Baxβ: A Constitutively Active Human Bax Isoform that Is under Tight Regulatory Control by the Proteasomal Degradation Mechanism  Nai Yang Fu, Sunil K. Sukumaran, Sze Yen Kerk, Victor C. Yu  Molecular Cell  Volume 33, Issue 1, Pages 15-29 (January 2009) DOI: 10.1016/j.molcel.2008.11.025 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Induction of a 24 kD Bax-Related Protein by Proteasome Inhibitor (A) A 24 kD Bax-related protein associates with MOAP-1 in cells treated with proteasome inhibitor. Equivalent lysates from 293T cells treated with vehicle (CT), 10 μM MG132, 10 μM EPOX, or the protein translation inhibitor cycloheximide (CHX, 50 μg/ml) for 14 hr were immunoprecipitated (IP) with MOAP-1 antibody, followed by immunoblotting (IB) with Bax antibody (bottom panel). Total lysates were probed for the indicated proteins (upper panels). (B) Detection of the 24 kD Bax-related protein by multiple Bax antibodies. Equivalent lysates from 293T cells with indicated treatment for 14 hr were analyzed by IP-western with indicated Bax antibodies (lower). Total lysates were probed for Actin. Epitope for individual Bax antibody is depicted (upper). (C) The 24 kD Bax-related protein is inducible by proteasome inhibitor in a variety of cell lines. Total lysates from the cells treated as in (B) were probed with the N20 antibody. (D) The 24 kD Bax-related protein is absent in the HCT116-Bax−/− cells. Lysates from HCT116-Bax+/− or Bax−/− cells treated as in (B) were probed with Bax (N20) or HSP60 antibody. Molecular Cell 2009 33, 15-29DOI: (10.1016/j.molcel.2008.11.025) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 The 24 kD Bax-Related Protein Is the Baxβ Isoform (A) Illustration of the splicing scheme of human Bax transcripts. The exons are indicated by boxes and identified by numbers. The number of nucleotides in each exon encoding Baxα protein is indicated at the top. The untranslated regions derived from exons 1 and 6 are not shown. Exons 4b, 5b, and 5c with number of nucleotides indicated represent the portion of intron 4 and intron 5 retained in certain splicing isoforms. 6a represents the portion of exon 6 skipped in Bax sigma. The predicted number of amino acids of the protein and number of nucleotides of the mRNA for each isoform are shown on the right. (B) Baxβ isoform is subjected to proteasomal degradation. 293T cells transfected with plasmids encoding the indicated Bax isoforms along with pEGFP were treated as indicated for 8 hr. Lysates were immunoblotted with myc or GFP antibody. (C) Inhibition of proteasome activity extends the half-life of Baxβ. Vehicle or MG132 was added to 293T cells transfected with plasmid encoding GST and myc-Baxβ prior to CHX (50 μg/ml) treatment. Equivalent lysates from the cells harvested at the indicated time points were immunoblotted with myc or GST antibody. (D)The relative amount of Baxβ at the indicated time points in (C) was quantified by densitometry. (E) Illustration depicting the primary structure of human Baxα and Baxβ proteins. (F) The C terminus of Baxβ mediates its degradation. 293T cells transfected with the plasmid encoding the indicated myc-tagged proteins were treated with vehicle or MG132 for 8 hr, followed by either direct immunoblotting of total cell lysates (upper) or IP-western using the indicated antibodies (lower). Heavy chain of IgG (∗) is indicated. (G) The proteasome-coupling function of the C terminus of Baxβ is transferable. 293T cells transfected with the plasmids expressing either GFP-Baxβ-CT or GST-Baxβ-CT in combination with pxj GST or pEGFP, respectively, were treated with vehicle or MG132 for 8 hr. Total lysates were immunoblotted with GFP or GST antibody. (H) Endogenous Baxβ protein is upregulated by proteasome inhibitor. Equivalent lysates from 293T cells treated as indicated for 14 hr were analyzed by either direct immunoblotting of total cell lysates (upper) or IP-western (lower) with the indicated antibodies. (I) The 24 kD Bax-related protein can be immunodepleted by the Baxβ antibodies. 