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

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 Bcl-rambo. 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-xL, 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-xL) 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, Bcl-rambo constitutes a novel type of pro-apoptotic Bcl-2 member that triggers cell death independently of its BH motifs.AF325209 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 Bcl-rambo. 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-xL, 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-xL) 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, Bcl-rambo constitutes a novel type of pro-apoptotic Bcl-2 member that triggers cell death independently of its BH motifs.AF325209 Bcl-2 homology amino acid(s) polymerase chain reaction hemagglutinin polyacrylamide gel electrophoresis phosphate-buffered solution membrane anchor vesicular stomatitis virus Apoptosis, the physiological process of cell death, is critical for modeling tissues and maintaining homeostasis in multicellular organisms (1Wyllie A.H. Kerr J.F. Currie A.R. Int. Rev. Cytol. 1980; 68: 251Crossref PubMed Scopus (6697) Google Scholar). Members of the Bcl-2 family of proteins are regulators of apoptosis that can be grouped into three sub-families (2Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4780) Google Scholar, 3Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3238) Google Scholar). Bcl-2 and several of its close relatives including Bcl-x, Bcl-w, andCaenorhabditis 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, mammalian Bad, Bik/Nbk, Blk, Hrk, Bid, Bim/Bod, Noxa, andC. elegans EGL-1 share homology only within the short BH3 motif and are therefore called BH3-only proteins (2Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4780) Google Scholar, 3Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3238) Google Scholar). 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 sub-family members would then determine whether the cell lives or dies (4Oltvai Z.N. Milliman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5838) Google Scholar, 5Oltvai Z.N. Korsmeyer S.J. Cell. 1994; 79: 189-192Abstract Full Text PDF PubMed Scopus (773) Google Scholar). Mutagenesis of Bcl-2 initially suggested that its ability to inhibit cell death required binding to a pro-apoptotic family member (6Yin X.M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1214) Google Scholar), but Bcl-xL mutants have been identified that do not bind Bax or Bak yet still block apoptosis (7Cheng E.H.Y. Levine B. Boise L.H. Thompson C.B. Hardwick J.M. Nature. 1996; 379: 554-556Crossref PubMed Scopus (443) Google Scholar). 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.DISCUSSIONIn this report we describe the cloning and the initial characterization of a novel Bcl-2 family member, Bcl-rambo. Bcl-rambo 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-xL, the BH-containing 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 (19Muchmore 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. Fesik S.W. Nature. 1996; 381: 335-341Crossref PubMed Scopus (1279) Google Scholar). Bcl-rambo appears to induce apoptosis, at least in part, independently of known mitochondrial signaling pathways. Overexpression of Bcl-xL (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 crelease, 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 (10Oda K. Arakawa H. Tanaka T. Matsuda K. Tanikawa C. Mori T. Nishimori H. Tamai K. Tokino T. Nakamura Y. Taya Y. Cell. 2000; 102: 849-862Abstract Full Text Full Text PDF PubMed Scopus (1017) Google Scholar).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. Apoptosis, the physiological process of cell death, is critical for modeling tissues and maintaining homeostasis in multicellular organisms (1Wyllie A.H. Kerr J.F. Currie A.R. Int. Rev. Cytol. 1980; 68: 251Crossref PubMed Scopus (6697) Google Scholar). Members of the Bcl-2 family of proteins are regulators of apoptosis that can be grouped into three sub-families (2Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4780) Google Scholar, 3Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3238) Google Scholar). Bcl-2 and several of its close relatives including Bcl-x, Bcl-w, andCaenorhabditis 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, mammalian Bad, Bik/Nbk, Blk, Hrk, Bid, Bim/Bod, Noxa, andC. elegans EGL-1 share homology only within the short BH3 motif and are therefore called BH3-only proteins (2Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4780) Google Scholar, 3Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3238) Google Scholar). 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 sub-family members would then determine whether the cell lives or dies (4Oltvai Z.N. Milliman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5838) Google Scholar, 5Oltvai Z.N. Korsmeyer S.J. Cell. 1994; 79: 189-192Abstract Full Text PDF PubMed Scopus (773) Google Scholar). Mutagenesis of Bcl-2 initially suggested that its ability to inhibit cell death required binding to a pro-apoptotic family member (6Yin X.M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1214) Google Scholar), but Bcl-xL mutants have been identified that do not bind Bax or Bak yet still block apoptosis (7Cheng E.H.Y. Levine B. Boise L.H. Thompson C.B. Hardwick J.M. Nature. 1996; 379: 554-556Crossref PubMed Scopus (443) Google Scholar). 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. DISCUSSIONIn this report we describe the cloning and the initial characterization of a novel Bcl-2 family member, Bcl-rambo. Bcl-rambo 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-xL, the BH-containing 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 (19Muchmore 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. Fesik S.W. Nature. 1996; 381: 335-341Crossref PubMed Scopus (1279) Google Scholar). Bcl-rambo appears to induce apoptosis, at least in part, independently of known mitochondrial signaling pathways. Overexpression of Bcl-xL (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 crelease, 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 (10Oda K. Arakawa H. Tanaka T. Matsuda K. Tanikawa C. Mori T. Nishimori H. Tamai K. Tokino T. Nakamura Y. Taya Y. Cell. 2000; 102: 849-862Abstract Full Text Full Text PDF PubMed Scopus (1017) Google Scholar).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. In this report we describe the cloning and the initial characterization of a novel Bcl-2 family member, Bcl-rambo. Bcl-rambo 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-xL, the BH-containing 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 (19Muchmore 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. Fesik S.W. Nature. 1996; 381: 335-341Crossref PubMed Scopus (1279) Google Scholar). Bcl-rambo appears to induce apoptosis, at least in part, independently of known mitochondrial signaling pathways. Overexpression of Bcl-xL (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 crelease, 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 (10Oda K. Arakawa H. Tanaka T. Matsuda K. Tanikawa C. Mori T. Nishimori H. Tamai K. Tokino T. Nakamura Y. Taya Y. Cell. 2000; 102: 849-862Abstract Full Text Full Text PDF PubMed Scopus (1017) Google Scholar). 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. 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.