Article15 January 1998free access Bim: a novel member of the Bcl-2 family that promotes apoptosis Liam O'Connor Liam O'Connor The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Victoria, 3050 Australia Search for more papers by this author Andreas Strasser Andreas Strasser The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Victoria, 3050 Australia Search for more papers by this author Lorraine A. O'Reilly Lorraine A. O'Reilly The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Victoria, 3050 Australia Search for more papers by this author George Hausmann George Hausmann The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Victoria, 3050 Australia Search for more papers by this author Jerry M. Adams Jerry M. Adams The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Victoria, 3050 Australia Search for more papers by this author Suzanne Cory Suzanne Cory The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Victoria, 3050 Australia Search for more papers by this author David C.S. Huang David C.S. Huang The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Victoria, 3050 Australia Search for more papers by this author Liam O'Connor Liam O'Connor The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Victoria, 3050 Australia Search for more papers by this author Andreas Strasser Andreas Strasser The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Victoria, 3050 Australia Search for more papers by this author Lorraine A. O'Reilly Lorraine A. O'Reilly The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Victoria, 3050 Australia Search for more papers by this author George Hausmann George Hausmann The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Victoria, 3050 Australia Search for more papers by this author Jerry M. Adams Jerry M. Adams The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Victoria, 3050 Australia Search for more papers by this author Suzanne Cory Suzanne Cory The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Victoria, 3050 Australia Search for more papers by this author David C.S. Huang David C.S. Huang The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Victoria, 3050 Australia Search for more papers by this author Author Information Liam O'Connor1, Andreas Strasser1, Lorraine A. O'Reilly1, George Hausmann1, Jerry M. Adams1, Suzanne Cory1 and David C.S. Huang1 1The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Victoria, 3050 Australia The EMBO Journal (1998)17:384-395https://doi.org/10.1093/emboj/17.2.384 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Certain members of the Bcl-2 family inhibit apoptosis while others facilitate this physiological process of cell death. An expression screen for proteins that bind to Bcl-2 yielded a small novel protein, denoted Bim, whose only similarity to any known protein is the short (nine amino acid) BH3 motif shared by most Bcl-2 homologues. Bim provokes apoptosis, and the BH3 region is required for Bcl-2 binding and for most of its cytotoxicity. Like Bcl-2, Bim possesses a hydrophobic C-terminus and localizes to intracytoplasmic membranes. Three Bim isoforms, probably generated by alternative splicing, all induce apoptosis, the shortest being the most potent. Wild-type Bcl-2 associates with Bim in vivo and modulates its death function, whereas Bcl-2 mutants that lack survival function do neither. Significantly, Bcl-xL and Bcl-w, the two closest homologues of Bcl-2, also bind to Bim and inhibit its activity, but more distant viral homologues, adenovirus E1B19K and Epstein–Barr virus BHRF-1, can do neither. Hence, Bim appears to act as a ‘death ligand’ which can only neutralize certain members of the pro-survival Bcl-2 sub-family. Introduction Apoptosis, the physiological process of cell death, is critical for modelling tissues and maintaining homeostasis in multicellular organisms (Kerr et al., 1972; Jacobson et al., 1997). The mechanism of this intrinsic suicide programme is under intense scrutiny. The executioners are a set of cysteine proteinases, termed caspases, that degrade critical cellular substrates (Nicholson