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

Abstract

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. 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. Apoptosis is essential for development and maintenance of tissue homeostasis and for elimination of harmful cells in multicellular organisms (1Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6191) Google Scholar). The Bcl-2 family proteins play an important role in the apoptotic pathway through regulation of the activity of caspases, which cleave vital cellular substrates to execute apoptosis (2Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-307Abstract Full Text PDF PubMed Scopus (2181) Google Scholar). The Bcl-2 family possesses both pro-apoptotic (Bax, Bak, Bcl-XS, Bad, Bik, Bid, Hrk, and Bim) and anti-apoptotic (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1) molecules (3Farrow S.N. Brown R. Curr. Opin. Genet. Dev. 1996; 6: 45-49Crossref PubMed Scopus (243) Google Scholar, 4Kroemer G. Nat. Med. 1997; 3: 614-620Crossref PubMed Scopus (1713) Google Scholar). The ratio of anti-apoptotic to pro-apoptotic molecules, such as Bcl-2/Bax, determines the cell response to an apoptotic signal (5Oltvai Z.N. Milliman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5865) Google Scholar). A striking characteristic of Bcl-2 family members is their propensity to form homodimers with themselves and heterodimers between pro- and anti-apoptotic family members (6Sato T. Hanada M. Bodrug S. Irie S. Iwama N. Boise L.H. Thompson C.B. Golemis E. Fong L. Wang H.-G. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9238-9242Crossref PubMed Scopus (592) Google Scholar, 7Sedlak T.W. Oltvai Z.N. Yang E. Wang K. Boise L.H. Thompson C.B. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7834-7838Crossref PubMed Scopus (784) Google Scholar). The Bcl-2 family members have homology clustered within four conserved Bcl-2 homology (BH) 1The abbreviations BHBcl-2 homologymAbmonoclonal antibodypAbpolyclonal antibodyZ-Aspbenzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzeneZ-VADbenzyloxycarbonyl-Val-Ala-Asp-CH2OC(O)-2,6-dichlorobenzeneZ-EVDbenzyloxycarbonyl-Glu-Val-Asp-CH2OC(O)-2,6-dichlorobenzeneDEVD-AMCacetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarinSTSstaurosporineIL-2interleukin 2WTwild typeGFPgreen fluorescent proteinDAPI4′,6-diamidino-2-phenylindole dihydrochlorideCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidPAGEpolyacrylamide gel electrophoresis domains: BH1, BH2, BH3, and BH4 (4Kroemer G. Nat. Med. 1997; 3: 614-620Crossref PubMed Scopus (1713) Google Scholar), in which only anti-apoptotic proteins, such as Bcl-2 and Bcl-XL, bear the NH2-terminal BH4 domain. Deletion of either BH1 or BH2 nullifies the ability of Bcl-2 to heterodimerize with Bax and suppress cell death (8Yin X.M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1217) Google Scholar). The BH3 domain of pro-apoptotic molecules such as Bax is required for its homodimerization, heterodimerization with Bcl-2 and Bcl-XL, and induction of apoptosis (9Chittenden T. Flemington C. Houghton A.B. Ebb R.G. Gallo G.J. Elangovan B. Chinnadurai G. Lutz R.J. EMBO J. 1995; 14: 5589-5596Crossref PubMed Scopus (438) Google Scholar, 10Zha H. Aime-Sempe C. Sato T. Reed J.C. J. Biol. Chem. 1996; 271: 7440-7444Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). The hydrophobic residues within the BH3 domain of Bax have been found to be critical for dimerization and apoptosis induction (11Wang K. Gross A. Waksman G. Korsmeyer S.J. Mol. Cell. Biol. 1998; 18: 6083-6089Crossref PubMed Scopus (189) Google Scholar). In contrast, deletion of the BH3 domain of Bcl-2 did not alter its anti-apoptotic function (12Reed J.C. Nature. 1997; 387: 773-776Crossref PubMed Scopus (1389) Google Scholar). Structural studies of a Bcl-XL monomer revealed that the BH1–4 domains corresponded to helices 1 to 7. Notably, the BH1, 2, and 3 domains are in close proximity and create a hydrophobic pocket that is presumably involved in interactions with BH3-containing peptides of the death promoters (13Muchmore S.W. Sattler M. Liang H. Meadows R.P. Harlan J.E. Yoon H.S. Nettesheim D. Chang B.S. Thompson C.B. Wong S.L. Ng S.L. Fesik S.W. Nature. 