of apoptosis, we have screened public data bases with a profile defining Bcl-2 family members. This search has yielded a novel BH4/BH3/BH1/BH2- containing protein, which we have denoted Bcl-rambo. Here we report its initial characterization. Apoptosis, the physiological process of cell death, is critical for modeling tissues and maintaining homeostasis in multicellular organisms (1). Members of the Bcl-2 family of proteins are regulators of apoptosis that can be grouped into three subfamilies (2, 3). Bcl-2 and several of its close relatives including Bcl-x, Bcl-w, and Caenorhabditis elegans CED-9 inhibit apoptosis, whereas structurally similar relatives such as Bax, Bak, Bok, and distant cousins such as Bik and Bim instead foster death. The Bcl-2 and Bax subfamilies share three of the four conserved Bcl-2 homology (BH) 1 sequence motifs. In contrast, * This work was supported in part by a grant of the Swiss National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This 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 GenBank TM /EBI Data Bank with accession number(s) AF325209. Present address: Dept. of Bioengineering, Tokyo Inst. of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. These authors contributed equally to this work. Supported by a grant of the Alliance des Recherches sur le Cancer. To whom correspondence should be addressed. Tel.: 41-21-692-5738; Fax: 41-21-692-5705; E-mail: Jurg.tschopp@ib.unil.ch. 1 The abbreviations used are: BH, Bcl-2 homology; aa, amino acid(s); PCR, polymerase chain reaction; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered solution; MA, membrane anchor; VSV, vesicular stomatitis virus. 19548 EXPERIMENTAL PROCEDURES Cloning and Sequences The expressed sequence tag clone T48205 encodes a short splice form of human Bcl-rambo that shares the N- terminal 1 200-amino acid (aa) of full-length Bcl-rambo. The expressed sequence tag clone AA190545 contains C-terminal 255 485-aa region. A cdna library was used to amplify the fragment containing the truncated part (aa 201 254) of Bcl-rambo using 5 -forward primer JT1108 (5 -CCT TCA CCA GCA CAG GCT TTG ACC G-3 ) and 3 -backward primer JT1109 (5 -GGA CCA CCT CCT CCA CTT CAG TAG G-3 ). The PCR product was cloned into PCR-Blunt (Invitrogen) in the counterclockwise direction of the vector. The NotI/BamHI fragment was inserted into the NotI and BamHI site of AA190545, which yielded the 78 485-aa fragment of Bcl-rambo. Double PCR was carried out to construct full-length Bcl-rambo. Two DNA fragments were amplified from the plasmid carrying Bcl-rambo (78 485) using 5 -primer JT1145 (5 -CAT ACC TCG AGG ACT ATT C-3 ) and 3 -primer flanked by NotI site JT1144 (5 -TAG CGG CCG CCT ATT TCT TTC TCA G-3 ), and T48205 using 5 -primer flanked by EcoRI site JT1143 (5 -AAA GAA TTC ATG GCG TCC TCT TCT AC-3 ) and 3 -primer JT1146 (5 -GAA TAG TCC TCG AGG TAT G-3 ), respectively. Both DNA fragments were mixed and amplified by PCR without primers followed by amplification with primers JT1143 and JT1144. The PCR product was cloned into PCR-Blunt, and the EcoRI/NotI fragment was subcloned into a modified version of PCR3 (Invitrogen) in frame with a N-terminal FLAG or HA peptide. Blast searches of GenBank TM revealed a 150,172- base pair human BAC clone (p1087l10) in the genomic data base derived from chromosome 22q11 (GenBank TM AC006285), which covers the complete Bcl-rambo gene. Plasmid Construction The DNA fragment corresponding to Bclrambo (1 201) was amplified by PCR from T48205 with 5 -forward This paper is available on line at http://www.jbc.org

