The N-terminus and alpha-5, alpha-6 helices of the pro-apoptotic protein Bax, modulate functional interactions with the anti-apoptotic protein Bcl-xL
© Parikh et al; licensee BioMed Central Ltd. 2007
Received: 16 November 2006
Accepted: 23 May 2007
Published: 23 May 2007
Bcl-2 family proteins are key regulators of mitochondrial integrity and comprise both pro- and anti-apoptotic proteins. Bax a pro-apoptotic member localizes as monomers in the cytosol of healthy cells and accumulates as oligomers in mitochondria of apoptotic cells. The Bcl-2 homology-3 (BH3) domain regulates interactions within the family, but regions other than BH3 are also critical for Bax function. Thus, the N-terminus has been variously implicated in targeting to mitochondria, interactions with BH3-only proteins as well as conformational changes linked to Bax activation. The transmembrane (TM) domains (α5-α6 helices in the core and α9 helix in the C-terminus) in Bax are implicated in localization to mitochondria and triggering cytotoxicity. Here we have investigated N-terminus modulation of TM function in the context of regulation by the anti-apoptotic protein Bcl-xL.
Deletion of 29 amino acids in the Bax N-terminus (Bax 30–192) caused constitutive accumulation at mitochondria and triggered high levels of cytotoxicity, not inhibited by Bcl-xL. Removal of the TM domains (Bax 30–105) abrogated mitochondrial localization but resulted in Bcl-xL regulated activation of endogenous Bax and Bax-Bak dependent apoptosis. Inclusion of the α5-α6 helices/TMI domain (Bax 30–146) phenocopied Bax 30–192 as it restored mitochondrial localization, Bcl-xL independent cytotoxicity and was not dependent on endogenous Bax-Bak. Inhibition of function and localization by Bcl-xL was restored in Bax 1–146, which included the TM1 domain. Regardless of regulation by Bcl-xL, all N-terminal deleted constructs immunoprecipitated Bcl-xLand converged on caspase-9 dependent apoptosis consistent with mitochondrial involvement in the apoptotic cascade. Sub-optimal sequence alignments of Bax and Bcl-xL indicated a sequence similarity between the α5–α6 helices of Bax and Bcl-xL. Alanine substitutions of three residues (T14A-S15A-S16A) in the N-terminus (Bax-Ala3) attenuated regulation by the serine-threonine kinase Akt/PKB but not by Bcl-xL indicative of distinct regulatory mechanisms.
Collectively, the analysis of Bax deletion constructs indicates that the N-terminus drives conformational changes facilitating inhibition of cytotoxicity by Bcl-xL. We speculate that the TM1 helices may serve as 'structural antagonists' for BH3-Bcl-xL interactions, with this function being regulated by the N-terminus in the intact protein.
Bcl-2 family proteins are central to the regulation of mitochondrial integrity and cell death pathways that converge on this organelle [1, 2]. This family comprises both pro-survival (Bcl-2, Bcl-xL, Mcl-1, Bcl-w) and pro-apoptotic (Bax, Bak, Bim, Bid, Bad) proteins. All members of this family are characterized by the presence of BH (Bcl-2 homology) domain(s) [3, 4]. Bax is a multidomain pro-apoptotic member of the Bcl-2 family, characterized by the presence of BH 1–3 domains. The BH3 domain regulates Bax homodimerization as well as heterodimerization with Bcl-2/Bcl-xL[5, 6], such that the Bax-BH3 domain docks into the hydrophobic groove formed by BH1, BH2 and BH3 domains of the anti-apoptotic proteins [7–9]. Selectivity in interactions between pro- and anti-apoptotic members of the Bcl-2 family has been reported .