1996; 381: 335-341Crossref PubMed Scopus (1286) Google Scholar, 14Sattler M. Liang H. Nettesheim D. Meadows R.P. Harlan J.E. Eberstadt M. Yoon H.S. Shuker S.B. Chang B.S. Minn A.J. Thompson C.B. Fesik S.W. Science. 1997; 275: 983-986Crossref PubMed Scopus (1291) Google Scholar). The BH4 domain is also indispensable for its ability to suppress cell death. However, the mechanism of the anti-apoptotic function of the BH4 domain is yet uncertain. It may be involved in regulation of apoptosis by interacting with Raf-1 kinase, calcineurin, and CED-4, resulting in phosphorylation of Bad, impairment of NF-AT signaling, and inhibition of caspase activation, respectively (15Wang H.G. Rapp U.R. Reed J.C. Cell. 1996; 87: 629-638Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 16Shibasaki F. Kondo E. Akagi T. McKeon F. Nature. 1997; 386: 728-731Crossref PubMed Scopus (333) Google Scholar, 17Huang D.C. Adams J.M. Cory S. EMBO J. 1998; 17: 1029-1039Crossref PubMed Scopus (200) Google Scholar). Bcl-2 homology monoclonal antibody polyclonal antibody benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene benzyloxycarbonyl-Val-Ala-Asp-CH2OC(O)-2,6-dichlorobenzene benzyloxycarbonyl-Glu-Val-Asp-CH2OC(O)-2,6-dichlorobenzene acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin staurosporine interleukin 2 wild type green fluorescent protein 4′,6-diamidino-2-phenylindole dihydrochloride 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid polyacrylamide gel electrophoresis Previous reports, together with our results, showed that Bcl-2 was cleaved by caspase-3-like proteases at D31AGD34↓A, removing the NH2-terminal 1–34 amino acids, which contains the BH4 domain. In the course of analyzing the ΔN mutant of Bcl-2, which mimicked the cleaved Bcl-2 fragment, we found that the NH2-terminal region of Bcl-2 was also required for heterodimerization with Bax and for its anti-apoptotic activity. Furthermore, site-directed mutagenesis identified that the hydrophobic residues in the BH4 domain of Bcl-2 were critical for dimerization with Bax and inhibition of cell death. Murine IL-2-dependent CTLL-2 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mml-glutamine, 100 μg/ml kanamycin, and 20 units/ml recombinant mouse IL-2. Human embryonic kidney 293T cells and human fibrosarcoma HT1080 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mml-glutamine, 100 μg/ml kanamycin. All cells were maintained at 37 °C in a 5% CO2 and 95% air. The pFLAG-CMV-2 vectors (Eastman Kodak Co.) containing murine wild-type bcl-X L(WT-bcl-X L) and the NH2-terminal truncation of bcl-X L(ΔN-bcl-X L, lacking the NH2-terminal 1–61 amino acids) were previously described (18Fujita N. Nagahashi A. Nagashima K. Rokudai S. Tsuruo T. Oncogene. 1998; 17: 1295-1304Crossref PubMed Scopus (145) Google Scholar). Murine wild-type bcl-2 (WT-bcl-2), the NH2-terminal truncation of bcl-2(ΔN-bcl-2, lacking the NH2-terminal 1–34 amino acids), and bax cDNAs were generated 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 in-frame into anEcoRI site of pc5XP vector, a pcDNA3 vector (Invitrogen) containing an Xpress epitope at the 5′ end, or the pFLAG-CMV-2 vector (Kodak). The pc5XP vector containing wild-type bcl-2cDNA or the pFLAG-CMV-2 vector containing wild-typebcl-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. The wild-type mouse Bcl-2 and mutant proteins were labeled with [35S]methionine usingin vitro transcription/translation system (Promega, Madison, WI). The translated proteins in reticulocyte lysate (2 μl) were incubated with active caspase-3, -6, or -7 in caspase assay buffer for 12 h at 37 °C (18Fujita N. Nagahashi A. Nagashima K. Rokudai S. Tsuruo T. Oncogene. 1998; 17: 1295-1304Crossref PubMed Scopus (145) Google Scholar). The reactions were applied to a 15–25% gradient polyacrylamide gel, followed by autoradiography. Experiments were performed as described previously (18Fujita N. Nagahashi A. Nagashima K. Rokudai S. Tsuruo T. Oncogene. 