Bcl-rambo, a Novel Member of the Bcl-2 Family 19549 FIG. 1. Sequence of Bcl-rambo and comparison to anti-apoptotic members of the Bcl-2 family. A, the predicted amino acid sequences of human Bcl-rambo with the predicted BH1, BH2, BH3, BH4, and membrane anchor (MA) region boxed are aligned to the sequences of Bcl-2, Bcl-x L, and Bcl-w. The locations of the two tandem repeats are indicated. Identical and similar amino acids are shown in black and gray blocks, respectively. B, alignment of the tandem repeats. C, exon-intron organization of the human Bcl-rambo gene. The positions of the start and stop codons as well as the BH domains are indicated. The non-coding region is shown in gray. primer containing EcoRI site JT993 (5 -AAA GAA TTC ATG GCG TCC TCT-3 ) and 3 -reverse primer containing NotI site JT994 (5 -TAG CGG CCG CTC ATA CCC AGC CAC C-3 ) and cloned into PCR-Blunt. Other deletion mutants were constructed by PCR amplification using fulllength Bcl-rambo as a template with 5 -forward primer flanked by EcoRI and 3 -reverse primer flanked by NotI as follows: Bcl-rambo (1 223), 5 -primer JT1143 and 3 -primer JT1244 (5 -AT AGC GGC CGC CTA GTC ATT GCT ATC TTC GT-3 ); Bcl-rambo (1 459), 5 -primer JT1143 and 3 -primer JT 1245 (5 -AT AGC GGC CGC CTA AGA CTT GCC CTC AGA C-3 ); Bcl-rambo (205 485), 5 -primer JT1251 (5 -AAA GAA TTC AGT CTT GAG TCA GAG G-3 ) and 3 -primer JT1144; Bcl-rambo (205 459), 5 -primer JT1251 and 3 -primer JT1245. The EcoRI/NotI fragments were subcloned into a modified version of PCR3 with N-terminal FLAG epitope: Bcl-rambo ( 207 456); the BHNo region was deleted according to standard procedures. Cell Lines and Reagents The cell lines used here were grown and maintained as previously described (8). zvad-fmk was purchased from Alexis. The antibody against Bcl-rambo (AL167) was generated in rabbits against bacterial recombinant Bcl-rambo encompassing aa 1 223. The antibody was affinity purified on immobilized antigen according to standard procedures. Northern Blots Northern blots (CLONTECH, Palo Alto, CA) were hybridized using random-primed radioactive probes in ExpressHyb buffer (CLONTECH) for about 3 h and then washed at room temperature for 40 min with several changes of 2 SSC/0.1% SDS followed by 0.1 SSC/0.05% SDS for 40 min at 50 C. The blots were probed human Bcl-rambo or actin (data not included). Transient Cell Transfection, Immunoprecipitation, and Western Blotting For co-immunoprecipitation experiments, approximately 3 10 5 293T cells/10-cm culture dish were transfected with the indicated expression plasmids by the calcium phosphate/hepes method (9). Cells were harvested 30 h after transfection and lysed in complete Nonidet P-40 lysis buffer (1% Nonidet P-40 lysis buffer with 20 mm Tris-HCl, ph 7.4, 150 mm NaCl, and protease inhibitor mixture (Complete, Roche Molecular Biochemicals) on ice. Postnuclear lysates were normalized for protein content, precleared on Sepharose 6B (Sigma) for 2 h at 4 C, and subsequently subjected to immunoprecipitation with anti-flag-m2-agarose beads (Sigma) for at least 2 h at 4 C on a rotating wheel. Immunoprecipitates were washed twice each with lysis buffer containing 1% or 0.05% Nonidet P-40, respectively, and washed beads were boiled in reducing sample buffer before analysis by SDS- PAGE and Western blotting. FLAG-, HA- and EE-tagged proteins were detected using anti-flag M2 (Sigma), the anti-ha (Babco) and anti-ee (Eurogentec) monoclonal antibodies, respectively. Cellular (endogenous) Bcl-rambo was detected in cell lysate (50 g) using an affinity-purified polyclonal rabbit antibody (AL167) directed against a recombinant protein encompassing aa 1 223 of human Bcl-rambo. Blots were revealed with peroxidase-coupled goat anti-mouse or anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories). Apoptosis Assay 293T cells (3 10 5 cells) were seeded on 60-mm culture plates the day before transfection and transiently co-transfected with 2 g of Bcl-rambo expression plasmids together with 0.2 g of EGFP expression vector (pegfp-c1, CLONTECH) or 0.5 g of pcmv-

