Disruption of Mcl-1·Bim Complex in Granzyme B-mediated Mitochondrial Apoptosis*

Recently, we reported the identification of a novel mitochondrial apoptotic pathway for granzyme B (GrB) (Han, J., Goldstein, L. A., Gastman, B. R., Froelich, C. J., Yin, X. M., and Rabinowich, H. (2004) J. Biol. Chem. 279, 22020–22029). The newly identified GrB-mediated mitochondrial cascade was initiated by the cleavage and subsequent degradation of Mcl-1, resulting in the release of mitochondrial Bim from Mcl-1 sequestration. To investigate the biological significance of Mcl-1 cleavage by GrB, we mapped the major GrB cleavage sites and evaluated the apoptotic potential of the cleavage products. GrB cleaves Mcl-1 after aspartic acid residues 117, 127, and 157, generating C-terminal fragments that all contain BH-1, BH-2, BH-3, and transmembrane domains. These fragments accumulate at an early apoptotic phase but are eliminated by further degradation during the apoptotic process. The major Mcl-1 C-terminal fragment generated by GrB (residues 118–350) was unable to induce or enhance apoptosis when transfected into tumor cells. Instead, this Mcl-1 C-terminal fragment maintained a partial protective capability against GrB-mediated apoptosis via its lower affinity to Bim. In comparison with ectopically expressed full-length Mcl-1, the stably transfected C-terminal fragments of Mcl-1 were less efficiently localized to the mitochondria. Knockdown of Mcl-1, as achieved by transfection with Mcl-1-specific short interfering RNA, resulted in a significant level of apoptosis in the absence of external apoptotic stimulation and, in addition, enhanced the susceptibility of breast carcinoma cells to GrB cytotoxicity. The significance of Bim in this GrB apoptotic cascade was indicated by the marked protection against GrB-mediated apoptosis endowed on these cells through Bim knockdown. Our studies suggest that the disruption of the Mcl-1·Bim complex by GrB initiates a major Bim-mediated cellular cytotoxic mechanism that requires the elimination of Mcl-1 following its initial cleavage.

and in the survival and homeostasis of mature lymphoid cells (4,5). Mcl-1 is an anti-apoptotic Bcl-2 family member with three putative Bcl-2 homology domains (BH1-3) (1,4). Other pro-survival Bcl-2 family members, including Bcl-2, Bcl-XL, and Bcl-w, also posses a BH4 domain, which is absent in Mcl-1 and A1/Bfl-1 (6). Because the BH4 domain is required for molecular interactions with other proteins (7)(8)(9), its absence in Mcl-1 suggests that this Bcl-2 member interacts with a different set of proteins as compared with Bcl-2 and Bcl-XL. We and others (5,10) have recently identified a Mcl-1 high affinity binding capacity for Bim, whereas its affinity for Bid, Bad, Bax, and Bak was low. By contrast, Bcl-2 displayed comparable binding to Bim and Bad (5). These findings suggest that the anti-apoptotic activity of different Bcl-2 family members depends on their selective interactions with other proteins, including various pro-death Bcl-2 members. Mcl-1 has a C-terminal hydrophobic domain that mediates its localization to membranous organelles (11,12). Because both Mcl-1 and Bim are mainly localized at the mitochondrial membrane (13), sequestration of Bim by Mcl-1 is expected to block the Bimmediated mitochondrial apoptotic cascade (10). Mcl-1 has a fast turnover rate and the shortest half-life among anti-apoptotic Bcl-2 family members (4). This rapid turnover may serve the apoptotic process, particularly in light of the finding that early elimination of Mcl-1 is required for a UV-mediated mitochondrial cascade in HeLa cells (14). Mcl-1 expression is highly regulated at both transcriptional and post-transcriptional levels. Its expression is dependent on environmental survival stimuli, mediated by various growth factors, such as IL-2, 1 IL-3, IL-4, IL-7, IL-13, and granulocyte-macrophage colonystimulating factor (5,(15)(16)(17)(18). Several post-transcriptional mechanisms have been implicated in its down-regulation, including decreased protein synthesis and various proteolytic activities that are mediated by proteasomes or caspases (5,19,20).
