Loss of Bif-1 suppresses Bax/Bak conformational change and mitochondrial apoptosis - PubMed (original) (raw)

Loss of Bif-1 suppresses Bax/Bak conformational change and mitochondrial apoptosis

Yoshinori Takahashi et al. Mol Cell Biol. 2005 Nov.

Abstract

Bif-1, a member of the endophilin B protein family, interacts with Bax and promotes interleukin-3 withdrawal-induced Bax conformational change and apoptosis when overexpressed in FL5.12 cells. Here, we provide evidence that Bif-1 plays a regulatory role in apoptotic activation of not only Bax but also Bak and appears to be involved in suppression of tumorigenesis. Inhibition of endogenous Bif-1 expression in HeLa cells by RNA interference abrogated the conformational change of Bax and Bak, cytochrome c release, and caspase 3 activation induced by various intrinsic death signals. Similar results were obtained in Bif-1 knockout mouse embryonic fibroblasts. While Bif-1 did not directly interact with Bak, it heterodimerized with Bax on mitochondria in intact cells, and this interaction was enhanced by apoptosis induction and preceded the Bax conformational change. Moreover, suppression of Bif-1 expression was associated with an enhanced ability of HeLa cells to form colonies in soft agar and tumors in nude mice. Taken together, these findings support the notion that Bif-1 is an important component of the mitochondrial pathway for apoptosis as a novel Bax/Bak activator, and loss of this proapoptotic molecule may contribute to tumorigenesis.

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Figures

FIG. 1.

FIG. 1.

Knockdown of Bif-1 prevents Bax conformational change. HeLa cells were transfected with siRNAs targeting Bif-1 (siBif-1) or control GFP (siGFP) and subjected to the following analyses. (A) Immunoblot analysis with anti-Bif-1 monoclonal antibody (Imgenex) revealed that Bif-1 protein expression was significantly reduced by transfection with siBif-1 duplexes. (B to D) SiRNA-transfected cells were exposed to either 1 μM ADR for 12 h, 0.5 μM STS for 6 h, or 1 mM methyl methanesulfonate (MMS) for 10 h. Bax conformational change was determined by immunoprecipitation (IP) with anti-Bax 6A7 monoclonal antibody (B), as well as by immunofluorescence staining (green) with anti-Bax N20 polyclonal antibody (C). The N20-positive cells in panel C were counted under fluorescence microscopy (D). The data shown are means plus standard deviations (n = 3). The nuclear morphology was examined by DAPI (blue) staining.

FIG. 2.

FIG. 2.

Knockdown of Bif-1 inhibits Bax translocation to mitochondria, cytochrome c release, caspase 3 activation, and PARP cleavage. (A) HeLa cells transfected with siGFP or siBif-1 were pretreated with 75 μM z-VAD-fmk for 30 min before incubation with 20 μM ActD for 4 h. The cells were stained with anti-cytochrome c (anti-cyt c) monoclonal antibody (red) plus anti-Bax polyclonal antiserum (green), and cells with released cytochrome c (indicated by red arrows) and mitochondrion-translocated Bax (indicated by green arrowheads) were counted (n > 500) under fluorescence microscopy and shown in bar graphs. (B) The siRNA-transfected HeLa cells were exposed to either DMSO as a control, 1 μM ADR for 12 h, or 0.5 μM STS for 6 h, and the caspase 3-like activity was measured by using the caspase 3 fluorometric assay kit (Sigma) according to the manufacturer's protocol. (C) HeLa cells with siGFP or siBif-1 were treated with 20 μM ActD for the indicated periods and subjected to SDS-PAGE/immunoblot analysis with anti-PARP antibody.

FIG. 3.

FIG. 3.

Knockdown of Bif-1 suppresses Bax conformational change and caspase 3 activity in ADR-treated HeLa cells. (A) HeLa cells were transfected with siRNAs targeting Bif-1 or GFP for 48 h and subjected to SDS-PAGE/immunoblot analysis. (B and C) HeLa cells transfected with siRNAs targeting Bif-1 or GFP were exposed to DMSO or 1 μM ADR for 12 h and subjected to immunoprecipitation (IP) with anti-Bax 6A7 monoclonal antibody and to caspase 3 activity assay. The error bars indicate standard deviations.

FIG. 4.

FIG. 4.

Knockout of Bif-1 in MEFs delays mitochondrial apoptosis. (A) Genomic organization of the mouse bif-1 gene, targeting construct, and targeted locus. The restriction enzyme sites shown are B (BamHI) and E (EcoRI). WT, wild type. (B) Immunoblot analysis of whole-cell lysates from Bif-1+/+, Bif-1+/−, and Bif-1−/− embryonic day 12.5 mouse embryos. (C) Bif-1+/+ and Bif-1−/− MEFs were cultured in the presence or absence of serum for 6 h and applied to immunostaining with anti-cytochrome c antibody. (D and E) Bif-1−/− and WT MEFs were cultured with or without serum for various times before caspase 3 activity analysis (D) and trypan blue dye exclusion assay (E). The error bars indicate standard deviations. (F) Bif-1−/− and Bif-1+/+ MEFs were treated with 1 μg/ml TUN for 24 h, 1 μM STS for 12 h, or DMSO as a control. Cell lysates were prepared in CHAPS lysis buffer and analyzed by SDS-PAGE/immunoblotting with the indicated antibodies. (G and H) Bif-1−/− MEFs were transfected with 10 μg of pIRES2-EGFP vector or pBif-1-IRES2-EGFP plasmid DNA for 40 h using the Nucleofector technology (Amaxa) and subjected to treatment with DMSO or 1 μM STS for 12 h. The percentage of apoptotic GFP-positive cells was determined by DAPI staining assay (G), and cell lysates were prepared and analyzed by SDS-PAGE/immunoblotting (H).