293T cells were treated as in (H). Equivalent cell lysates were immunoprecipitated with Baxβ antibodies or control myc antibody. The depleted supernatants were immunoblotted with the 2D2 antibody. (J) The mRNA levels of Baxβ are not affected by proteasome inhibitor. The relative Baxβ mRNA levels in control, and treated 293T cells were analyzed by real-time PCR. Error bars indicate SEM. (K) Endogenous Baxβ protein is upregulated in a variety of cell lines upon proteasome inhibition. The cells were treated and analyzed by IP-western as in (H). (L) Proteasome inhibition induces accumulation of ubiquitinated forms of endogenous Baxβ. HCT116-Bax+/− or Bax−/− cells were treated as in (H). Lysates were immunoprecipitated with Baxβ antibody, followed by immunoblotting with ubiquitin (Ubi) antibody. Molecular Cell 2009 33, 15-29DOI: (10.1016/j.molcel.2008.11.025) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Spontaneous Targeting and Integration of Native Baxβ to Mitochondria (A) Baxβ localizes mainly in mitochondria. MCF-7 cells were transfected with plasmid encoding the indicated GFP-tagged Bax isoforms or mutants for 4 hr in OptiMEM medium. Four to five hours after replacement with complete medium, the cells were stained with MitoTracker Red. (B) Baxβ associates with isolated mitochondria. On the left, the input for IVT HA-tagged Baxα and Baxβ. A nonspecific band (∗) in the rabbit reticulate lysate (R. lysate) is indicated. On the right, mitochondria (mito) isolated from COS-1 cells were incubated with IVT Baxα and Baxβ, followed by pelleting by centrifugation and washing twice. Mitochondrial extracts were immunoblotted with HA or VDAC antibody. The blot probed with HA antibody was exposed for 2 or 20 min. (C) Baxβ integrates into mitochondrial membrane. The indicated IVT proteins were incubated with mitochondria isolated from COS-1 cells. Integration of proteins into mitochondria membrane was determined by alkali wash assay. Molecular Cell 2009 33, 15-29DOI: (10.1016/j.molcel.2008.11.025) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Native Baxβ Is Constitutively in Active Form and Is a Potent Inducer of Cytochrome c Release from Mitochondria (A) Baxβ is constitutively in an active conformation. The indicated IVT myc-tagged Bax proteins were immunoprecipitated with the N20 antibody in the absence or presence of the detergent NP40, followed by immunoblotting with myc antibody. (B) Confocal analysis of conformation states of Bax isoforms and mutants. MCF-7 cells transfected with the plasmid encoding various GFP-tagged Bax proteins were immunostained for active form of Bax (Act Bax) with the N20 antibody (red). (C) Baxβ spontaneously forms oligomers. The oligomerization of IVT Baxα or Baxβ proteins was analyzed by gel exclusion column. The molecular weights for standard proteins were labeled above the fraction numbers. (D and E) Baxβ induces cytochrome c and SMAC release. MCF-7 transfected with indicated GFP-Bax expression plasmids was stained with anti-cytochrome c (Cyto c) (D) or SMAC (E) antibody. (F) Baxβ induces caspase-3 activation. MCF-7 cells stably expressing procaspase-3 were transfected with the plasmid encoding GFP-Baxβ. The cells were stained with anti-cleaved casp3 (red) and anti-cytochrome c (blue) antibodies. (G) Baxβ potently induces cytochrome c release from isolated mitochondria. Mitochondria isolated from HCT116-Bax+/− cells were incubated with IVT proteins. The mitochondria were pelleted by centrifugation. The supernatants (Sup) and mitochondrial pellets were immunoblotted with cytochrome c or HSP60 antibody. Molecular Cell 2009 33, 15-29DOI: (10.1016/j.molcel.2008.11.025) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Stable Expression of Baxβ Restores the Sensitivity of HCT116-Bax−/− Cells to Multiple Apoptotic Stimuli (A) Expression of Bax proteins in various stable HCT116 lines. RIPA lysates from the cells treated with vehicle or MG132 for 12 hr were probed with Bax, Mcl-1, or HSP60 antibody. (B) Induction of morphologic changes by TRAIL. The images were captured before or after treatment with 100 ng/ml TRAIL for 4 hr. (C) Induction of cytochrome c release by TRAIL. Cells treated with TRAIL were stained with cytochrome c antibody (red). DNA was visualized by Hoechst 33342 (blue). (D) Induction of DNA fragmentation by TRAIL. Cells treated with 100 ng/ml TRAIL for 8 hr were fixed and analyzed by PI staining. (E) Induction of cleavage of Casp6, Casp7, and PARP by TRAIL. Cell extracts were prepared at the indicated time points after treatment with 100 ng/ml TRAIL. Immunoblotting was performed to analyze the cleavage of Casp6 (C-Casp6), Casp7 (C-Casp7), and PARP. (F) Induction of DNA fragmentation by INDO and THA. The indicated cell lines treated with 300 mM INDO or 1 μM THA for the indicated time periods were fixed and analyzed by PI staining. Error bars indicate SEM. Molecular Cell 2009 33, 15-29DOI: (10.1016/j.molcel.2008.11.025) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Apoptotic