NH2-terminal BH4 Domain of Bcl-2 Is Functional for Heterodimerization with Bax and Inhibition of Apoptosis*

The Bcl-2 family proteins comprise pro-apoptotic as well as anti-apoptotic members. Heterodimerization between members of the Bcl-2 family proteins is a key event in the regulation of apoptosis. We report here that Bcl-2 protein was selectively cleaved by active caspase-3-like proteases in CTLL-2 cell apoptosis in response to interleukin-2 deprivation. Structural and functional analyses of the cleaved fragment revealed that the NH2-terminal region of Bcl-2 (1–34 amid acids) was required for its anti-apoptotic activity and heterodimerization with pro-apoptotic Bax protein. Site-directed mutagenesis of the NH2-terminal region showed that substitutions of hydrophobic residues of BH4 domain resulted in the loss of ability to form a heterodimer with Bax. Particularly instructive was that the V15E mutant of Bcl-2, which completely lost the ability to form a heterodimer with Bax, failed to inhibit Bax- and staurosporine-induced apoptosis. Our results suggest that the BH4 domain of Bcl-2 is critical for its heterodimerization with Bax and for exhibiting anti-apoptotic activity. Therefore, agents interferring with the critical residues of the BH4 domain may provide a new strategy in cancer therapy by impairing Bcl-2 function.

ated by reverse transcription-polymerase chain reaction with CTLL-2 mRNA as the template. The polymerase chain reaction products were cloned into a pCRII vector (Invitrogen, San Diego) and subcloned inframe into an EcoRI site of pc5XP vector, a pcDNA3 vector (Invitrogen) containing an Xpress epitope at the 5Ј end, or the pFLAG-CMV-2 vector (Kodak). The pc5XP vector containing wild-type bcl-2 cDNA or the pFLAG-CMV-2 vector containing wild-type bcl-2 cDNA was employed as the template for mutagenesis using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) Substitutions of amino acids (V15E, Y21D, S24E, W30S, D31A, D34A, and D36A) in bcl-2 cDNA were accomplished by converting the corresponding amino acid codon GTG, TAT, TCA, TGG, GAT, GAT, or GAC to GAG, GAT, GAA, TCG, GCT, GCT, or GCC, respectively. The proper construction of all plasmids was confirmed by DNA sequencing.
In Vitro Cleavage Assay-The wild-type mouse Bcl-2 and mutant proteins were labeled with [ 35 S]methionine using in vitro transcription/ translation system (Promega, Madison, WI). The translated proteins in reticulocyte lysate (2 l) were incubated with active caspase-3, -6, or -7 in caspase assay buffer for 12 h at 37°C (18). The reactions were applied to a 15-25% gradient polyacrylamide gel, followed by autoradiography.
Transient Transfection -Experiments were performed as described previously (18). Briefly, 293T cells or HT1080 cells were grown to about 80% confluence in 60-mm plates before transfection. Five g of the pFLAG-CMV-2 vectors containing mock, WT-bcl-2, ⌬N-bcl-2, WT-bcl-X L , ⌬N-bcl-X L , or bcl-2 mutant cDNAs were transiently transfected into cells using a Superfect transfection reagent according to the manufacturer's instruction (Qiagen, Hilden, Germany). The transfected cells were lysed 24 h later, and the cell lysates were used for Western blot analysis and co-immunoprecipitation assay. To investigate the effects of Bcl-2 mutants on the inhibition of Bax-induced apoptosis, each Bcl-2 mutant plasmid (1 g) was co-transfected into 293T cells with 1 g of pc5XP-Bax and 1 g of pcDNA3 vector expressing enhanced GFP protein. To investigate the effects of Bcl-2 mutants on the inhibition of STS-induced apoptosis, each Bcl-2 mutant plasmid (1.5 g) was cotransfected into HT1080 cells with 1 g of pcDNA3 vector expressing enhanced GFP protein. The proportion of apoptotic cells were determined in GFP-positive cells that showed nuclear condensation and fragmentation confirmed by DAPI staining.
Measurement of Caspase Activity-293T cells co-transfected with Bax and Bcl-2 mutants were harvested and lysed in lysis buffer containing 10 mM HEPES (pH 7.4), 2 mM EDTA, 0.1% CHAPS, and 5 mM dithiothreitol. The cell lysates were obtained by centrifugation at 4°C for 30 min at 40,000 rpm. Twenty g of the cell lysate were used to incubate with 20 M fluorogenic substrate DEVD-AMC (Peptide Institute, Osaka, Japan) in caspase assay buffer (20 mM HEPES (pH 7.4), 10% glycerol, and 2 mM dithiothreitol) for 1 h at 37°C. The AMC released from the fluorogenic substrates was excited at 380 nm, and the emission was measured at 460 nm using a Hitachi fluorescence spectrophotometer, model F-2000 (Hitachi, Tokyo, Japan).