We reported that Mcl-1 is a direct substrate for GrB (10). Based on this observation and the identification of the high affinity of Mcl-1 for Bim, we proposed that the GrB-mediated degradation of Mcl-1 may free sequestered Bim and therefore allows for the execution of a potent Bim-mediated mitochondrial apoptotic cascade. However, other Mcl-1-related pro-survival Bcl-2 members, Bcl-2 and Bcl-XL, are converted into pro-apoptotic effectors upon their cleavage by caspase activity (21,22). In the current study, we investigated the apoptotic nature of GrB-cleaved Mcl-1 products, as well as the significance of a complex between Bim and Mcl-1 or its GrB-generated C-terminal cleavage products for cellular survival.
Cell Lines, Cell Lysates, and Cell Extracts-Jurkat T leukemic cells were grown in RPMI 1640 medium containing 10% fetal calf serum, 20 mM HEPES, 2 mM L-glutamine, and 100 units/ml each of penicillin and streptomycin. HeLa, breast carcinoma CAMA-1, and colon cancer Hct116 cells were grown in Dulbecco's modified Eagle's medium containing 15% fetal calf serum, 20 mM L-glutamine, and 100 units/ml each of penicillin and streptomycin. The cell lysates were prepared with 1% Nonidet P-40, 20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 10 g/ml aprotinin. To prepare cell extracts for GrB or caspase-3 reactions, cultured cells were washed twice with phosphate-buffered saline and then resuspended in ice-cold buffer (20 mM HEPES, pH 7.0, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, 250 mM sucrose, and protease inhibitors). After incubation on ice for 20 min, the cells (2.5 ϫ 10 6 /0.5 ml) were disrupted by Dounce homogenization. The nuclei were removed by centrifugation at 650 ϫ g for 10 min at 4°C. Cellular extracts were obtained as the supernatants resulting from centrifugation at 14,000 ϫ g at 4°C for 30 min.
Cellular Fractionation and Mitochondria Purification-To obtain an enriched mitochondrial fraction, Jurkat or HeLa cells were suspended in mitochondrial buffer (MIB) composed of 0.3 M sucrose, 10 mM MOPS, 1 mM EDTA, and 4 mM KH 2 PO 4 , pH 7.4, and lysed by Dounce homogenization as described previously (10). Briefly, the nuclei and debris were removed by a 10-min centrifugation at 650 ϫ g, and a pellet containing mitochondria was obtained by two successive spins at 10,000 ϫ g for 12 min. To obtain the S-100 fraction, the postnuclear supernatant was further centrifuged at 100,000 ϫ g for 1 h at 4°C. To obtain the enriched mitochondrial fraction, the mitochondria containing pellet was resuspended in MIB and layered on a Percoll gradient consisting of four layers of 10, 18, 30, and 70% Percoll in MIB. After centrifugation for 30 min at 15,000 ϫ g, the mitochondrial fraction was collected at the 30/70 interface. Mitochondria were diluted in MIB containing 1 mg/ml bovine serum albumin (at least a 10-fold dilution required to remove Percoll). The mitochondrial pellet was obtained by a 40-min spin at 20,000 ϫ g and used immediately. Purity was assessed by electron microscopy and by enzyme marker analysis (23). For enzyme analysis, the following enzymes were assayed: aryl sulfatase (lysosomes/granules); N-acetyl-␤-D-glucosaminidase, ␣-L-fucosidase, and ␤-glucoronidase (lysosome); lactate dehydrogenase (cytosol); cytochrome oxidase or monoamine oxidase (mitochondria); thiamine pyrophosphatase (Golgi); NADH oxidase (endoplasmic reticulum); and dipeptidyl peptidase IV (plasma membrane). The purity was assessed at 95%, with ϳ5% or less contamination from the microsomal fraction.
Molecular Cloning of Human BimEL-Production of human BimEL cDNA clones was as described previously (10).