and Thornberry, 1997). The regulatory machinery that governs the activation of the caspases is less well understood, but a central role is played by the Bcl-2 family (Cory, 1995; Korsmeyer, 1995; White, 1996; Jacobson, 1997; Kroemer, 1997; Reed, 1997). Bcl-2 itself was the first intracellular regulator of apoptosis to be identified (Vaux et al., 1988), and high levels enhance cell survival under diverse cytotoxic conditions (Sentman et al., 1991; Strasser et al., 1991). Other cellular homologues, such as Bcl-xL (Boise et al., 1993) and Bcl-w (Gibson et al., 1996), also enhance cell survival, as do more distantly related viral homologues, such as the adenovirus E1B19K protein (White et al., 1992) and Epstein–Barr virus (EBV) BHRF-1 (Henderson et al., 1993). Remarkably, the family also includes members such as Bax (Oltvai et al., 1993) and Bak (Chittenden et al., 1995b; Farrow et al., 1995; Kiefer et al., 1995) which antagonize the activity of the pro-survival proteins and provoke apoptosis when expressed at high concentrations. The ability of the pro-survival and anti-survival family members to form heterodimers makes it possible 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 (Oltvai et al., 1993; Oltvai and Korsmeyer, 1994). Mutagenesis of Bcl-2 initially suggested that its ability to inhibit cell death required binding to a pro-apoptotic family member (Yin et al., 1994), but Bcl-xL mutants have been identified that do not bind Bax or Bak yet still block apoptosis (Cheng et al., 1996). Thus, it remains unclear whether the ability to associate with other family members is central to regulating apoptosis. The homology between members of the Bcl-2 family is greatest within four small segments, designated Bcl-2 homology (BH) regions (Yin et al., 1994; Chittenden et al., 1995a; Gibson et al., 1996; Zha et al., 1996), some of which contribute to the interactions between Bcl-2 family members. The N-terminal BH4 domain is restricted to some antagonists of apoptosis, while BH1, BH2 and BH3 can be found in both sub-families. Association of a pro-survival with a pro-apoptotic protein, such as Bcl-2 (or Bcl-xL) with Bax (or Bak), requires the BH1 and BH2 domains of the former (Yin et al., 1994; Hanada et al., 1995; Sedlak et al., 1995) and the BH3 domain of the latter (Chittenden et al., 1995a; Simonian et al., 1996; Zha et al., 1996). In the tertiary structure of Bcl-xL, the BH1, BH2 and BH3 domains form an elongated hydrophobic cleft (Muchmore et al., 1996) along which the amphipathic helix formed by BH3 domains of the pro-apoptotic proteins can bind (Sattler et al., 1997). The importance of the BH3 region for facilitating apoptosis has been underscored by the discovery of several BH3-containing proteins: Bik/Nbk (Boyd et al., 1995; Han et al., 1996), Bid (Wang et al., 1996) and Hrk/DP5 (Imaizumi et al., 1997; Inohara et al., 1997), which are otherwise unrelated to the Bcl-2 family but are potent activators of apoptosis when overexpressed. To search for additional regulators of apoptosis, we have screened a cDNA expression library with a Bcl-2 protein probe. This interaction screen has yielded a novel BH3-containing protein, which we have denoted Bim. Three Bim isoforms all promote apoptosis but differ in potency. Bim interacts with some but not all Bcl-2 family members that promote cell survival, and only those pro-survival relatives that bind to it can neutralize its cytotoxicity. Bim therefore appears to represent a death ligand with a specificity restricted to certain pro-survival members of the Bcl-2 family. Results Isolation of a novel gene encoding a Bcl-2-binding protein In an attempt to identify novel proteins that bind to Bcl-2, we used recombinant human Bcl-2 protein, labelled with 32P (Blanar and Rutter, 1992), to screen a bacteriophage λ cDNA expression library constructed from the p53−/− T lymphoma cell line KO52DA20 (Strasser et al., 1994). A screen of 106 clones yielded five independent clones that encoded the same novel protein, which we named Bim, for Bcl-2 interacting mediator of cell death. Sequence analysis