Western Blot Analysis-Cells were solubilized under reduced conditions with lysis buffer containing 0.5% Nonidet P-40. The cell lysates were subjected to SDS-polyacrylamide gel electrophoresis. The electrophoresed proteins were transblotted onto a nitrocellulose membrane. After blocking, the membranes were incubated with an anti-mouse Bcl-2 mAb (Pharmingen, San Diego, CA), an anti-human Bcl-2 pAb (Calbiochem), an anti-human Bax pAb (Upstate Biotechnology, Lake Placid, NY), and an anti-FLAG M5 mAb (Kodak). The membranes were then incubated with peroxidase-conjugated second antibody followed by developing with an enhanced chemiluminescence (ECL) mixture (Amersham Pharmacia Biotech).

RESULTS
Bcl-2 Cleavage during CTLL-2 Cell Apoptosis-We previously reported that IL-2 deprivation could induce apoptosis in IL-2-dependent CTLL-2 cells (18). When we examined the expression of Bcl-2, we found an additional band (23 kDa) crossreactive to the specific anti-mouse Bcl-2 mAb was observed in apoptotic CTLL-2 cells cultured in medium containing 0 or 0.01 ng/ml IL-2 but not in viable CTLL-2 cells cultured in medium containing 0.1 or 1 ng/ml IL-2 ( Fig. 1A). Because caspases were activated during CTLL-2 cell apoptosis, we examined whether this novel 23-kDa band was a cleaved product of Bcl-2. The addition of the cell-permeable caspase inhibitor Z-VAD, Z-EVD, or Z-Asp to CTLL-2 cells cultured in IL-2-depleted medium suppressed the activation of caspase-3-like proteases (data not shown) and blocked the appearance of the 23-kDa band (Fig.  1B), indicating that the novel 23-kDa protein was indeed a cleaved Bcl-2 fragment.
To identify the still unclear cleavage sites in mouse Bcl-2 protein, we constructed several Bcl-2 mutant cDNAs in which the aspartic acid codon at 31, 34, or 36 was converted to the alanine codon (D31A, D34A, or D36A, respectively) because the caspase-3-like proteases specifically cleaved its substrates at DXXD2 motif (in which X may be any amino acid). The wildtype and mutant Bcl-2 proteins were produced by in vitro transcription/translation in reticulocyte lysates and were incubated with recombinant active human caspase-3, -6, or -7. As shown in Fig. 1C, the wild-type Bcl-2 was cleaved by caspase-3, -6, and -7, whereas the D31A and D34A mutants, but not the D36A mutant, could block the cleavage by caspase-3. This suggested that Bcl-2 protein was cleaved at the site D 31 AGD 34 2A. Similar to Bcl-2, Bcl-X L was also cleaved by caspase-3 at the site of HLAD 61 2S and SSLD 76 2A (18). Both Bcl-2 and Bcl-X L lost the NH 2 -terminal region containing the BH4 domain, which is only conserved in anti-apoptotic Bcl-2 family proteins.  8), or D36A mutant protein (lanes 9 and 10) were incubated with 10 g/ml active human caspase-3, -6, or -7, as indicated, for 12 h at 37°C. Each reaction was electrophoresed in a 15-25% gradient polyacrylamide gel, followed by autoradiography.

Requirement of the NH 2 -terminal Region of Bcl-2 for Inhibition of Bax-induced Caspase
Activation-To investigate the biological function of the NH 2 -terminal region of Bcl-2, we tested the effect of the NH 2 -terminal deleted Bcl-2 mutant (⌬N-Bcl-2) ( Fig. 2A) on Bax-induced apoptosis. The pFLAG-CMV-2 vector encoding either mock, WT-bcl-2, or ⌬N-bcl-2 was co-transfected with the pc5XP vector containing bax cDNA and the pcDNA3 vector containing GFP cDNA into 293T cells. After transfection for 8 h, the activity of caspase-3-like proteases and the extent of apoptosis were assessed using the fluorogeniclabeled tetrapeptide DEVD-AMC and DAPI staining, respectively. Wild-type Bcl-2 could counteract Bax-induced caspase activation, whereas ⌬N-Bcl-2 could not inhibit Bax-induced caspase activation (Fig. 2B) and apoptotic cell death (Fig. 2C), similar to mock transfection, indicating that the NH 2 -terminal region of Bcl-2 was essential for exhibiting its anti-apoptotic function. ⌬N-Bcl-2 might not have the ability to promote apoptosis as effectively as Bax because the overexpression of ⌬N-

Bcl-2 alone could not induce apoptosis in 293T cells (data not shown).