Transfection-Hct116 cells were washed in cold phosphate-buffered saline and resuspended in electroporation buffer (Amaxa) at a final concentration of 3 ϫ 10 7 cells/ml. Five g of linearized plasmid DNA were mixed with 0.1 ml of cell suspension, transferred to a 2.0-mm electroporation cuvette, and nucleofected with an Amaxa Nucleofector apparatus (Amaxa, www.amaxa.com) utilizing program T20, according to the manufacturer's directions. CAMA-1 cells were transfected by the GenePorter Transfection Reagent (Gene Therapy Systems Inc., San Diego, CA) according to the manufacturer's directions. Geneticin-resistant cell lines were grown in the presence of G418 (1500 g/ml). Geneticin-resistant clonal cell lines were generated by dakocytomation (1 cell/well) utilizing a MOFLO high speed cell sorter and Summit Software. Transient transfection of CAMA-1 cells was carried out with Mcl-1 recombinant plasmids (see above) and the EGFP control plasmid pEGFP-C2 (BD Biosciences) using GenePorter transfection reagent following the manufacturer's protocol. Gly 128 -Pro 133 ) and the same reverse primer as WT Mcl-1. PCR amplicons were gel-purified as described previously (10), digested with the restriction enzymes BamHI and SalI, and subsequently ligated into the BamHI and SalI sites of the vector pGEX-4T-1 (Amersham Biosciences). Sequence integrity of recombinant pGEX-4T-1 clones was determined as above.
Production of GST-Mcl-1 Fusion Proteins-E. coli BL21 cells (Novagen) were transformed with recombinant pGEX-4T-1 Mcl-1 plasmids and plated out on LB agar-ampicillin plates as directed by the manufacturer. Four colonies were randomly picked for each Mcl-1 transformant, and each colony was grown overnight in 3 ml of LB-ampicillin medium. Three ml of 2ϫYT-ampicillin medium (ϫ4) was inoculated with 45 l of overnight culture for each colony and cells were grown at 37°C to A 600 ϭ 0.6 -0.8 (ϳ2.5 h). Isopropyl ␤-D-thiogalactoside was added to a final concentration of 1 mM, and the cultures were incubated for an additional 3-4 h at 37°C. Bacterial pellets (2200 ϫ g for 30 min at 4°C) were extracted, and GST-Mcl-1 fusion proteins were isolated following the manufacturer's protocol (Amersham Biosciences) for Mi-croSpin GST purification modules except that 16 units of Benzonase (Sigma) and 3 l of Protease Inhibitor Mixture Set III (Novagen) were added to the pellet extraction buffer. GST-Mcl-1 fusion proteins eluted from the microspin columns were concentrated and underwent buffer exchange using YM-10 Microcon centrifugal filter devices (Millipore).
RNAi Using Mcl-1, Bim, and Lamin siRNAs-Short interfering RNAs were obtained as duplexes in purified and desalted form (Option C) from Dharmacon. The three siRNAs had the following sense strand sequences: Mcl-1, 5Ј-GAAACGCGGUAAUCGGACUdTdT-3Ј; Bim, 5Ј-GACCGAGAAGGUAGACAAUUGdTdT-3Ј; and Lamin, 5Ј-CUGGACU-UCCAGAAGAACAdTdT-3Ј. CAMA-1 cells (2.5 ϫ 10 5 ) were plated in a 6-well plate and following 24 h (at ϳ30% confluency) were transfected with 200 nM siRNA in Opti-MEM medium (Invitrogen) without fetal calf serum using Oligofectamine reagent (Invitrogen) according to the manufacturer's transfection protocol. After 4 h, fetal calf serum was added to a final concentration of 10%. At 40 h, the medium over the cells was adjusted to 1 ml before the addition of an apoptotic agent.
Release of Mitochondrial Apoptogenic Proteins-Purified mitochondria (50 g of protein) were incubated with various doses of recombi- His-BimL was then added for a co-incubation at 37°C for 30 min. Mitochondria were pelleted by centrifugation at 10,000 ϫ g for 10 min. The resulting supernatants or mitochondria were mixed with SDS sample buffer, resolved by SDS-PAGE, and analyzed by immunoblotting for the presence of mitochondrial apoptogenic proteins, cytochrome c, and AIF.