of the bim cDNAs revealed three variants of the coding region, apparently produced by alternative splicing, that we designated bimEL, bimL and bimS (Figure 1A). RT–PCR on mRNA from KO52DA20 cells gave PCR products of the sizes expected for each of these transcripts, although bimS was in low yield (data not shown). The predicted BimEL, BimL and BimS proteins comprise 196, 140 and 110 amino acid residues (Figure 1B). Hybridizing human fetal spleen and peripheral blood cDNA libraries with a mouse bim cDNA yielded human cDNAs encoding BimL and BimEL. Human BimEL is a protein of 198 residues, 89% identical to its mouse counterpart (Figure 1C), and human BimL (138 residues) is 85% identical to mouse BimL. Figure 1.Isolation of cDNAs encoding three isoforms of Bim: BimEL, BimL and BimS. (A) Open reading frames of five independent clones isolated by screening a cDNA expression library with recombinant Bcl-2 protein. Dotted lines indicate putative splices. (B) Relationship of the three Bim isoforms. The black box denotes the BH3 homology region and the hatched box the predicted hydrophobic region. Regions specific to the larger splice variants are shaded. (C) Sequence alignment of the mouse and human BimEL polypeptide sequences using the GCG ‘BESTFIT’ program, identical residues appear on a dark background. The BH3 homology region and the C-terminal hydrophobic region predicted by the Kyte–Doolittle algorithm are boxed. The arrows indicate residues present only in the longer isoforms. Since the nucleotide sequences of the mouse and human cDNAs diverged 5′ of the predicted initiating ATG and there are stop codons in all three reading frames upstream of the human open reading frame, that start codon is likely to be correct. Download figure Download PowerPoint Bim has no substantial homology with any protein in current databases. However, scrutiny of its sequence (Figure 1C) revealed a stretch of nine amino acids corresponding to a BH3 homology region (Boyd et al., 1995; Chittenden et al., 1995a). The BH3 region of Bcl-xL and a peptide corresponding to this region of Bak have each been shown to form part of an amphipathic α helix (Sattler et al., 1997). Plotting the BH3 region of Bim as a helical wheel revealed that it was also amphipathic (data not shown). Apart from the BH3 region, the Bim sequence is unrelated to that of any other BH3-containing protein; it contains no other BH region, nor indeed any other known functional motif. The protein does, however, have a C-terminal hydrophobic region (Figure 1C). Such regions are found in most members of the Bcl-2 family and appear to be important for their localization to intracytoplasmic membranes (Kroemer, 1997). Northern blot analysis showed that bim was expressed in a number of B- and T-lymphoid cell lines, although not in the myeloid line FDC-P1(Figure 2). A major transcript of 5.7 kb and minor transcripts of 3.8, 3.0 and 1.4 kb were detected. Neither the level nor relative abundance of these transcripts changed significantly in KO52DA20 cells induced to undergo apoptosis by treatment with dexamethasone (Figure 2, compare lanes 1 and 2, and lanes 3 and 4) or exposure to γ-radiation (compare lanes 3 and 5). In addition, overexpression of bcl-2 in several of the lines did not affect bim mRNA levels (Figure 2). Figure 2.Expression of bim RNA in haemopoietic cell lines. Northern blot analysis of poly(A)+ RNA, using a mouse bim cDNA probe. The RNAs were derived from the following mouse lines: T-lymphomas KO52DA20 (lanes 1–5), WEHI 703 (lane 6), WEHI 707 (lane 7) and WEHI 7.1 (lane 8), B-lymphomas CH1 (lanes 9 and 10) and WEHI 231 (lanes 11 and 12), pre-B-lymphoma WEHI 415 (lane 13), T-hybridoma B6.2.16 BW2 (lanes 14 and 15) and myeloid progenitor FDC-P1 (lane 16). Those lines that harbour a bcl-2 expression vector or transgene are indicated by a plus sign. Certain RNAs were isolated from cells exposed to cytotoxic conditions: 1 μM dexamethasone (lanes 2 and 4), γ-irradiation (10 Gy) (lane 5). Lanes from a single autoradiograph have been electronically arranged in order. Download figure