Requirement of the NH 2 -terminal Region of Bcl-2 for Heterodimerization with Bax-To further investigate the molecular mechanisms by which ⌬N-Bcl-2 lost its apoptosis inhibitory function, we examined its ability to bind to Bax in vivo. The binding ability of ⌬N-Bcl-X L to Bax was also investigated. Sequential immunoprecipitation and Western blot analysis were performed using cell extracts from the transfected 293T cells expressing FLAG-tagged wild-type and ⌬N-mutant constructs of Bcl-2 and Bcl-X L to determine whether deletion of the NH 2 -terminal region disrupted their heterodimerization with endogenous Bax. After transfection for 24 h, the size and stability of the corresponding proteins were confirmed by examining extracts from the transfected cells by Western blot analysis using an anti-FLAG M5 mAb (Fig. 3A, upper panel). The extracts from each transfectant had comparable amounts of Bax protein before immunoprecipitation experiments, as confirmed by Western blot analysis using an anti-human Bax pAb (Fig.  3A, lower panel). When these FLAG-tagged proteins were immunoprecipitated with an anti-FLAG M2 mAb and then subjected to Western blot analysis using an anti-Bax pAb, both wild-type Bcl-2 and Bcl-X L proteins bound to Bax. In contrast, the NH 2 -terminal-deleted mutants of Bcl-2 and Bcl-X L failed to interact with Bax (Fig. 3B). These results indicate that the NH 2 -terminal regions of Bcl-2 (amino acids 1-34) and Bcl-X L (amino acids 1-61) are necessary for heterodimerization with Bax.
Identification of Residues Critical for Heterodimerization with Bax-The NH 2 -terminal regions of Bcl-2 and Bcl-X L contain the BH4 domain that is only conserved in the anti-apoptotic Bcl-2 family proteins (Fig. 4A). To investigate the specific, conserved sequence of the BH4 domain in the NH 2terminal region that is critical for the ability of Bcl-2 to form a heterodimer with Bax, we generated four site-directed point mutants in which the most conserved hydrophobic residues (Val 15 , Tyr 21 , and Trp 30 ) or the Raf-1 kinase target residue within ␣1 amphipathic helix of BH4 domain (Ser 24 ) (13) were converted to charged residues, as shown in Fig. 4A. These Bcl-2 mutants were transfected into 293T cells, and we analyzed their ability to bind to Bax in vivo. The transfection of the mutant Bcl-2 proteins did not affect the expression level of endogenous Bax in 293T cells (Fig. 4B, lower panel). Interestingly, the V15E mutant completely abrogated the ability to interact with Bax like ⌬N-Bcl-2 did (Fig. 4C, lanes 4 and 3,  respectively). Other amino acid replacements, such as Y21D and W30S mutants, also displayed reduced binding ability to Bax (Fig. 4C, lanes 5 and 7, respectively). In contrast, the S24E mutant displayed the same binding capability with Bax as WT-Bcl-2 did (Fig. 4C, lanes 6 and 2, respectively). These results suggest that the hydrophobic surface within BH4 domain is critical for Bcl-2 to interact with Bax.

BH4 Mutants That Disrupt the Heterodimerization with Bax Failed to Suppress Bax-and STS-induced Apoptosis-Because
Bax has been reported to form homodimers on the mitochondria membrane to initiate caspase activation and apoptosis, we further investigated whether the BH4 mutant (V15E), which lost the ability to bind to Bax, could inhibit caspase activation and apoptosis. Because the overexpression of V15E mutant itself could not induce apoptosis in 293T cells (data not shown), we examined its apoptosis-regulatory effect on Bax-induced apoptosis. The pFLAG-CMV-2 vector containing WT-bcl-2, ⌬Nbcl-2, or V15E mutant was co-transfected with the pc5XP vector containing wild-type bax cDNA and the pcDNA3 vector containing GFP cDNA into 293T cells. After incubation for 8 h, the apoptotic cells were counted among the GFP-positive cells with nuclear fragmentation confirmed by DAPI staining, as shown in Fig. 5B. Expression of wild-type Bcl-2 inhibited Baxinduced apoptosis. In contrast, both the V15E mutant and ⌬N-Bcl-2 failed to protect cells from undergoing Bax-induced apoptosis (Fig. 5A). Furthermore, we examined whether V15E mutant lost the apoptosis-inhibitory function in STS-induced apoptosis. After transfection of the pFLAG-CMV-2 vector containing WT-bcl-2, ⌬N-bcl-2, or V15E mutant in HT1080 cells, the transfected cells were treated with 1 M STS for 8 h. As shown in Fig. 5C, V15E mutant and ⌬N-Bcl-2 could not suppress the STS-induced apoptosis in HT1080 cells, whereas wild-type Bcl-2 protected cells from undergoing apoptosis. Thus, the Val 15 residue exhibited a critical role in modulating the structure and function of BH4 domain of Bcl-2.