FIG. 5.

FIG. 5.

Loss of Bif-1 delays Bax activation, cytochrome c release, caspase 3 activation, and cell death in MEFs induced by TUN. (A to C) Bif-1−/− and wild-type (WT) MEFs were treated with 1 μg/ml TUN for the indicated times and subjected to trypan blue dye exclusion (A) and caspase 3 activity (B) assays and immunofluorescence staining with anti-cytochrome c (cyt c) antibody, and cells with released cytochrome c were counted (n > 300) by fluorescence microscopy (C) analyses. The error bars indicate standard deviations. (D) MEFs were treated with 1 μg/ml TUN for various times prior to preparation of cell lysates in CHAPS lysis buffer and immunoprecipitation (IP) with anti-Bax 6A7 monoclonal antibody or anti-Bax polyclonal rabbit antiserum. The resulting immune complexes and total-cell lysates were analyzed by SDS-PAGE/immunoblotting with the indicated antibodies. (E) Bif-1−/− and Bif-1+/+ MEFs were treated with 1 μg/ml TUN for 0 or 24 h and subjected to subcellular fractionation. The resulting cytosol and mitochondrion fractions were analyzed by SDS-PAGE/immunoblotting with anti-Bax antibody to determine Bax translocation. In addition, the mitochondrion fraction was treated with 0.1 M Na2CO3 (pH 11.5) before SDS-PAGE/immunoblot analysis to examine the membrane-integrated (alkali-resistant) Bax protein. (F) Isolated mouse liver mitochondria were incubated in the absence (−) or presence (+) of 10 nM tBid, 200 nM Bif-1, or a combination of both for 30 min at 30°C. After centrifugation, the resulting supernatant (Sup) and pellet fractions were subjected to SDS-PAGE/immunoblot analysis with antibodies specific for cytochrome c (Cyt c) or COX IV.

FIG. 6.

FIG. 6.

Apoptosis induction enhances Bif-1 interaction with Bax on mitochondria in intact cells as visualized by BiFC analysis. COS-7 cells were cotransfected with pcDNA3-YN154-Bif-1 (YN-Bif-1) and pcDNA3-YC155-Bax (YC-Bax) or pcDNA3-YC155-Bif-1 (YC-Bif-1) plasmid at 37°C for 21 h and then switched to 30°C for 3 h to promote YFP (green) fluorophore maturation. After incubation with 20 nM of MitoTracker (red) for 30 min, the cells were exposed to 1 μM STS in the presence of 50 μM z-VAD-fmk for 3 h and analyzed by confocal microscopy.

FIG. 7.

FIG. 7.

Bif-1 contributes to not only Bax, but also Bak activation. (A) siBif-1-expressing HeLa clones or control cells were transfected with the indicated amounts of plasmid DNA encoding GFP, GFP-Bax, or GFP-BaxΔS mutant for 24 h prior to trypan blue dye exclusion assay. The error bars indicate standard deviations. (B) HeLa clones were transfected with 0.5 μg GFP- or GFP-Bax-producing plasmids for 24 h and treated with 1 μM ADR or control DMSO for 8 h. After being stained with DAPI, the apoptotic GFP-positive cells were counted (n > 300). (C) HeLa cells transfected with the indicated siRNAs were treated with 20 μM ActD for the indicated periods of time and subjected to SDS-PAGE/immunoblot analysis with the indicated antibodies. (D) HeLa cells transfected with siGFP or siBif-1 were treated with 4 μg/ml TUN for 24 h prior to the preparation of cell lysates in CHAPS lysis buffer. Immunoprecipitation (IP) was performed with anti-Bak monoclonal antibody (Ab-2) or anti-Bax 6A7 monoclonal antibody, and the resulting immune complexes and total-cell lysates were analyzed by SDS-PAGE/immunoblotting with anti-Bax and anti-Bak polyclonal antibodies. (E) Bif-1−/− and Bif-1+/+ MEFs were treated with 1 μg/ml TUN for 24 h, 1 μM STS for 12 h, or DMSO as a control. Cell lysates were prepared in CHAPS lysis buffer and subjected to immunoprecipitation with anti-Bak monoclonal antibody (Ab-2). The resulting immune complexes, as well as total-cell lysates, were analyzed by SDS-PAGE/immunoblotting with anti-Bak polyclonal antibody.

FIG. 8.

FIG. 8.

Inhibition of Bif-1 expression enhances the tumorigenicity of HeLa cells. (A) siBif-1 stable clones or control HeLa cells were seeded at 125 to 1,000 cells/well in triplicate in six-well plates in 0.3% agar over a 0.6% agar layer and cultured for 2 to 3 weeks. After being stained with 1 mg/ml MTT in growth medium, the colonies were photographed and counted. (B) Stable HeLa transfectants were harvested, suspended in PBS, and injected subcutaneously into the right and left flanks (10 × 106 cells/flank) of female nude mice (five mice/cell line). The tumor size was monitored daily using calipers. Statistical significance was determined at P < 0.05 (*). The error bars indicate standard deviations. (C) Cultures of logarithmically growing HeLa transfectants were subjected to BrdU incorporation and DNA content analysis by flow cytometry. FITC, fluorescein isothiocyanate.

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