In Vitro Transcription-Translation-Mcl-1, Mcl-1⌬N127, Mcl-1⌬N117, and BimEL cDNAs were expressed in the TNT T7 transcription-translation reticulocyte lysate system (Promega). Each coupled transcription-translation reaction contained 1 g of plasmid DNA in a final volume of 50 l in a methionine-free reticulocyte lysate reaction mixture supplemented with 35 S-labeled methionine according to the manufacturer's instructions. After incubation at 30°C for 90 min, the reaction products were immediately used or stored at Ϫ70°C.
In Vitro Cleavage Reaction with Caspase-3 or GrB-In vitro cleavage reactions were performed in total volume of 20 l. The reaction buffer consisted of 20 mM HEPES, pH 7.4, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 20% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 10 g/ml aprotinin. Each reaction also contained 1 l of reticulocyte lysate containing 35 S-labeled Mcl-1, or BimEL, and also reticulocyte lysate minus plasmid in the presence or the absence of recombinant caspase-3 (5-100 nM) or GrB (33-330 nM) for 20 min at 37°C. The reactions were terminated by addition of SDS loading buffer and boiling for 5 min.
Immunoprecipitation-For Mcl-1 and Bim immunoprecipitation experiments, the cells (5-10 ϫ 10 6 ) and mitochondria (200 g of protein) were lysed in 1% CHAPS buffer (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 1% CHAPS). The lysates were precleared with protein A-or G-Sepharose beads, and incubated with anti-Mcl-1 or anti-Bim Abs at 4°C for 4 h. The immune complexes were then precipitated with protein A-or G-Sepharose beads at 4°C overnight. The pellets were washed four times with the appropriate lysis buffer and boiled for 5 min in SDS sample buffer.
Western Blot Analysis-Proteins in cell lysates, cell extracts, mitochondria, or S-100 were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes, as described previously (25). Following probing with a specific primary Ab and horseradish peroxidaseconjugated secondary Ab, the protein bands were detected by enhanced chemiluminescence (Pierce).
Flow Cytometry-Cytofluorometric analyses of apoptosis were performed by co-staining with propidium iodide and fluorescein isothiocyanate-annexin V conjugates (Becton-Dickenson). The staining was performed according to the manufacturer's procedures, assessed by a Beckman Coulter Epics XL-MCL, and analyzed with the EXPO32 software.

Elimination of Mcl-1 during Caspase-3 and GrB-mediated
Apoptosis-We recently reported the identification of a novel mitochondrial cascade for GrB-mediated apoptosis that encompasses the release of Bim from sequestration by mitochondrial Mcl-1 (10). Our reported studies suggest that the disruption of the Mcl-1⅐Bim complex is mediated by cleavage of Mcl-1 by GrB or caspase-3. Examples for the observed down-regulation in Mcl-1 expression are depicted in Fig. 1. Thus, in breast carci-noma CAMA-1 cells that serve as targets for cytotoxicity mediated by treatment with purified GrB and Ad, expression of Mcl-1 is significantly reduced as early as 6 h following treatment (Fig. 1A). In this cytotoxicity system, Ad substitutes for perforin as an endosomolytic agent that following the cellular internalization of GrB, facilitates its release into the cytoplasm (26,27). Reduction in the level of Mcl-1 expression has also been observed in VP-16-treated cells, where the activity of caspases is stimulated. However, in etoposide-treated cells, the down-regulation in Mcl-1 expression is not efficiently blocked by a potent caspase inhibitor (Fig. 1B), suggesting that other proteolytic activities may also be involved in the observed elim-  (24). Thus, mutant Mcl-1 cDNAs that encode single residue conversions (Asp 3 Ala) at one of the five sites as well as a mutant for both residues 127 and 157 were produced. All Mcl-1 cDNAs were ligated into the mammalian expression vector pCR3.1. The mutated Mcl-1 plasmids were transcribed and translated by an in vitro system, and the products were treated with recombinant caspase-3 or GrB. As determined by autoradiography ( Fig. 2A, top panel) or by immunoblotting (bottom panel), Mcl-1 cleavage by caspase-3 was mapped to aspartic acid residues 127 and 157. Mcl-1 cleavage by caspase-3 was completely blocked in the Mcl-1 mutant for both the 127 and 157 aspartic acid residues. These cleavage sites for caspase-3 were recently reported by two independent groups (19,20). GrB cleavage sites were mapped to aspartic acid residues 117, 127, and 157 (Fig. 2B). As demonstrated by immunoblotting (Fig.  2B, bottom panel), the major cleavage site for GrB is aspartic acid 117, because the D117A mutation blocked significantly the processing of Mcl-1, whereas D127A, D157A, and the double mutant of D127A/D157A were significantly less efficient in blocking the loss in full-length Mcl-1.