Changes in Bax conformation and localization are characteristic of many cells undergoing apoptosis [11–13]. In dying cells a conformationally distinct form of (activated) Bax accumulates at the mitochondrial outer membrane triggering the release of apoptogenic intermediates sequestered in the mitochondrial intermembrane space . Several studies have yielded insights on the molecular mechanisms regulating Bax activation [15–18] and the N-terminus is implicated in this function. Controlled cleavage of the Bax N-terminus by calpain, a Ca2+-dependent cysteine protease generates a dominantly active form of Bax [19–21]. Short forms of Bax deleted of the first 28 or 32 amino acids are highly cytotoxic, largely associated with mitochondrial membrane and poorly inhibited by ectopically expressed Bcl-2 and Bcl-xL [22, 23]. Two splice variants Bax ψ and Bax κ lack 19 amino acids present at the N-terminal of Bax-α and are potent inducers of cytotoxicity when ectopically expressed in cells [24, 25].
Specific regions in the N and C terminus are implicated in the regulated mitochondrial targeting of Bax [26–28]. However, recombinant Bax protein lacking these regions retains mitochondrial targeting and insertion capabilities [27, 29, 30] because of two putative transmembrane (TM) regions, one encompassing the α5–α6 helices (amino acids 110–146) and the second the α9 helix (amino acids 172–188) at the C-terminus . These are referred to as TM1 and TM2 respectively in this study. Recent experiments have shown that both TM1 and TM2 span the lipid bilayer in the membrane fraction of cells undergoing apoptosis . Additionally other reports have identified a role for the α5–α6 helices in mitochondrial membrane insertion and cytochrome c release [29, 33]. In the present study the TM1 domain and N-terminus have been characterized in the context of regulation of activity by Bcl-xL. Specifically, the experiments suggest that interactions between the N-terminus and TM1 region in intact Bax, modulate susceptibility to inhibition by Bcl-xL.
Apoptosis triggered by a Bax N-terminal deletion mutant (Bax 30–192) is poorly inhibited by the anti apoptotic proteins, Bcl-2 and Bcl-xL
Percent viability with tissue specific GAL4 drivers
Percent viability and extent of wing defect with vg-GAL 4 for additional UAS transgenic lines
% viability vg-GAL4
% viable flies strong wing defect
% viable flies mild wing defect
% viable flies no wing defect
Apoptosis triggered by transmembrane -1 (TM1) deletion mutants is inhibited by Bcl-xL
We then tested the deletion mutants for regulation by Bcl-xL in the HEK cell line as the larger size and adherent nature of the cells facilitates assessment of apoptotic damage and subcellular distribution of GFP-tagged proteins. Bax induced apoptosis was effectively blocked by Bcl-xL; apoptosis triggered by Bax 30–192 was poorly inhibited, and apoptosis triggered by Bax 30–105 was blocked by ectopically expressed Bcl-xL in this cell line (Figure 3D). Jurkat cells express lower levels of Bax protein compared to the HEK cell line arguing against interactions with endogenous Bax accounting for the responses of the deletion mutants.
Bcl-xL regulation of Bax 30–192 and Bax 30–105 colocalization with Bid and Bim
In the absence of the N-terminus, the TM1 domain attenuates inhibition by Bcl-xL but triggers caspase-9 dependent apoptosis
A construct that included the TM1 domain comprising the α5–α6 helices, but not the N-terminal 29 amino acids (Bax 30–146) (Figure 5A) triggered Bcl-xL independent cytotoxicity (Figure 5B) indicating that the TM1 domain interfered with the anti-apoptotic function of Bcl-xL. Since Bax function converges on the activation of caspase-9  we analyzed apoptotic damage induced by Bax, Bax 30–146 or Bax 30–105 when co-expressed with a construct that is a dominant-negative inhibitor of caspase-9 (DNC9) [37, 38]. Apoptosis triggered by all the mutants tested, including Bax 30–192 (data not shown) was blocked by the co-transfection of DNC9 in HEK cells (Figure 5C) and in the Jurkat cell line (data not shown). The data show that all the constructs tested activate a caspase-9 mediated apoptotic cascade as has been established for Bax.