Interestingly, when examining the amount of Bcl-2 protein in transfected 293T cells, we found that the co-transfection of bax cDNA reduced the expression level of exogenous ⌬N-Bcl-2 and V15E mutant (Fig. 6, lower panel, lanes 2 and 3), as compared with that of WT-Bcl-2 (Fig. 6, lower panel, lane 1). Similarly, the amount of exogenous Bax, but not the endogenous Bax, was also decreased in ⌬N-Bcl-2 and V15E mutant transfectants (Fig. 6, middle panel, lanes 2 and 3). To elucidate the reason for the decreased expression of both Bax and mutant Bcl-2 proteins in the transfected cells, we performed the above co-transfection experiments in the presence of the caspase inhibitor Z-Asp. Z-Asp could recover the exogenous protein level of both ⌬N-Bcl-2, V15E mutant (Fig. 6, lower panel, lanes 5 and  6) and Bax (Fig. 6, middle panel, lanes 5 and 6). However, the cysteine protease inhibitor Z-LLH and calpain inhibitor E-64 had no such effects (data not shown). These data suggested that the reduced expression of exogenous ⌬N-Bcl-2, V15E mutant, or Bax was caused by rapid cell death in the ⌬N-Bcl-2and V15E mutant-transfected cells because of the inability of ⌬N-Bcl-2 and V15E mutant to suppress Bax-induced apoptosis, as shown in Fig. 5. To rule out that the reduced binding abilities of ⌬N-Bcl-2 and V15E mutant with Bax are not due to the decreased expression of these proteins in 293T cells, we tested the abilities of ⌬N-Bcl-2 and V15E mutant to form heterodimers with Bax in the presence of Z-Asp. As shown in Fig.  6, wild-type Bcl-2 could bind both exogenous Xpress-tagged Bax and endogenous Bax (upper panel, lanes 1 and 4). In contrast, the ⌬N-Bcl-2 and V15E mutant lost their abilities to form heterodimers with Bax even in the presence of Z-Asp (Fig.  6, upper panel, lanes 5 and 6). Taken together, our results provide an instructive example that the V15E mutant could not inhibit apoptosis due to the reduced capacity to dimerize with Bax.

DISCUSSION
The Bcl-2 protein plays an essential role in preventing cell death. Its anti-apoptotic activity is regulated through association with Bcl-2 homologous and nonhomologous proteins and also by phosphorylation at the serine residue (12). Site-directed mutagenesis studies of Bcl-2 and Bcl-X L revealed at least three discrete functional regions. The first region (transmembrane domain) is a hydrophobic domain at the COOH terminus that confers membrane anchorage; deletion of this region reduces but does not eliminate Bcl-2 activity (19). The second region (BH1 and BH2 domains) is critical for both their anti-apoptotic function and the capacity to heterodimerize with pro-apoptotic Bcl-2 family proteins like Bax or Bak (7,8,20). Almost all BH1 or BH2 mutants that disrupt heterodimerization with Bax lose the death repressor function. The third region is located at the NH 2 terminus. The NH 2 -terminal deletion mutants not only failed to prevent apoptosis (21) but also functioned as transdominant inhibitors of wild-type Bcl-2 (19,22), although its mechanism is unknown.