Kinetics of Elimination of Mcl-1 Cleavage Products-Cleavage of other anti-apoptotic Bcl-2 members, such as Bcl-2 and Bcl-XL, results in their conversion to pro-apoptotic proteins (21,22). Caspase cleavage of either Bcl-2 or Bcl-XL removes from each of these proteins an N-terminal BH4 domain that may be required for their antiapoptotic activity. Mcl-1 does not possess a BH4 domain, and cleavage by GrB or caspase-3 removes a 117-(GrB-only), 127-, or 157-residue N-terminal fragment, producing a C-terminal Mcl-1 fragment(s) that contains the transmembrane and BH1-3 domains (Fig. 2C). To gain a better insight into the apoptotic significance of the cleavage products, we determined their fate during the reaction of Mcl-1 with caspase-3 or GrB. The C-terminal cleavage products of Mcl-1 represented by residues 158 -350 and 118 -350 that are generated by caspase-3 and GrB, respectively, appear to accumulate during the initial phase of the reaction but start to decline within 2 h of enzymatic exposure (Fig. 3A). To generate a reaction setting that mimics the intracellular condi-  (Fig. 3B). In addition to its effect on the degradation of fulllength Mcl-1, GrB also accelerated the degradation of the Mcl-1 deletion mutants, because each contains at least one GrB cleavage site at residue Asp 157 . These observations also suggest that the major cleavage products of Mcl-1 generated by either caspase-3 or GrB are being further degraded by proteolytic activity present in the cell extract during an advanced apoptotic process.
The Apoptotic Response to GrB Is Significantly Attenuated by Stable Transfection of Full-length Mcl-1-To assess the significance of the expression of Mcl-1 and its cleavage products on survival, we transiently co-transfected the breast carcinoma CAMA-1 cell line with plasmids that express full-length Mcl-1, Mcl-1⌬N117 (GrB-cleaved C-terminal product), Mcl-1⌬N127 (caspase-3-cleaved C-terminal product), and a control plasmid encoding EGFP at a 10:1 ratio, respectively. As judged by EGFP/annexin V flow cytometry, we did not observe induction of apoptotic death in successfully transfected cells (EGFP-positive) (results not shown). To analyze the significance of the presence of Mcl-1 and its cleaved forms for survival under better defined conditions, we stably transfected colon carcinoma Hct116 and breast carcinoma CAMA-1 cell lines with the plasmids encoding these different Mcl-1 forms. We obtained a comparable number of geneticin-resistant clones from each of the cell lines (ϳ40 clones for each plasmid) and assessed the levels of Mcl-1 expression by Western blotting (Fig. 4A). The majority of the transfected clones demonstrated equal levels of expression of the transfected proteins. Geneticin-resistant Hct116 or CAMA-1 clonal cell lines that were confirmed by immunoblotting to overexpress the aforementioned Mcl-1-related proteins at equal levels were assessed for susceptibility to death mediated by GrB/Ad. A significant reduction in the apoptotic response to GrB/Ad as assessed by annexin V/propidium iodide was detected in each of the tumor cell lines transfected with full-length Mcl-1 (Fig. 4, B and C). However, overexpression of the Mcl-1 fragments that correspond to the caspase-3 and GrB C-terminal cleavage products provided a reduced protective effect from GrB-mediated cytotoxicity (Fig. 4, B and C). The two cells lines included in this analysis (CAMA-1 or Hct116) were sensitive to GrB-mediated apoptosis, but demonstrated different levels of phosphatidylserine externalization (Fig. 4, B and C). Differential detection of phosphatidylserine exposure has been reported to be cell type-specific and in various cells dependent on caspase activity or intracellular ATP levels (29 -31). Thus, in CAMA-1 cells, where only few cells were stained by annexin V, GrB may not directly activate intracellular molecules that are involved in phosphatidylserine externalization. Similar to the observations in transient transfected cells, overexpression of the C-terminal Mcl-1 fragments in either the Hct116 or CAMA-1 cell line was not associated with induction of apoptosis.