Download PowerPoint Bim localizes to cytoplasmic membranes The presence of the C-terminal hydrophobic domain in Bim prompted us to investigate its subcellular localization. L929 fibroblasts were transiently transfected with an expression vector encoding BimL tagged with an N-terminal EE epitope, and the permeabilized cells were stained with an anti-EE monoclonal antibody. Confocal microscopy revealed that BimL had a granular cytoplasmic distribution, consistent with an association with intracellular membranes (Figure 3A). When we also introduced the bimL vector into L929 cells stably expressing human Bcl-2, the similarity of the anti-EE staining pattern of these cells (Figure 3C) to that of those expressing BimL alone (Figure 3A) demonstrated that high concentrations of Bcl-2 did not perturb the localization of BimL. The pattern of BimL staining was similar to that reported for Bcl-2 (Monaghan et al., 1992; Krajewski et al., 1993; Lithgow et al., 1994), and overlaying the images obtained from the same cells stained with anti-Bcl-2 (Figure 3B) and anti-EE (Figure 3C) antibodies showed that the two proteins co-localized (Figure 3D). This co-localization does not simply reflect binding of Bim to Bcl-2 since a mutant form of Bim lacking the BH3 region, and therefore incapable of binding to Bcl-2 (see below), localized similarly (Figure 3E). Figure 3.Localization of Bim protein to intracellular membranes. (A) Single optical sections of L929 fibroblasts transiently co-transfected with EE-tagged BimL and baculovirus p35, then stained with the anti-EE antibody. (B) and (C) Confocal images of L929 cells stably co-expressing human Bcl-2 and EE-tagged BimL stained with anti-human Bcl-2 antibody (B) or anti-EE antibody (C). (D) Overlay of images (B) and (C). Co-localization of antibody staining is indicated by yellow fluorescence. (E) Confocal image of L929 cells transiently expressing human Bcl-2 and EE-tagged Bim ΔBH3 stained with anti-EE antibody. (F) Subcellular fractionation of lysates from FDC-P1 cells expressing EE-BimL and Bcl-2. Lysates from equivalent numbers of unfractionated cells (whole) and of subcellular fractions (nuclear, cytoplasmic or membrane), were resolved by SDS–PAGE and immunoblotted using the anti-human Bcl-2 (upper panels) or anti-EE (lower panels) monoclonal antibodies. Download figure Download PowerPoint Subcellular fractionation studies in FDC-P1 cells (see below) were consistent with these observations. Immunoblotting of fractionated lysates obtained from cell lines overexpressing human Bcl-2 and either EE-tagged BimL or EE-tagged BimL lacking the BH3 region showed that all three proteins were present in the nuclear and membrane fractions but not the cytosolic fraction (Figure 3F and data not shown). Together with the microscopic data, these results suggest that Bim localizes to intracytoplasmic membranes, independently of its association with Bcl-2. Overexpression of Bim kills cells by a pathway requiring caspases Other known ‘BH3-only’ proteins (Bik/Nbk, Bid and Hrk/DP5) provoke apoptosis when highly expressed (Boyd et al., 1995; Han et al., 1996; Wang et al., 1996; Imaizumi et al., 1997; Inohara et al., 1997). We therefore tested whether Bim is cytotoxic by transiently transfecting 293T human embryonal kidney cells with a plasmid encoding EE-BimL. The viability of the transfected cells was determined by flow cytometric analysis of permeabilized cells stained with anti-EE antibody and the DNA-intercalating dye propidium iodide (PI). Whereas almost all untransfected cells or those transfected with an empty vector remained viable after 24 h, many of those expressing Bim (i.e. EE antibody-positive) contained sub-diploid DNA (Figure 4A). Indeed, by 3 days, 90% of the cells expressing BimL were dead (Figure 4B). The extent of cell death increased with the amount of bim DNA transfected (black bars, Figure 4C). Figure 4.Bim induces apoptosis which can be inhibited by the general caspase inhibitor p35 and Bcl-2 but not by CrmA. (A) Flow cytometric DNA analysis (see Materials and methods) of 293T cells transfected 24 