Stimuli Upregulate Baxβ through Inhibition of Its Ubiquitination Process (A and B) Upregulation of Baxβ protein by TRAIL and THA coincides with caspase activation. Lysates from the HCT116-Baxβ cells treated with 100 ng/ml TRAIL (A) or 2 μM THA (B) for the indicated time periods were immunoprecipitated with Baxβ antibody B1, followed by immunoblotting with Bax antibody 2D2. Cleaved Casp7 and Casp9 in the lysates were monitored with their cleaved form-specific antibodies. (C) Upregulation of Baxβ protein by TRAIL is independent of caspase activation. z-VAD or QVD (10 μM) was added to HCT116-Baxβ cells prior to the treatment with 100 ng/ml TRAIL for 3 hr. Levels of Baxβ were determined as in (A). Cleaved Casp7 and PARP in the lysates were monitored with their cleaved form-specific antibodies. (D) Apoptotic stimuli inhibit ubiquitination of Baxβ. HCT116-Baxβ cells were treated with 100 ng/ml TRAIL for 1 hr, 300 mM INDO for 10 hr, or 2 μM THA for 10 hr before MG132 was added and the cells were incubated for another 6 hr. Equivalent lysates were immunoprecipitated with Baxβ antibody, followed by immunoblotting with ubiquitin (Ubi) antibody. The blot was stripped and probed again with Bax antibody. Total lysates were also probed with ubiquitin antibody. Molecular Cell 2009 33, 15-29DOI: (10.1016/j.molcel.2008.11.025) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 Baxβ Associates with Baxα and Synergizes with Baxα to Promote Bax-Dependent Apoptosis Signaling (A) Baxα can be activated by Baxβ. Myc-Baxα was in vitro translated alone or together with myc-Baxβ. The conformation of the translated proteins was analyzed by the N20 antibody in the absence or presence of NP40. (B) Baxβ is more potent in inducing cytochrome c release in mitochondria prepared from HC116-Bax+/− than those from HCT116-Bax−/− cells. Mitochondria isolated from HC116-Bax+/− (Bax+/− Mito) or Bax−/− (Bax−/− Mito) cells were incubated with Baxβ, followed by centrifugation. The supernatants (Sup) and pellets were immunoblotted with cytochrome c or HSP60 antibody. (C) Baxβ synergizes with Baxα to stimulate release of cytochrome c. Mitochondria isolated from HC116-Bax−/− cells were incubated with Baxα, Baxβ, or VDAC (as a control). The release of cytochrome c was analyzed as in (B). (D) Baxβ associates with Baxα. 293T cells transfected with the plasmids encoding the indicated Bax proteins were resuspended in CHAPS buffer. Immunoprecipitation and immunoblotting were performed with the indicated antibodies. (E) TRAIL induces upregulation of Baxβ and promotes its association with Baxα. HCT116-Bax+/−Baxβ cells treated with 100 ng/ml TRAIL for 1 or 3 hr were resuspended in CHAPS buffer. Immunoprecipitation and immunoblotting were performed with the indicated antibodies. For immunoprecipitation with the N20 antibody, 50 μl of 10% NP40 was added to 500 μl of cell lysates. (F) Stable expression of Baxβ enhances the sensitivity of HCT116-Bax+/−cells to TRAIL. The HCT116-Bax+/− vector control cells (Bax+/−) or HCT116-Bax+/− cells stably expressing Baxβ (Bax+/−Baxβ) were treated with TRAIL for 8 hr. DNA fragmentation was analyzed by flow cytometry. Error bars indicate SEM. (G) Baxβ shRNAs efficiently deplete Baxβ mRNA. Baxβ mRNA levels in HCT116-Bax+/− cells stably expressing Baxβ shRNA-1 or shRNA-2 or the corresponding mutants (shRNA-1-m or shRNA-2-m) were analyzed by real-time PCR. Error bars indicate SEM. (H) The Baxβ protein is selectively and efficiently reduced in HCT116-Bax+/−Baxβ shRNA cells. Baxα protein levels in the indicated resting cells were analyzed by immunoblotting with the N20 antibody (left). After treatment of cells with MG132 for 24 hr, Baxβ protein levels were determined by immunoprecipitation with Baxβ antibody B1, followed by immunoblotting with Bax antibody 2D2 (right). Total lysates were probed for HSP60. (I) Baxβ knockdown cells display reduced sensitivity in undergoing DNA fragmentation induced by TRAIL. Cells treated with 10–50 ng/ml TRAIL for 12 hr were fixed and analyzed by PI staining. Error bars indicate SEM. (J) Baxβ knockdown cells are less sensitive to the apoptotic effect of TRAIL. Lysates from the cells treated with 20 ng/ml TRAIL for the indicated time periods were prepared. Bax activation was analyzed by immunoprecipitation with the N20 antibody. Total lysates were probed for Baxα and the cleaved forms of Casp6 and PARP. Molecular Cell 2009 33, 15-29DOI: (10.1016/j.molcel.2008.11.025) Copyright © 2009 Elsevier Inc. Terms and Conditions