1998; 17: 1295-1304Crossref PubMed Scopus (145) Google Scholar). Briefly, 293T cells or HT1080 cells were grown to about 80% confluence in 60-mm plates before transfection. Five μg of the pFLAG-CMV-2 vectors containing mock, WT-bcl-2, ΔN-bcl-2, WT-bcl-X L, ΔN-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 μg) was co-transfected into 293T cells with 1 μg of pc5XP-Bax and 1 μg 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 μg) was co-transfected into HT1080 cells with 1 μg of pcDNA3 vector expressing enhanced GFP protein. The proportion of apoptotic cells were determined in GFP-positive cells that showed nuclear condensation and confirmed by 293T cells co-transfected with Bax and Bcl-2 mutants were and lysed in buffer containing 2 and The cell lysates were by at °C for at μg of the cell lysate were used to with 20 in caspase assay buffer 10% and 2 for 1 h at 37 The the substrates was at and the was at using a were with buffer containing The cell lysates were to gel The proteins were a the were incubated with an Bcl-2 San an Bcl-2 an Bax and an (Kodak). The were incubated with antibody followed by with an enhanced were lysed in buffer 1 The cell lysates were incubated with for 2 with buffer 1 proteins were to followed by Western blot We previously that apoptosis in IL-2-dependent CTLL-2 cells (18Fujita N. Nagahashi A. Nagashima K. Rokudai S. Tsuruo T. Oncogene. 1998; 17: 1295-1304Crossref PubMed Scopus (145) Google Scholar). we the of Bcl-2, we found an to the Bcl-2 was in apoptotic CTLL-2 cells cultured in medium containing or not in CTLL-2 cells cultured in medium containing or 1 1 were CTLL-2 cell apoptosis, we was a cleaved of The of the caspase or to CTLL-2 cells cultured in medium the of caspase-3-like proteases not and the of the 1 that the protein was a cleaved Bcl-2 To the in mouse Bcl-2 we Bcl-2 mutant cDNAs in which the acid codon at or was to the codon D34A, or the caspase-3-like proteases cleaved its substrates at which may be amino The wild-type and mutant Bcl-2 proteins were by in in reticulocyte lysates and were incubated with recombinant active human caspase-3, -6, or in the wild-type Bcl-2 was cleaved by caspase-3, -6, and the and not the the by that Bcl-2 protein was cleaved at the site to Bcl-2, Bcl-XL was also cleaved by at the site of and (18Fujita N. Nagahashi A. Nagashima K. Rokudai S. Tsuruo T. Oncogene. 1998; 17: 1295-1304Crossref PubMed Scopus (145) Google Scholar). Bcl-2 and Bcl-XL lost the NH2-terminal region containing the BH4 which is only conserved in anti-apoptotic Bcl-2 family To investigate the function of the NH2-terminal region of Bcl-2, we the of the NH2-terminal Bcl-2 mutant 2 on Bax-induced apoptosis. The pFLAG-CMV-2 vector either mock, WT-bcl-2, or was co-transfected with the pc5XP vector containing bax cDNA and the pcDNA3 vector containing GFP cDNA into 293T transfection for the activity of caspase-3-like proteases and the of apoptosis were using the and respectively. Bcl-2 Bax-induced caspase activation, not inhibit Bax-induced caspase 2 and apoptotic cell death 2 to that the NH2-terminal region of Bcl-2 was essential for exhibiting its anti-apoptotic function. not have the ability to apoptosis as as Bax the of not apoptosis in 293T cells not To investigate the by which lost its apoptosis we its ability to to Bax in The ability of to Bax was also and Western blot analysis were performed using cell the transfected 293T cells expressing wild-type and of Bcl-2 and Bcl-XL to deletion of the NH2-terminal region their heterodimerization with Bax. transfection for 24 the and of the corresponding proteins were confirmed by the transfected cells by Western blot analysis using an 3 The each of Bax protein before as confirmed by Western blot analysis using an Bax 3 proteins were with an and to Western blot analysis using an both wild-type Bcl-2 and Bcl-XL proteins to Bax. In contrast, the mutants of Bcl-2 and Bcl-XL failed to with Bax 3 results that the NH2-terminal of Bcl-2 acids and Bcl-XL acids are for heterodimerization with Bax. The NH2-terminal of Bcl-2 and Bcl-XL the BH4 domain that is only conserved in the anti-apoptotic Bcl-2 family proteins To investigate the conserved of the BH4 domain in the NH2-terminal region that is critical for the ability of Bcl-2 to form a heterodimer with Bax, we generated four site-directed mutants in which the conserved hydrophobic residues and or the Raf-1 within of BH4 domain (13Muchmore S.W. Sattler M. Liang H. Meadows R.P. Harlan J.E. Yoon H.S. Nettesheim D. Chang B.S. Thompson C.B. Wong S.L. Ng S.L. Fesik S.W. Nature. 