19550 Bcl-rambo, a Novel Member of the Bcl-2 Family FIG. 2. Expression of Bcl-rambo. Northern blots of various human tissues (A) and tumor cell lines (B) (CLONTECH) were probed with a random-primed 32 P- labeled cdna fragment of the N-terminal part of Bcl-rambo. B, expression of Bclrambo protein in various human and mouse (mu) cell lines. Western blot analysis was performed with an affinity-purified antibody (AL167) generated against the Bcl-2 homology region of Bcl-rambo (aa 1 223,); Jurkat and EL-4 (T cell leukemias); A20, Raji, Ramos, Bjab, and Raw (B cell lymphomas); THP-1, U937, and K562 (monocytic cell lines); HeLa (cervical carcinoma); HEK293 and 293T (embryonic kidney cell line). D, various FLAG-tagged Bcl-rambo expression plasmids (aa 1 485, 1 223, 205 485) were transfected into 293T cells, and cell extracts were assayed using the anti-flag antibody M2 (left panel) or the polyclonal antibody against Bcl-rambo (right panel). -galactosidase expression vector. Postnuclear lysates were used to measure fluorescence intensity of EGFP, -galactosidase activity, and caspase-3 activity. Caspase-3 activity was measured by mixing 10 l of cell lysate (30 40 g of protein) with 100 l of reaction buffer (10 mm Tris-HCl, ph 7.4, 0.1% CHAPS, 2 mm MgCl 2,1mM dithiothreitol, 5 mm EGTA, and 150 mm NaCl) containing 50 M of a fluorogenic caspase-3 substrate (Ac-DEVD-AMC, Alexis). The mixture was incubated for 60 min in an enzyme-linked immunosorbent assay titer plate and fluorescence was measured in a Fluoroskan enzyme-linked immunosorbent assay reader (excitation 355 nm, emission 460 nm). The caspase-3 activity (fold induction) was normalized with the fluorescence intensity of EGFP or -galactosidase activity, respectively. Subcellular Localization 293T cells or HeLa cells (1 106 cells, 100-mm dish) were seeded the day before transfection. 293T cells were transfected with 0.5 g of FLAG-tagged Bcl-rambo constructs or FLAGtagged Bcl-2 plus 9.5 g of empty vectors for 12 h, whereas HeLa cells were transfected with 5 g of FLAG-tagged Bcl-rambo constructs plus 5 g of empty vectors for 12 h. Cells were collected from four dishes and washed twice with PBS. The cells were disrupted in 250 mm sucrose, 10 mm HEPES-OH (ph 7.4), 1 mm EDTA, 1 mm EGTA, and protease inhibitor mixture by dounce homogenizer (20 strokes), and resultant cell lysates were centrifuged (800 g, 10 min) to remove unbroken cells and nuclei. Postnuclear lysates were centrifuged (10,000 g, 10 min) to yield heavy membrane fractions enriched with mitochondria. The supernatants were then centrifuged (100,000 g, 60 min) to separate cytosolic fraction (S100) and light membrane fractions (P100). Proteins were separated by 10% SDS-PAGE and blotted with anti-flag and anti-bcl-2 antibodies. Cytochrome c Release For cytochrome c release, cells were incubated for 10 min on ice in 20 mm HEPES, ph 7.5, 10 mm KCl, 15 mm MgCl 2,1mM EDTA, and 1 mm EGTA containing protease inhibitors and then lysed with 15 strokes in a syringe (gauge G22). The lysate was incubated for 10 min on ice and then centrifuged. Supernatants and pellets were resuspended in reducing Laemmli s buffer and probed for the presence of cytochrome c by Western blotting (PharMingen). Immunostaining