As we have previously reported, endogenous Mcl-1 localizes mainly to the outer mitochondrial membrane (10). Following subcellular fractionation to cytosolic S-100 and purified mitochondrial fractions, we assessed the localization of the stably transfected full-length and C-terminal fragments  (Fig. 5, left top panel). In contrast to fulllength Mcl-1, the ectopically expressed C-terminal fragments of Mcl-1 accumulated mainly in the S-100 cytosolic fraction (Fig. 5, right top panel). Of note, cell equivalent loading for the various cell fractions (extract, S-100, and mitochondria) is confirmed by the expression levels of ␤-actin (cytosolic marker) and Cox IV (mitochondrial marker) as demonstrated by reprobing of the same membranes (Fig. 5, bottom panels). This inefficient mitochondrial subcellular localization of the Mcl-1 GrB cleavage product may contribute to its ineffective protection against GrB-mediated apoptosis.
Binding between Bim and Mcl-1 Cleavage Products-The high affinity binding between Mcl-1 and Bim may be at the crux of its anti-apoptotic effect, which is most likely accomplished through the sequestration of this potent pro-apoptotic protein, Bim (5,10). We therefore assessed the ability of the Mcl-1 cleavage products to bind Bim. The ability of 35 Slabeled in vitro translated Mcl-1 or in vitro translated 35 Slabeled recombinant Mcl-1 proteins corresponding to the caspase-3 or GrB generated C-terminal cleavage products to bind cold in vitro translated BimEL was assessed by coimmunoprecipitation utilizing Bim-specific Ab (Fig. 6). Both Mcl-1 cleavage products, generated by either GrB or caspase-3, were co-immunoprecipitated with BimEL. Yet, as judged by the efficiency of the co-immunoprecipitation, the affinity of full-length Mcl-1 for Bim was higher than that of the cleavage products for Bim; full-length Mcl-1 was completely depleted from the supernatant by Bim immunoprecipitation, whereas only partial co-precipitation with BimEL was observed for the Mcl-1 cleavage products.

Mcl-1 Cleavage Products Maintain Partial Capability to Inhibit Bim from Mediating the Release of Mitochondrial
Apoptogenic Proteins-To investigate the ability of the Mcl-1 C-terminal cleavage products to inhibit Bim apoptotic function, we applied His-BimL protein to purified mitochondria in the absence or presence of GST-Mcl-1 (Fig. 7A), GST-Mcl-1⌬N127 (Fig. 7B), or GST-Mcl-1⌬N117 (Fig. 7C) and assessed the release of cytochrome c and AIF. Recombinant proteins corresponding to Mcl-1 C-terminal cleavage products generated by caspase-3 or GrB could prevent His-BimL from mediating the release of cytochrome c and AIF. A slightly reduced efficiency in the inhibitory effects of the fragments as compared with full-length Mcl-1 was observed in regard to the release of cytochrome c, but not AIF. The difference between full-length GST-Mcl-1 (Fig. 7A) and the GST-Mcl-1 fragments (Fig. 7, B and C)  achieve a similar effect. These findings suggest that the Mcl-1 C-terminal fragments generated by either GrB or caspase-3 can at least partially inhibit Bim-mediated cytochrome c release from purified mitochondria.