h previously with EE-bimL plasmid (0.5 μg). (B) Kinetics of apoptosis elicited by EE-bimL plasmid (0.5 μg), assessed as in (A). (C) Cell viability 48 h after transfection with 0.1, 0.2 or 0.5 μg of EE-bimL plasmid alone (black bars) or together with 0.5 μg of wild-type or mutant p35 or crmA plasmid (grey bars). (D) Cell viability 48 h after transfection with 0.1, 0.2 or 0.5 μg of EE-bimL plasmid together with 0.5 μg of the indicated wild-type or mutant bcl-2 plasmids. (C) and (D) show the percentage of viable Bim-expressing cells, determined by DNA FACS analysis, as in (A), and are the means ± SD of three or more independent experiments. Download figure Download PowerPoint The cells expressing Bim appeared to die by apoptosis, as assessed by cell morphology and the generation of sub-diploid DNA (Figure 4A). As expected, the death process required activation of caspases, because co-expression of baculovirus p35, a competitive inhibitor of many types of caspases (Bump et al., 1995), antagonized Bim-induced cell death, whereas an inactive (D87E) mutant p35 (Xue and Horvitz, 1995) did not (Figure 4C). Since CrmA, a potent inhibitor of caspases 1 and 8 (ICE and FLICE) (Orth et al., 1996; Srinivasula et al., 1996), was not effective (Figure 4C), these particular caspases do not appear to play an essential role. Numerous failed attempts to generate lines that stably express Bim suggested that it is toxic to diverse cell types. Those repeatedly tested include haemopoietic lines (FDC-P1, CH1, Jurkat, SKW6 and B6.2.16BW2), fibroblastoid lines (Rat-1, NIH-3T3 and L929) and an epithelial line (293). The cells were electroporated with a vector encoding antibiotic resistance and either EE- or FLAG-tagged BimL, but no drug-selected line that expressed Bim emerged. A vector encoding untagged Bim also failed to generate viable clones. We quantified the cytotoxicity of Bim by colony assays on transfected L929 fibroblasts. Cells transfected with the EE-BimL vector yielded only one-fifth as many antibiotic-resistant colonies as those transfected with the control vector, and when six of the EE–BimL-transfected, drug-resistant colonies were expanded, only one contained any Bim and the level was very low (Table I and data not shown). Thus, Bim suppresses clonogenicity, and expression above a relatively low threshold is incompatible with prolonged cell viability. Table 1. Bim inhibition of L929 colony growth is abrogated by Bcl-2 Cell line Construct Cloning efficiency No. of antibiotic-resistant clones expressing Bim L929 control 1.0 0/6 bimL 0.21 ± 0.04 1/6 bimEL 0.19 ± 0.05 1/6 bimS 0.11 ± 0.03 0/6 bimL ΔBH3 0.69 ± 0.07 6/6 L929 bcl-2 control 1.0 0/6 bimL 0.64 ± 0.07 4/6 Parental L929 fibroblasts and a cloned derivative that stably expresses human Bcl-2 (L929 bcl-2) were co-transfected with a plasmid conferring antibiotic resistance with or without encoding various forms of Bim. After 48 h, antibiotic selection was added and the number of colonies were scored after 14–18 days. The data shown are means ± SD of at least four independent experiments. Bim cytotoxicity can be abrogated by wild-type Bcl-2 but not inactive mutants Co-expression experiments established that Bcl-2 could block cell death induced by BimL (Figure 4D). In 293T cells transiently transfected with both the bcl-2 and bimL plasmids, relatively few cells died, even with a high concentration of bimL DNA (compare the fourth bar in Figure 4C with the third in Figure 4D). The cytotoxicity of Bim, however, could not be countered by mutant forms of Bcl-2 rendered inactive by deletion of the BH4 homology region (ΔBH4) (Borner et al., 1994; D.C.S.Huang, J.M.Adams and S.Cory, submitted), or by a point mutation in its BH1 (G145E) or BH2 (W188A) region (Yin et al., 1994) (Figure 4D). Thus the ability to antagonize Bim-induced cell death required a functional Bcl-2 molecule. High levels of Bcl-2 allowed stable expression of BimL. Indeed, when retrovirally infected L929 clones overexpressing Bcl-2 were transfected with the EE-BimL vector, the frequency of antibiotic-resistant colonies approached