and Confocal Laser Scanning Microscopy The day before transfection, HeLa cells were plated at 10 20% confluency on 20-mm glass coverslips in 5.5-cm culture plates. Cells were transfected the following day by the calcium phosphate/bes method and harvested 36 h after onset of transfection. Cells on coverslips were washed twice with cold PBS, fixed for 12 min in 4% paraformaldehyde at room temperature, washed twice with cold PBS, and permeabilized in PBS with 0.1% saponin (PBSS) overnight at 4 C. Coverslips were blocked for 30 min at room temperature with 5% milk in PBSS (PBSSM), stained with primary antibody anti-bcl-rambo AL167 (1 g/ml) for 1 h at room temperature, washed three times with PBSS and stained with Alexa-488-labeled (Molecular Probes) secondary anti-rabbit antibody diluted 1/100 in PBSSM for 1hinthedark. For mitochondrial staining, cells were incubated for 1 h in the presence of anti-smac/diablo antibodies (Alexis) and subsequently with the anti-mouse Alexa-546 secondary antibody. Finally, coverslips were washed three times with PBS and mounted with FluorSave reagent (Calbiochem) on microscope slides. Confocal microscopy was performed on a Zeiss Axiovert 100 microscope (Zeiss Laser Scanning Microscope 510). RESULTS Identification and Sequence of Bcl-rambo Using the prf.bcl-2 profile 2 as a search tool, we screened public data bases for novel Bcl-2 homologues and identified Bcl-rambo. Subsequent DNA sequencing and identification of additional expressed sequence tags allowed the isolation of the full-length human Bcl-rambo cdnas. The open reading frame of Bcl- 2 F. Martinon, K. Hofmann, and J. Tschopp, unpublished data.

Bcl-rambo, a Novel Member of the Bcl-2 Family 19551 FIG. 3. Subcellular localization of Bcl-rambo. A, HeLa cells were transfected with plasmids encoding full-length Bcl-rambo (1 485) or Bcl-rambo lacking the C-terminal membrane anchor domain ( MA, 1 459), and their subcellular localization was determined 48 h after transfection by confocal laser microscopy using the anti-bcl-rambo antibody AL167 or an antibody detecting Smac/DIABLO. The overlay of Bcl-rambo fluorescence (green) and Smac/DIABLO (red) is shown in yellow. Bar, 10 m. B, 293T cells and HeLa cells were transfected with the various Bcl-rambo constructs (see Fig. 5), and subcellular localization was analyzed by separating heavy membranes (HM, including mitochondria), light membranes (LM), and cytosol. As marker protein, VSV-tagged Bcl-2 was co-transfected in 293T cells and detected using an anti-flag antibody. In both cell lines, endogenous (end) Bcl-2 protein was detected with an anti-bcl-2 antibody. rambo encodes a protein of 485 amino acids (Fig. 1A). The methionine initiation codon is preceded by a typical consensus Kozak sequence and hydrophobicity analysis predicts the presence of a C-terminal transmembrane domain. Comparison of protein sequences among different Bcl-2 proteins indicated that Bcl-rambo contained all four BH (BH1, -2, -3 and -4) motifs that identify anti-apoptotic Bcl-2 members (Fig. 1A). Although in most other Bcl-2 members containing more than one BH domain the Bcl-2 homology region is followed by the C-terminal membrane anchor, these two regions are separated in Bclrambo by a stretch of more than 250 aa that show no obvious sequence homology with any other protein in the data base; we named this domain BHNo domain because it contains no BH motif. Of note is the presence of two distinct short tandem repeats (repeat A and repeat B, Fig. 1B) within the BHNo domain. The presence of a zebrafish homologue containing the BHNo domain (data not shown) suggests