GrB Susceptibility of Breast Carcinoma Cells Is Enhanced by siRNA Silencing of Mcl-1 and Inhibited by Bim Silencing-Our results (Fig. 3) suggest that cleavage of Mcl-1 by either GrB or caspase activity leads to an eventual elimination of Mcl-1. To investigate the effects of Mcl-1 elimination on breast carcinoma cell viability and their susceptibility to GrB-mediated apoptosis, we subjected CAMA-1 cells to RNAi for Mcl-1, Bim, or Lamin A/C. Successful knockdown of these genes was con-firmed by immunoblotting at 40 -48 h post-transfection with the specific siRNA (Fig. 8A). At 40 h post-transfection, the cells were treated with GrB/Ad and assessed 6 h later for viability by flow cytometry of annexin V and propidium iodide staining (Fig. 8B). Silencing of Mcl-1 alone, but not of Bim or Lamin A/C, reduced the viability of CAMA-1 cells even in the absence of external apoptotic stimulation. This increase in background apoptosis may relate to the ability of Mcl-1 to sequester and/or neutralize Bim and probably other mediators of apoptosis. Furthermore, elimination of Mcl-1 enhanced the susceptibility of CAMA-1 cells to GrB/Ad-mediated apoptosis. Such an increased susceptibility in the absence of Mcl-1 has been reported for other apoptotic inducers, including UV and TRAIL, and therefore is not GrB-specific (14,32). However, apoptotic activity of GrB in CAMA-1 cells was predominantly mediated by the Mcl-1 high affinity binding partner, Bim, because it was significantly blocked in cells transfected with Bim-specific siRNA (Fig. 8B). Control silencing of Lamin A/C did not have any effect on the response of these cells to GrB/Ad. These results underscore the important contribution of the Mcl-1⅐Bim cascade in CAMA-1 cell susceptibility to GrB-mediated apoptosis and further emphasize the importance of Mcl-1 elimination in the execution of this apoptotic response. DISCUSSION Mitochondrial disruption has been established as a key event during GrB-mediated apoptosis, but the exact mechanism for GrB function has remained unclear (33,34). Recent studies have identified Bid as a direct substrate for GrB and therefore as a direct link to the mitochondrial apoptotic cascade mediated by Bax or Bak (35)(36)(37). Although in a cell-free system GrB-cleaved Bid is a potent inducer for the release of mitochondrial apoptotic proteins (38), a recent study questions whether a direct cleavage of Bid by GrB occurs under physiologic conditions (34,39). Furthermore, in response to GrB treatment, embryonic fibroblasts from Bid Ϫ/Ϫ mice display disrupted mitochondrial transmembrane potentials (40). These studies implied that cytosolic mediators other than Bid may act as a link between GrB and the mitochondria. Our recent studies (10) have identified Mcl-1 as a direct substrate for GrB and as a high affinity binding partner for Bim. We proposed that the Mcl-1⅐Bim cooperation may constitute an alternative mitochondrial apoptotic pathway that is activated directly by GrB, independent of Bid. In the current study, we investigated the functional mechanism and biological significance of this novel GrB-mediated mitochondrial apoptotic cascade.
Caspase cleavage of the pro-survival Bcl-2 members, Bcl-2 and Bcl-XL, converts them into death effector proteins that further amplify the apoptotic cascade (21,22). In contrast to Mcl-1, Bcl-2 and Bcl-XL are not susceptible to GrB activity. The current study has mapped the GrB cleavage sites of Mcl-1 to aspartic acid residues 117, 127, and 157 and confirmed the recently reported caspase-3 cleavage sites of Mcl-1 at aspartic acid residues 127 and 157 (19,20). Interestingly, cleavage of Mcl-1 by either caspase-3 or GrB activities resembles the caspase-3 cleavage of Bcl-2 and Bcl-XL in its removal of an N-terminal fragment while producing a C-terminal protein that contains the BH1-3 domains. Therefore, we investigated the apoptotic nature of the Mcl-1 C-terminal fragments generated by either caspase-3 or GrB activity. Whereas stable transfection of full-length Mcl-1 endowed significant protection from apoptosis on GrB-susceptible tumor cells, only mild (CAMA-1) to moderate (Hct116) protection was observed in these tumor cells when they were stably transfected with plasmids encoding the C-terminal protein fragments generated by GrB or caspase-3. Furthermore, ectopic expression of the cleavage products did not induce apoptosis in colon or breast carcinoma cell lines and did not enhance apoptosis mediated in these cells by cytotoxic drugs. 2 Thus, our findings suggest that the caspase-3 and GrB Mcl-1 C-terminal cleavage products are functionally different from the caspase-cleaved Bcl-2 or Bcl-XL fragments.