Sub-cellular fractionation of HEK cells (Figure 5D) transfected with the relevant constructs showed that Bax is present in both the cytosolic and mitochondrial fractions, whereas increased amounts of Bax 30–146 was detected in the mitochondrial fractions (Figure 5D, upper and middle panel). In many experiments, the distribution of Bax 30–146 and Bax 30–192 was skewed to the mitochondrial fraction (not shown). Expectedly, Bax 30–105 was consistently detected only in the cytosolic fraction. The mitochondrial matrix protein Cox-4 was used to ascertain purity of the fractions.
Bcl-xL associates with but does not regulate the localization of N-terminal deletion constructs
Ectopically expressed Bcl-xL immunoprecipitated cotransfected Bax, Bax 30–146 or Bax 30–105 (Figure 6D) indicating that association with Bcl-xLis not impaired in the absence of the first twenty nine residues in Bax protein.
The Bax N-terminus regulates inhibition by Bcl-xL
Phosphorylation dependent modifications that change protein conformation regulate the apoptotic function of Bcl-2 family proteins. Bax-induced apoptosis is inhibited by the serine-threonine kinase Akt (Figure 7F and 7H). This regulation was compromised in a Bax construct (Bax-Ala3) with alanine substitutions of the Ser/Thr residues (T14, S15, S16) in the N-terminus which was refractory to inhibition by Akt (Figure 7F and 7H). The modification in the N-terminus however, did not interfere with Bcl-xL inhibition of Bax-Ala3 induced apoptosis (Figure 7G and 7I).
The N-terminus regulates sensitivity to inductive stimuli and inhibition by Bcl-xL
The data thus far suggested that apoptosis triggered by Bax 30–146 and Bax 30–192 should not depend on endogenous Bax or Bak, whereas Bax 30–105 induced apoptosis may depend on these proteins. MEF deficient for both Bax and Bak were therefore tested for susceptibility to apoptosis by the Bax deletion mutant constructs. Confirming these predictions Bax and Bax 30–192 but not Bax 30–105 triggered apoptosis in Bax/Bak deficient cells (Figure 8F).
TM1 helices of full-length Bax are structural antagonists to Bcl-xL
The BH3 domain is critical to several interactions within the Bcl-2 family [5–9] and the anti-apoptotic proteins Bcl-2 and Bcl-xL regulate Bax via interactions involving this domain [5, 42]. However, mutations in the Bax-BH3 domain which disrupt interactions with pro-survival proteins do not always disrupt regulation by the latter [17, 43–45]. The N-terminus of Bax is implicated in the regulation of its function by Bcl-2/Bcl-xL and activator BH3-only proteins [[15, 22, 23] and ]. Here we show that the BH3 and TM1 domains are adequate for association with Bcl-xL and localization to mitochondria respectively, but an intact N-terminus plays a non-redundant role in the regulation of Bax cytotoxicity by Bcl-xL.
In contrast to Bax [11, 15, 26], the distributions of Bax 30–192 or Bax 30–146 were largely unchanged by Bcl-xL (Figure 6B–C). The TM1 domain/α5–α6 helices (amino acids 110–146) in Bax bring about membrane insertion and cytochrome c release from mitochondria in cell-free assays [29, 33]. The substitution of charged residues in the α5–α6 helices of Bax resulted in its constitutive localization to mitochondria, akin to gain-of-function mutants and increased binding to Bcl-xL . Despite consistent differences from Bax in regulation by Bcl-xL, the N-terminal deletion mutants immunoprecipitated ectopically expressed Bcl-xL (Figure 6D). This is not unexpected as the association of apoptotic with anti-apoptotic proteins does not culminate in the inhibition of pro-apoptotic activity [17, 43–45, 48]. Further, Bax 30–192 triggered caspase-9 dependent apoptosis and localized with Bid and Bim conventionally associated with regulated activity of Bax [15, 48, 49] a distribution largely unchanged by Bcl-xL. In our experiments the effect of the TM1 domain was mitigated by the inclusion of the N-terminal region i.e. Bax-1–146 (Figure 7).