that Bcl-rambo is highly evolutionarily conserved. Using the cdna of Bcl-rambo, a human BAC clone from chromosome 22q11 was identified in the human genomic data base covering the Bcl-rambo sequence. Comparison with the cdna shows that the coding region is encompassed by six exons, displaying the typical genetic structure of Bcl-2 family members (Fig. 1C). Interestingly, the C-terminal part of the BH2 domain, the large BHNo insert, and the membrane anchor domain are encoded by a single exon. Expression and Subcellular Localization of Bcl-rambo The expression of Bcl-rambo mrna in diverse human tissues was examined. As shown in Fig. 2A, a Bcl-rambo transcript of 4.1 kilobases was detected in all tissues studied. Highest mrna levels were found in heart, pancreas, and placenta. Two weaker bands of 2.1 and 1.2 kilobases may correspond to alternatively spliced forms. Human cell lines of different origin expressed Bcl-rambo mrna at various levels (Fig. 2B) in agreement with the observation that the Bcl-rambo protein was detectable in all cell lines tested (Fig. 2C). Bcl-rambo has a predicted molecular mass of 55 kda. Hence, the apparent high molecular mass of 85 kda deduced from SDS-PAGE was surprising. Expression of various fragments of Bcl-rambo indicated that the unusual migration property of the protein was caused by the BHNo domain that exhibited an apparent molecular mass of 52 kda as compared with its predicted molecular mass of 30 kda (Fig. 2D). Based on confocal microscopic analysis of HeLa cells transfected with a Bcl-rambo expression plasmid, Bcl-rambo protein was found to be cytosolic, localizing to intracellular organelles in a pattern typical of Bcl-2 family proteins (Fig. 3A). Identical results were obtained when 293T cells were analyzed (data not shown). The majority of Bcl-rambo co-localized with the IAP inhibitor Smac/DIABLO (11, 12) to mitochondria. When the C-terminal membrane anchor was removed, the punctate cytoplasmic staining disappeared and was replaced by a diffuse nuclear staining (Fig. 3A). Thus, in agreement with the crucial role of this region in the subcellular distribution of other Bcl-2 family proteins (reviewed in Refs. 2 and 3), the apparent mitochondrial localization of Bcl-rambo is dictated by its hydrophobic C terminus. The subcellular localization studies were confirmed by cellular fractionation studies (Fig. 3B). Full-length Bcl-rambo and Bcl-rambo lacking the BHNo domain ( 207 459; BHNo in Fig. 5) were both recovered in heavy membrane fractions containing mitochondria, whereas the full-length Bcl-rambo lacking the membrane anchor domain was present mostly in the fraction containing soluble proteins. An identical subcellular fractionation pattern was found with Bcl-2. Absence of Interaction with Other Bcl-2 Members Prosurvival and antisurvival Bcl-2 family members are known to frequently form heterodimers (reviewed in Ref. 13). We thus investigated interactions between Bcl-rambo and different anti- and pro-apoptotic Bcl-2 family proteins using the 293T overexpression system. As shown in Fig. 4A, there was no interaction found with the pro-apoptotic members Bax, Bak, Bik, Bid, Bim, Bad, and Bcl-rambo, whereas the interaction of these BH3-only members with Bcl-x L (Fig. 4C) was readily demonstrable. Of interest, the co-expression of some of these proapoptotic Bcl-2 members with Bcl-rambo significantly decreased their expression (Fig. 4A). To demonstrate that the lack of interactions between Bcl-rambo and pro-apoptotic Bcl-2 members was not due to increased cell death of transfected cells, the experiments were repeated in the presence of