that obtained with the control vector, and four of six colonies analysed contained moderate to high levels of Bim (Table I and data not shown). Similarly, using FDC-P1 clones overexpressing wild-type Bcl-2 (but not mutant Bcl-2), we could readily establish sub-clones expressing intermediate to high levels of BimL (Figure 5A). When grown in the presence of interleukin-3 (IL-3), all were indistinguishable in growth characteristics and morphology from the parental FDC-P1 cells or those bearing Bcl-2 alone. However, when deprived of IL-3 or irradiated, cells expressing Bcl-2 and a moderate or high level of Bim died more rapidly than those expressing Bcl-2 alone (Figure 5B). Since each clone had the same level of Bcl-2 (not shown), their sensitivity to apoptosis presumably reflects the ratio of the pro-death protein Bim to the pro-survival protein Bcl-2. Figure 5.Bim antagonizes the anti-apoptotic activity of Bcl-2 in a dose-dependent fashion. (A) Immunofluorescence staining of cloned FDC-P1 cell lines stably expressing Bcl-2 alone (dashed line) or co-expressing Bcl-2 and varying levels of EE-BimL (solid lines). (B) Viability of these clones when cultured in the absence of IL-3 or after exposure to γ-irradiation (10 Gy). Cell viability was assessed by vital dye exclusion. Data shown are means ± SD of at least three experiments and are representative of results obtained with at least three independent lines of each genotype. Download figure Download PowerPoint The three isoforms of Bim all interact with Bcl-2 in vivo but vary in cytotoxicity We next explored whether all isoforms of Bim were equivalent. An FDC-P1 clone expressing human Bcl-2 was transfected with vectors expressing BimEL, BimL or BimS, and antibiotic-resistant clones that expressed the same amount of each isoform were selected for further analysis (Figure 6A). To test for association with Bcl-2, immunoprecipitates prepared from cell lysates using a monoclonal antibody specific for human Bcl-2 were fractionated electrophoretically and blotted with anti-EE antibody. Each of the Bim isoforms clearly bound to Bcl-2 (Figure 6B). However, when the transfectants were deprived of IL-3 or subjected to γ-irradiation, it became evident that BimS antagonized Bcl-2 more effectively than BimL, while BimEL was the least potent (Figures 6C). In addition, BimS suppressed L929 colony formation more effectively than BimL or BimEL (Table I). Thus, although all three Bim isoforms can bind to Bcl-2, they vary in cytotoxicity, BimS being the most potent. Figure 6.Different killing activities of the three Bim isoforms. (A) Immunofluorescence staining, performed in parallel, of cloned FDC-P1 lines expressing Bcl-2 alone (dotted) or Bcl-2 plus EE-tagged BimL, BimEL or BimS (solid lines). (B) Association of EE-tagged BimS, BimL and BimEL with Bcl-2 demonstrated by anti-EE immunoblots of immunoprecipitates prepared with anti-human Bcl-2 monoclonal antibody from FDC-P1 cells expressing the indicated proteins. The 25 kDa non-specific band in the fourth lane, indicated by an asterisk, was not consistently seen. (C) Effect of Bim isoforms on viability of FDC-P1 cells expressing Bcl-2, after removal of growth factor or exposure to irradiation. All data were obtained on lines that expressed equivalent levels of the introduced proteins (see A). Those shown are means ± SD of at least three experiments representative of the results obtained with at least two independent lines of each genotype. Download figure Download PowerPoint Bim binds to and antagonizes Bcl-xL and Bcl-w but not viral Bcl-2 homologues Bim also associates with certain other anti-apoptotic Bcl-2 family members. Immunoprecipitation of lysates from 35S-labelled 293T cells transiently co-transfected with the relevant vectors revealed binding to Bcl-xL, although not to a mutant (mt 7) that lacks pro-survival activity, nor to two mutants (mt 1 and mt 15) that retain significant anti-apoptotic activity but cannot bind to Bax (Cheng et al., 1996) (Figure 7A). Binding to Bcl-xL and Bcl-w was confirmed by immunoprecipitation followed by Western