The anti-apoptotic proteins Bcl-2 and Bcl-xL are not functionally equivalent and the differences in the regulation of Bax 30–105 (Figure 3C) likely reflect differences in affinities of the mutant for the anti-apoptotic proteins. Such differences in functional interactions between pro-survival members and the pro-apoptotic or BH3 only proteins (BOPs) have been previously reported [17, 50]. The BOPs are thought to be critical mediators of the activation of Bax/Bak although the mechanism underlying this activation is unresolved. Thus, 'activator" BOPs (Bid, Bim and Puma) have been reported to directly bind Bax and Bak triggering activation of the latter and are assisted in this function by "derepressor" BOP (Noxa, Bad etc.) which bind anti-apoptotic proteins . On the other hand there is compelling evidence that BOP trigger the activation of Bax principally by binding Bcl-2/Mcl-1/Bcl-xL  obviating a requirement for direct interactions with Bax/Bak except in some conditions . Bax 30–105 fulfills several criteria associated with BH3 domain only proteins triggering activation of endogenous Bax (which colocalized with Bid and Bim); Bcl-xL dependence and in the requirement for endogenous Bax-Bak for apoptotic function (Figure 3C–D, 4E–F and 8F).
We do not know the mechanism underlying the lack of regulation of Bax 30–192 and Bax 30–146 by Bcl-xL. The constitutive association of Bax 30–192 and 30–146 with Bcl-xL may arise from structural changes in the deletion mutants culminating in a greater affinity (compared to Bax) for the anti-apoptotic protein. This may permit the mutants to sequester ectopically expressed Bcl-xL. On the other hand the association may impose conformational changes on Bcl-xL such that the latter is rendered functionally inactive and thereby unable to regulate apoptotic damage. That the pattern of Bid distribution in cells expressing Bax or Bax 30–105 changes on co-expressing Bcl-xL but not in cells that express Bax 30–192 provides some support for this possibility.
Our experiments extend previous observations that the Bax N-terminus is an essential component in the regulation of Bax function by anti-apoptotic proteins such as Bcl-xL. Furthermore we propose that an intramolecular interaction between the N-terminus and the TM1 may be the mechanism underlying this regulation.
Cells and reagents
Jurkat, HEK and COS-7 cell lines and the Bax-Bak deficient MEF were maintained in culture in medium supplemented with antibiotics, glutamine (Invitrogen-Gibco, Carlsbad, CA) and 5 or 10% fetal calf serum (Hyclone, Logan, UT). Antibodies were procured from the following sources: to GFP and Bim from BD Biosciences (Franklin Lakes, NJ); to Bax and Bcl-2 from Santa Cruz (Santa Cruz, CA); to Cox-4 and and Alexa Fluor 555/Cy5 conjugated secondary antibodies from Invitrogen-Molecular Probes; to Bid from Cell Signaling Technology; to Bcl-xL from Abcam (Cambridge, UK); actin and clone 6A7 from Neomarker (Fremont, CA) respectively. Protein G agarose beads were from Pierce Biotechnology (Rockford, IL). Mouse or Rabbit True-Blot HRP (Ebiosciences, San Diego, CA) was used in the western blot analysis of immunoprecipitated proteins. All other chemicals were obtained from Calbiochem (San Diego, CA) or Invitrogen.