the Pan-caspase inhibitor z-vad-fmk; the outcome, however, was identical. We further tested interactions between Bcl-rambo and several anti-apoptotic Bcl-2 members (Bcl-2, Bcl-x L, Bcl-w, A1, MCL-1, E1B-19K, and BHRF1) (Fig. 4B); but again no interaction was observed. Bcl-rambo also did not interact with Bcl-2 family members only when the Bcl-2 homology region of Bcl-rambo (aa 1 223) was expressed, and moreover, no Bclrambo homodimerization was detected (data not shown). Induction of Apoptosis by Bcl-rambo The ability of Bclrambo to regulate apoptosis in mammalian cells was investigated. In 293T cells, transfection with expression vectors encoding Bcl-rambo resulted in significant caspase-3 activation and cell death (Fig. 5A). The pro-apoptotic effect of Bcl-rambo was specific because transfection of the empty plasmid did not affect cell survival. Overexpression of the N-terminal Bcl-2

19552 Bcl-rambo, a Novel Member of the Bcl-2 Family FIG. 4. No association of Bcl-rambo with various Bcl-2 family members. 293 T cells were transiently transfected with FLAG- (A) or HA-tagged (B) Bcl-rambo together with FLAG-tagged anti-apoptotic Bcl-2 members or EE-tagged pro-apoptotic Bcl-2 members in the presence of z-vad-fmk. The postnuclear lysates were immunoprecipitated with anti-flag M2-agarose and blotted with anti-ee or anti-ha antibody, respectively. C, control experiment: 293T cells were transfected with FLAG-tagged Bcl-x L together with EE-tagged Bax, Bak, Bik, Bid, Bim, or Bad for 22 h in the presence of z-vad-fmk. The postnuclear lysates were immunoprecipitated with anti-flag M2-agarose and blotted with an anti-ee antibody. FIG. 5.Bcl-rambo induces apoptosis via its C-terminal domain. A, 293T cells were transfected with 2 g of various Bcl-rambo constructs (right panel) together with 0.2 g of the EGFP expression vector (pegfp-c1) for 42 h. The activity of caspase-3 and the fluorescence intensity of EGFP in duplicate determinations was measured. The caspase-3 activity (fold increase) was normalized with the fluorescence intensity of transfected EGFP. Protein expression was assessed in cell extracts by Western blot analysis using the anti-flag antibody M2. B, release of cytochrome c from mitochondria was determined in 293T cells transfected with Bcl-rambo and for control purposes with Bax and Bak. homology region (aa 1 223 or 1 203) had little effect on cell viability, whereas the expression of the C-terminal BHNo domain (aa 205 485) induced slightly higher caspase-3 activation than that observed with the full-length protein despite identical protein expression (Fig. 5A). The pro-apoptotic activity of Bcl-rambo and its BHNo domain was completely dependent on the C-terminal membrane anchor (MA) as its removal resulted in the complete loss of cell death activity. We therefore investigated whether the pro-apoptotic activity of the C-terminal region of Bcl-rambo was solely due to its MA region or whether

Bcl-rambo, a Novel Member of the Bcl-2 Family 19553 FIG. 6. Bcl-rambo-induced cell death is blocked by IAPs. A, 293 T cells were transfected with 2 g of Bcl-rambo plasmid and 0.5 g of expression vectors encoding various inhibitors of apoptosis together with 0.5 g ofthe -galactosidase expression vector for 27 h. B, asina but 293 T cells were transfected with 2 g of Bcl-rambo encoding aa 205 485 (BHNo domain and membrane anchor domain). The activity of caspase-3 and -galactosidase in duplicate determinations was measured. The caspase-3 activity (fold increase) was normalized based on the -galactosidase activity. the presence of the BHNo domain was also required. A mutant protein