Growing evidence has demonstrated that Bax may dominantly regulate caspase activation (23). The ability of Bax to localize to mitochondrial membranes (24,25) and to form homodimers (23) may relate to its ability to form distinct ionconductive channels (26,27) and consequently to induce mitochondrial dysfunction, caspase activation, and cell death (11). The structural features of Bax and Bcl-2 that allow them to participate in homo-and heterodimerization phenomena are markedly different, despite their amino acid sequence similarity. Deletional analysis studies indicate that BH3 is a critical domain of Bax for both homo-and heterodimerization (9,10). This is also consistent with the capacity of Bax-derived BH3 peptides to block homo-and heterodimerization with Bcl-2 family members (28). BH3-deleted Bax molecules also showed impaired killing activity (7,9,29). Some cell death activators in Bcl-2 family contain only the BH3 domain but not BH1, BH2, or BH4 (30). In contrast, mutations within the BH3 domain do not significantly affect the anti-apoptotic functions of Bcl-2. Consistent with this, a genetic approach with Bcl-2-deficient and Bax-deficient mice also suggested that Bax could function independently of Bcl-2 (31). Thus, Bax seems to dominantly induce apoptosis by its own activity rather than by blocking the anti-apoptotic activity of Bcl-2 or Bcl-X L , whereas Bcl-2 and Bcl-X L inversely inhibit apoptosis through forming heterodimers with Bax or other types of proteins ranging from protein kinases and phosphatases to proteins that bind caspases (15)(16)(17)20).
Previous results and the present report showed that the cleavage of Bcl-2 (both in human and mouse Bcl-2) by caspases ( Fig. 1) (22,32) removed the NH 2 -terminal region of Bcl-2 (amino acid , which contains the BH4 domain that is conserved among the anti-apoptotic Bcl-2 family members. Our data revealed that the NH 2 -terminal deletion mutant (⌬N-Bcl-2), which mimics the cleaved Bcl-2 fragment, could not suppress Bax-and STS-induced apoptosis in transfected 293T cells and HT1080 cells, respectively (Figs. 2 and 5). Although the NH 2 -terminal region is critical for anti-apoptotic Bcl-2 family proteins to prevent cells from undergoing apoptosis, the molecular mechanisms by which loss of NH 2 -terminal region affect the activity of Bcl-2 has not been fully understood. Our data reported herein showed that deletion of the NH 2 -terminal region of Bcl-2 and Bcl-X L abrogated their heterodimerization with Bax (Fig. 3B) in mammalian cells in vivo and thus lost their apoptosis-inhibitory activity, suggesting that the heterodimerization of Bcl-2 with Bax depends on not only the BH1-2 but also BH4 domains. This is consistent with the previous reports showed a requirement for NH 2 -terminal region of Bcl-2 for binding to Bax (6) and Bad (33) in a yeast two-hybrid system.
The structural analysis of the Bcl-X L monomer promoted a molecular model of the BH4 domain within the NH 2 -terminal region of Bcl-2, which revealed an amphipathic helix (␣1 helix) on the surface that forms extensive hydrophobic interactions with ␣2, ␣5, and ␣6 (13). Through mutagenesis analysis of the BH4 domain in Bcl-2, we revealed several critical amino acids responsible for modulating the conformation of BH4 domain and affecting its function. The V15E mutant, which replaced a hydrophobic residue with a charged residue, proved most instructive because it lost the ability to heterodimerize with Bax ( Fig. 4) as well as its anti-apoptotic activity (Fig. 5), consistent with the fact that the ability of V15G mutant of Bcl-2 to inhibit staurosporine-induced apoptosis in fibroblasts was totally abrogated (34). W30S and Y21D mutants also displayed altered capacities to heterodimerize with Bax, although less dramatically than V15E mutant. In contrast, substitution of the serine phosphorylation site S24E displayed a normal pattern of heterodimerization with Bax (Fig. 4). This may rule out the possibility that the altered Bax binding ability of the above point mutations is due to a close location to the phosphorylation site. Taken together, these results suggest that the maintaining the amphipathic nature of the ␣1 helix in BH4 domain is critical for Bcl-2 to form a heterodimer with Bax and to protect cells from killing. Without hydrophobic surface of BH4, the ␣1 helix cannot dock on the backside of the Bcl-2 protein and probably the protein alters its conformation so that the hydrophobic pocket, which normally binds the BH3 domain of Bax, is altered. Thus, introducing a charged residue on this hydrophobic face appears to be particularly deleterious to the anti-apoptotic function of Bcl-2. This may indicate that hydrophobic interactions at the base of the binding pocket of Bcl-2 are more important than electrostatic interactions.
These results indicate that the BH4 domain, besides the BH1 and BH2 domains, of Bcl-2 is also essential for heterodimerization with Bax. Our data supported that the interaction of Bcl-2 with Bax is critical for exhibiting its anti-apoptotic activity, whereas we could not rule out that other Bcl-2-interacted proteins may be also involved in BH4 domain function. Our findings provide an opportunity to develop new agents to promote tumor cell apoptosis by interrupting the function of the BH4 domain.