A mammalian expression vector encoding murine full-length Bax was the kind gift of Peter Vandenabeele (University of Gent, Belgium). Bax-GFP and the deletion mutants were generated by PCR amplification and subcloning into the pEGFP-N1 vector (BD Clontech, Mountain View, CA) with GFP in-frame at the C- terminus. The following primers were used to generate the Bax and Bax mutants: Bax/Bax 1–171/Bax 1–146 forward 5' CCG CTC GAG ATG GAC GGG TCC GGG 3' Bax 30–192/Bax 30–171/Bax 30–146/Bax 30–105 forward 5' CCG CTC GAG ATG TTC ATC CAG GAT 3' Bax/Bax 30–192 reverse 5' CCC AAG CTT GCC CAT CTT CTT CCA 3' Bax 30–171/Bax 1–171 reverse 5' CCC AAG CTT CTG CCA TGT GGG 3' Bax 30–146/Bax 1–146 reverse 5'CCC AAG CTT CTC ACG GAG GAA 3' Bax 30–105 reverse 5' CCC AAG CTT GAA GTT GCC ATC 3'. The murine Bcl-2 plasmid was obtained from Upstate (Charlottesville, VA). The dominant negative caspase 9 construct was the kind gift of C. Vincenz (University of Michigan Medical School, Ann Arbor, MI) and the human Bcl-xL plasmid was from Richard J. Youle (National Institutes of Health, Bethesda, MD). CA-Akt was from Upstate (Charlottesville, VA). Monomeric RFP (mRFP) plasmid was originally from Roger Y. Tsien (University of California, San Diego, CA) and obtained through Satyajit Mayor (National Centre for Biological Sciences, Bangalore, India). Bcl-xL was subcloned into the pUSEAmp mammalian expression vector and was also subcloned at the 3' end of mRFP under the CMV promoter. Mito-DsRed was obtained from V. Sriram (National Centre for Biological Sciences, Bangalore, India). The sequence of all constructs was verified by automated sequencing (Microsynth, Balgach, Switzerland). The alanine substitution mutant Bax-Ala3 (substituted on T14, S15 and S16) was generated at Everogen (Moscow, Russia).
Transient transfections and assay for nuclear damage
Jurkat and 2B4 cells were transfected by electroporation. Briefly 3–5 million cells were electroporated at 250 V and 960 μF and cultured for 12 hours prior to analysis. HEK and COS-7 cells were plated at 0.4–0.5 million cells/ml and MEF at 0.3 million/ml 12–15 hours prior to transfection. Cells were transfected using lipofectaime 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Cos-7 cells were transfected with 0.2 μg of Bax constructs and 2 μg Bcl-xL. 12–15 hours later staurosporine was added at a final concentration of 1 μM and the cultures continued for an additional 3–4 hours before analysis.
Transfected cells were harvested and stained for 3 minutes at ambient temperatures in the dark using the Hoechst 33342 (1 μg/ml) and assessed for apoptotic nuclear damage. GFP positive cells were viewed under the UV filter of the fluorescence microscope and nuclear morphology scored for a minimum of 200 cells in each experimental condition. In a minimum of two experiments in each set of data samples were scored double-blind. Statistical significance of the differences between groups was assessed by the Student's t-test and is indicated by an * in the figures.
The protocol followed has been previously described . Briefly, 1 million cells were incubated in 100 μl homogenization buffer (250 mM sucrose, 20 mM Hepes, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, pH 7.2 supplemented with 2 μg/ml aprotinin, leupeptin and pepstatin) for 15 min on ice. Cells were mechanically disrupted by passing through a 26-gauge needle 15 times followed by a 30-gauge needle 15 times. Cell lysates were centrifuged at 750 g for 10 min at 4°C to remove unlysed cells and nuclei. The supernatant was centrifuged at 12,000 g for 20 min at 4°C to obtain the pellet enriched in mitochondria and the supernatant enriched for cytosolic proteins. Equivalent volumes of cytosolic and mitochondrial fractions were used for western blot analysis to assess the relative distribution of Bax and the mutants. The densitometry analysis of western blots was performed using Image Gauge software Version-3 (Fujifilm Science, Japan)
In each group two million cells were harvested and lysed in 500 μl of CHAPS lysis buffer (1% CHAPS in 50 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, pH7.4 supplemented with 2 μg/ml aprotinin, leupeptin, pepstatin, 1 mM PMSF, 1 mM NaF and 1 mM Na3OV4) by 3 freeze thaw cycles in liquid nitrogen and 37°C followed by an incubation (30 minutes) at 4°C on a cell rotator. The lysate was centrifuged at 3000 rpm to remove debris and unlysed cells. The supernatant was incubated with 10 μg of antibody to Bcl-xLat 4°C for 1 hour on a cell mixer. 80 μl of washed Protein-G agarose beads were then added to the sample and mixed by gentle rotation for another 2 hours at 4°C. Beads were pelleted by centrifugation at 1700 rpm and subsequently washed 4 times with chilled PBS. After the final wash the beads-complex was pelleted by centrifugation at 2500 rpm and boiled in SDS lysis buffer for 10 minutes before western blot analysis.