was generated where the MA region was directly fused to the BH domain, thus lacking the BHNo region ( BHNo). Bcl-rambo BHNo induced apoptosis when overexpressed although with reduced efficiency (Fig. 5A). We conclude that the MA as well as the BHNo domain are important for optimal pro-apoptotic activity. Bax and Bak induce apoptosis by causing cytochrome c release from mitochondria which then binds to APAF-1 resulting in caspase-9 activation and apoptosis. Similar to Bax and Bak, overexpression of Bcl-rambo resulted in the appearance of cytochrome c in the cytosol (Fig. 5B) indicating that mitochondrial damage occurs during Bcl-rambo-induced cell death. Bcl-rambo-induced cell death was caspase-dependent and was completely blocked by the Pan-caspase-inhibitor zvad-fmk (data not shown). In contrast, co-expression of c-flip (14), a dominant negative form (DN) of FADD (15), CrmA (16) (Fig. 6), suggests that Bcl-rambo induces cell death independent of the death receptor pathway. Moreover, Bcl-x L as well as DN-caspase-9 (17) only marginally prevented Bcl-rambo-induced cell killing indicating that cell death could still proceed in the absence of an active cytochrome c-apaf1-caspase-9 pathway. Only IAPs (18) displayed a strong inhibitory effect, in particular X-IAP and to a lesser extend c-iap1 and c-iap2. These IAP members have been shown to bind and inactivate caspase-3, -6, and to a lesser degree caspase-9, but not caspase-1, -6, -8, or -10 (18). DISCUSSION In this report we describe the cloning and the initial characterization of a novel Bcl-2 family member, Bcl-rambo. Bclrambo contains all four conserved BH motifs and a C-terminal membrane anchor and is therefore predicted to be a member of the anti-apoptotic Bcl-2 subfamily. Unlike other members of this subfamily, such as Bcl-2 or Bcl-x L, the BHcontaining domain is separated from the membrane anchor by a unique 250-aa-long region. This C-terminal BHNo domain, which shares no significant sequence homology with other proteins in the data base, reveals very little information about a possible domain structure and function. The only notable feature is the presence of two short tandem repeats that both are rich in serines. One is located at a position where Bcl-2 members usually contain the membrane anchor domain, whereas the longer second repeat unit is found further downstream immediately preceding the transmembrane domain of Bcl-rambo (see Fig. 1). Surprisingly, it is this BHNo domain and not the Bcl-2 homology region that more efficiently induces cell death upon overexpression. It is currently unclear how this segment causes apoptosis. Bcl-rambo is localized to mitochondria, and because removal of the C-terminal membrane anchor results not only in the loss of mitochondrial localization but also of its cell death activity, it is likely that Bcl-rambo initiates the cell death pathway by inflicting damage to mitochondria. Indeed, cytochrome c release was detected upon Bcl-rambo overexpression. For the pro-apoptotic Bcl-2 members such as Bax, it was proposed that they cause mitochondrial outer membrane damage by forming channels in agreement with their structural homology with bacterial toxins (19). Bclrambo appears to induce apoptosis, at least in part, independently of known mitochondrial signaling pathways. Overexpression of Bcl-x L (Fig. 6) and Bcl-2 (data not shown), which are known to efficiently block apoptosis induced by pro-apoptotic Bcl-2 members by inhibiting cytochrome c release, had little effect on Bcl-rambo-mediated cell death. Moreover, a dominant-negative form of caspase-9, which blocks apoptosis induced by