The protective function of Mcl-1 is mediated at the mitochondrial outer membrane, the subcellular localization site for both Mcl-1 and Bim (10,13). Thus, the inefficient protection by the Mcl-1 C-terminal cleavage products can be partly explained by their altered ability to target the mitochondria. Recent studies have suggested that Mcl-1 functions at an apical point in the mitochondrial apoptotic pathway, and its elimination is required for mitochondrial apoptotic events to take place (14). Thus, in UV-mediated apoptosis in HeLa cells, elimination of Mcl-1 occurs prior to Bax translocation, Bax and Bak oligomerization, and cytochrome c release (14). This cascade of events fits with our proposal that Mcl-1 has a role in the regulation of mitochondrial events by sequestering Bim, thereby preventing the activation of the aforementioned mitochondrial apoptotic events. Because cleavage of Mcl-1 by GrB does not completely abolish the sequestration of Bim, we reasoned that a proteolytic mechanism(s) other than via GrB and caspases may be involved in the further degradation of the Mcl-1 cleavage products. Indeed, we obtained evidence that the Mcl-1 cleavage products are further degraded by proteolytic activity present in the cell extract. Such activity may be mediated by the proteasome, because several studies have documented increased stability of Mcl-1 in the presence of proteasome inhibitors (14,41,42). To investigate the significance of ptotic cascades, knockdown of Mcl-1 is very likely to increase the sensitivity of the cells to an array of apoptotic agents, including GrB. Indeed, it has recently been reported that Mcl-1 knockdown was associated with an increased sensitivity to UV or TRAIL (14,32). In the context of the current study, Mcl-1 knockdown may be viewed as a model for cells that undergo Mcl-1 elimination. Thus, in addition to increased susceptibility to apoptotic agents, knockdown of Mcl-1 may also free Bim and other apoptotic Bcl-2 proteins from being sequestered by Mcl-1. Such a scenario may explain the significant level of apoptosis (24%) detected following Mcl-1 siRNA transfection in the absence of an external apoptotic signal. Recently, an example of apoptosis induction by Bcl-2 knockdown was reported (43). Silencing of Bcl-2 induced p53-mediated apoptosis that occurred without stimulation by genotoxic drugs, and that was not seen in the controls or in isogenic clonal cells deficient in p53. The increased level of background apoptosis we observed in cells treated with Mcl-1 siRNA suggests a role for Mcl-1 in the regulation of cell survival under homeostatic conditions. The biological importance of the Mcl-1⅐Bim cascade in GrB function was further underscored by the significant block in the response of CAMA-1 cells to GrB/Ad in the absence of Bim. Because GrB has multiple potential entry points for initiation of a caspase-dependent apoptotic cascade (34,44), it was surprising that the absence of Bim arrested, at least within the time frame of the first 6 h of exposure to GrB/Ad, the apoptotic response seen in controls.
In summary, our studies suggest that the disruption of Mcl-1⅐Bim complex is initiated by GrB cleavage of Mcl-1. The GrBgenerated Mcl-1 C-terminal fragment(s) exerts a variable but partial protective effect against GrB-mediated apoptosis by maintaining limited ability to bind and sequester Bim. The inefficient protective effect of this GrB-cleaved Mcl-1 fragment is most likely due to the combination of its altered subcellular localization to the mitochondria and reduced binding to Bim. Based on these findings and the studies involving Mcl-1 or Bim knockdown, we propose that the disruption of the Mcl-1⅐Bim complex by GrB initiates a major Bim-mediated cellular cytotoxic mechanism that requires the elimination of Mcl-1 following its initial cleavage.