Immunochemistry and confocal imaging
Cells washed with PBS and fixed in 4% paraformaldehyde for 15 minutes at ambient temperature were permeabilized with 0.2% CHAPS in PBS (permeabilization buffer) for 30 minutes on ice and blocked for 1 hour on ice with 5% BSA diluted in permeabilization buffer. Cells were incubated with primary antibodies diluted in blocking buffer for 1–2 hours on ice followed by a wash with permeabilization buffer. The cells were finally incubated with Alexa Fluor 555 and/or Cy5 conjugated secondary antibodies diluted in blocking buffer for 1 hour on ice. Cells were also counterstained with Hoechst 33342 before the final wash and imaged.
Cells were imaged using a Bio-Rad MRC 1024 laser scanning confocal microscope equipped with Argon-Krypton laser and 60X, NA 1.4 objective lens. Fluorescence images of cells were recorded with sequential excitation and emission conditions using appropriate optics. The acquired images were processed for co-localization analysis using Image J1.34s software. For the analysis of endogenous Bid, Bim or Bax, samples were imaged using a LSM 510 Meta laser scanning confocal microscope (Carl Zeiss, Jena, Germany) equipped with Argon, Helium-Neon lasers and 63X, NA 1.4 objective. The fluorescence images for GFP tagged, Alexa Fluor 555 and Cy5 labeled proteins were acquired with sequential excitation (488 nm, 543 nm and 633 nm respectively) and emission conditions using appropriate optics. The nuclear morphology images were acquired using two photon excitation of single plane with 750 nm pulsed laser (Spectra-Physics, CA). Images were processed for co-localization analysis using Image J1.34s and LSM Meta (Carl Zeiss, Jena, Germany) software.
Transgenic flies and genetic interaction studies
Bax-GFP, Bax 30–192-GFP and Bcl-2 were cloned in the UAS vector pUAST. The resulting plasmids were injected into yw; +; Ki P(Δ2-3ry+) embryos using standard procedures. Several independent transformant lines were obtained by P-element transformation for each transgene. A minimum of two lines in each case were characterized for further analysis. Bax-GFP insertion was mapped to chromosomes III and I for BA64III and BA13I transgenic lines respectively. Bax 30–192-GFP insertion was mapped to chromosome II and I for DBA8II and DBA8IA fly lines respectively. Bcl-2 insertion was mapped to chromosome I and III for BC57I and BC24III fly lines respectively. A single copy transformant fly line for each transgene was crossed to flies carrying vestigial (vg)-GAL4, twist-GAL4, eyeless-GAL4, scalloped-GAL4 or collagen-GAL4. Flies were raised on standard Drosophila medium at 25°C.
We acknowledge M.K. Mathew (NCBS) for comments and discussion. The Bax-Bak double knock out MEF were generously gifted by Dr. Anthony G. Letai, Dana Farber Cancer Institute, MA, USA and were obtained from Dr. TR Santhosh Kumar, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India. We are grateful to several laboratories and colleagues for the generous gifts of reagents used in this study. We acknowledge H. Krishnamurthy and the Imaging and Flowcytometry Facility at NCBS.
NP and VD are funded by fellowships from the Council of Scientific and Industrial Research, India and the Indian Council of Medical Research, India respectively. The study was funded by a Senior Research Fellowship in Biomedical Sciences from the Wellcome Trust, UK to RS and AS.
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