Apaf-1, showed only a moderate inhibitory effect suggesting that Bcl-rambo death activity is not dependent on the classical mitochondrial pathway. This conclusion is supported by the absence of interaction with more than 10 different Bcl-2 pro- and anti-apoptotic family members tested. Similarly, FLIP and a dominant-negative form of FADD, both potent inhibitors of the death receptor signaling pathway, do not block the Bcl-rambo-induced caspase-3 activation. Only inhibitors that interfere with the activity of the downstream caspase-3, such as members of the IAP family, are effective at blocking Bcl-rambo death activity. The p53-induced protein p53aip1 is another example of a protein that is localized within mitochondria and that triggers apoptosis with an unknown mechanism (10). Taken together, Bcl-2-rambo, via its BHNo and MA domain, is likely to trigger death via an as yet to be discovered signaling pathway that joins other pro-apoptotic pathways at the level of caspase-3. It will be interesting to determine whether the Bcl-2 homology region of Bcl-rambo, carrying all four BH domains, also has an anti-apoptotic effect for example by inhibiting the pro-apoptotic activity of the BHNo domain. Identification of Bcl-rambo may therefore provide further insight into the complex mechanism of cell death regulation by the Bcl-2 family members. Acknowledgments We are grateful to Drs. David Huang and Andreas Strasser for providing us with many of the cdna clones employed in this study. We thank Drs. Chris Benedict, Margot Thome, and Kim Burns for helpful comments. REFERENCES 1. Wyllie, A. H., Kerr, J. F., and Currie, A. R. (1980) Int. Rev. Cytol. 68, 251 251 2. Adams, J. M., and Cory, S. (1998) Science 281, 1322 1326

19554 Bcl-rambo, a Novel Member of the Bcl-2 Family 3. Gross, A., McDonnell, J. M., and Korsmeyer, S. J. (1999) Genes Dev. 13, 1899 1911 4. Oltvai, Z. N., Milliman, C. L., and Korsmeyer, S. J. (1993) Cell 74, 609 619 5. Oltvai, Z. N., and Korsmeyer, S. J. (1994) Cell 79, 189 192 6. Yin, X. M., Oltvai, Z. N., and Korsmeyer, S. J. (1994) Nature 369, 321 323 7. Cheng, E. H. Y., Levine, B., Boise, L. H., Thompson, C. B., and Hardwick, J. M. (1996) Nature 379, 554 556 8. Thome, M., Martinon, F., Hofmann, K., Rubio, V., Steiner, V., Schneider, P., Mattmann, C., and Tschopp, J. (1999) J. Biol. Chem. 274, 9962 9968 9. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1999) in Current Protocols in Molecular Biology (Chanda, V. B., ed) Vol. 2, John Wiley and Sons, Inc. 10. Oda, K., Arakawa, H., Tanaka, T., Matsuda, K., Tanikawa, C., Mori, T., Nishimori, H., Tamai, K., Tokino, T., Nakamura, Y., and Taya, Y. (2000) Cell 102, 849 862 11. Du, C., Fang, M., Li, Y., Li, L., and Wang, X. (2000) Cell 102, 33 42 12. Verhagen, A. M., Ekert, P. G., Pakusch, M., Silke, J., Connolly, L. M., Reid, G. E., Moritz, R. L., Simpson, R. J., and Vaux, D. L. (2000) Cell 102, 43 53 13. Kelekar, A., and Thompson, C. B. (1998) Trends Cell Biol. 8, 324 330 14. Tschopp, J., Irmler, M., and Thome, M. (1998) Curr. Opin. Immunol. 10, 552 558 15. Newton, K., Harris, A. W., Bath, M. L., Smith, K. G. C., and Strasser, A. (1998) EMBO J. 17, 706 718 16. Zhou, Q., Snipas, S., Orth, K., Muzio, M., Dixit, V. M., and Salvesen, G. S. (1997) J. Biol. Chem. 272, 7797 7800 17. Pan, G., O Rourke, K., and Dixit, V. M. (1998) J. Biol. Chem. 273, 5841 5845 18. Deveraux, Q. L., and Reed, J. C. (1999) Genes Dev. 13, 239 252 19. Muchmore, S. W., Sattler, M., Liang, H., Meadows, R. P., Harlan, J. E., Yoon, H. S., Nettesheim, D., Chang, B. S., Thompson, C. B., Wong, S. L., Ng, S. L., and Fesik, S. W. (1996) Nature 381, 335 341