blot analysis (Figure 7B). Not all mediators of cell survival associate with Bim, however. Under the same conditions, Bim did not bind to either of two virally encoded Bcl-2 homologues, the adenovirus E1B19K protein and the EBV BHRF-1 protein (Figure 7B). Figure 7.Bim binds to and antagonizes Bcl-xL or Bcl-w but not E1B19K. (A) Lysates of 35S-labelled 293T cells transiently co-transfected with the plasmids encoding the indicated proteins were immunoprecipitated with anti-EE antibody, and the EE-BimL-containing complexes were fractionated by SDS–PAGE. (B) Lysates from parental 293T cells or 293T cells co-expressing EE-tagged BimL and FLAG-tagged Bcl-xL, Bcl-w, E1B19K or BHRF-1 were immunoblotted directly or after immunoprecipitation, as indicated. (C and D) 293T cells were transiently transfected with a vector control (unfilled bar) or with 0.1, 0.2 or 0.5 μg of EE-BimL plasmid, either alone (black bars) or together with 0.5 μg of plasmids encoding wild-type or mutant Bcl-xL (C), Bcl-w or E1B19K protein (D) (grey bars). The flow cytometric analysis was as described in the legend to Figure 4. Data shown are means ± SD of three or more independent experiments. (E) Lysates of 35S-labelled 293T cells transiently co-transfected with plasmids encoding the indicated proteins were immunoprecipitated with either anti-FLAG or anti-EE antibody, and the resulting complexes were fractionated by SDS–PAGE. Download figure Download PowerPoint Functional tests mirrored the binding properties of the various Bcl-2 homologues. When transiently co-expressed with Bim in 293T cells, Bcl-xL and Bcl-w countered Bim toxicity as effectively as Bcl-2 (compare Figures 7C and D with Figure 4D). In contrast, little inhibition was observed with comparable levels of the mutant Bcl-xL proteins (Figure 7C) or the adenovirus E1B19K protein (Figure 7D). These data suggest that Bcl-2-like inhibitors of apoptosis must bind to Bim to inhibit its action. Bim does not interact with any pro-apoptotic family member tested. No interaction of Bim with Bax could be observed under conditions in which Bim–Bcl-xL association was readily detectable (Figure 7E). In other experiments (not shown) in which we co-expressed FLAG-tagged Bim with a range of other EE-tagged pro-apoptotic family members, we failed to detect any Bim homodimers or any interaction of Bim with Bak, Bad, Bik or Bid. The BH3 region is essential for interaction of Bim with pro-survival Bcl-2 family members and for most of its ability to promote apoptosis Since the BH3 region of several death-promoting proteins is essential for their activity (see Introduction), we tested a bimL mutant lacking the BH3 region. In transiently transfected 293T cells, the mutant protein (ΔBH3) was readily detected by immunoblotting but it did not bind to Bcl-xL (Figure 8A), or to Bcl-2 or Bcl-w (data not shown). Figure 8.The BH3 homology region of Bim is required for binding to and inhibiting pro-survival Bcl-2 homologues. (A) Immunoblot showing that Bcl-xL associates with wild-type BimL but not with the ΔBH3 mutant. (B) Immunofluorescence staining of cloned FDC-P1 lines expressing Bcl-2 alone (dotted) or with EE-BimL or EE-Bim ΔBH3 (solid lines), and of EE-Bim ΔBH3 in the parental FDC-P1 cells (broken line). (C) Viability of FDC-P1 clones expressing the indicated proteins (see B) was assessed by vital dye exclusion. Data shown are means ± SD of at least three experiments and are representative of results obtained with at least three independent lines of each genotype. Download figure Download PowerPoint In some biological assays, the ΔBH3 mutant of Bim appeared inert. In contrast to wild-type Bim, it was easy to establish lines expressing BimL ΔBH3 from FDC-P1 (Figure 8B) or L929 cells (Table I and data not shown). Moreover, BimL ΔBH3 did not impair the viability of the FDC-P1 cells in either the presence or absence of Bcl-2 (Figure 8C). Finally, 293T cells transiently transfected with BimL ΔBH3 exhibited high viability (not shown). These results indicate that the BH3 region is critical for Bim to promote apoptosis and suggest that