1996; 381: 335-341Crossref PubMed Scopus (1286) Google were to as in A. Bcl-2 mutants were transfected into 293T and we their ability to to Bax in The transfection of the mutant Bcl-2 proteins did not the of Bax in 293T cells the V15E mutant completely the ability to with Bax did and amino acid such as and also ability to Bax and In contrast, the mutant the with Bax as did and 2, results suggest that the hydrophobic within BH4 domain is critical for Bcl-2 to with Bax. Bax been to form homodimers on the to caspase and apoptosis, we the BH4 mutant which lost the ability to to Bax, inhibit caspase and apoptosis. the of V15E mutant not apoptosis in 293T cells not we its on Bax-induced apoptosis. The pFLAG-CMV-2 vector containing WT-bcl-2, ΔN-bcl-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 for the apoptotic cells were the GFP-positive cells with nuclear confirmed by as in B. of wild-type Bcl-2 Bax-induced apoptosis. In contrast, both the V15E mutant and failed to cells Bax-induced apoptosis Furthermore, we V15E mutant lost the function in STS-induced apoptosis. transfection of the pFLAG-CMV-2 vector containing WT-bcl-2, ΔN-bcl-2, or V15E mutant in HT1080 the transfected cells were with 1 for in V15E mutant and not suppress the STS-induced apoptosis in HT1080 wild-type Bcl-2 cells apoptosis. the a critical role in the and function of BH4 domain of the of Bcl-2 protein in transfected 293T we found that the cDNA the of and V15E mutant 2 and as with that of the of Bax, not the Bax, was also in and V15E mutant 2 To the for the of both Bax and mutant Bcl-2 proteins in the transfected we performed the in the of the caspase the protein of both V15E mutant and and Bax and However, the and such effects not that the of V15E or Bax was by cell death in the and V15E cells of the of and V15E mutant to suppress Bax-induced apoptosis, as in To that the of and V15E mutant with Bax are not to the of proteins in 293T we the of and V15E mutant to form heterodimers with Bax in the of in wild-type Bcl-2 both Bax and Bax 1 and In contrast, the and V15E mutant lost their to form heterodimers with Bax in the of and our results provide an instructive that the V15E mutant not inhibit apoptosis to the to with Bax. The Bcl-2 protein an essential role in cell death. anti-apoptotic activity is through with Bcl-2 and proteins and also by phosphorylation at the (12Reed J.C. Nature. 1997; 387: 773-776Crossref PubMed Scopus (1389) Google Scholar). Site-directed mutagenesis studies of Bcl-2 and Bcl-XL revealed at functional The region is a hydrophobic domain at the that deletion of region not Bcl-2 activity Mol. Cell. Biol. 1996; PubMed Google Scholar). The region and BH2 is critical for both their anti-apoptotic function and the to heterodimerize with pro-apoptotic Bcl-2 family proteins Bax or T.W. Oltvai Z.N. Yang E. Wang K. Boise L.H. Thompson C.B. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7834-7838Crossref PubMed Scopus (784) Google Scholar, X.M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1217) Google Scholar, B. Boise L.H. Thompson C.B. J.M. Nature. 1996; PubMed Scopus Google Scholar). all BH1 or BH2 mutants that heterodimerization with Bax the death function. The region is at the The NH2-terminal deletion mutants not only failed to apoptosis C. C. M. E. J.C. J. J. Biol. 1994; PubMed Scopus Google also as of wild-type Bcl-2 Mol. Cell. Biol. 1996; PubMed Google Scholar, R.J. R. A. K. J.M. Science. 1997; PubMed Scopus Google Scholar), its mechanism is that Bax may caspase A. J. Korsmeyer S.J. EMBO J. 1998; 17: PubMed Scopus Google Scholar). The ability of Bax to to R.J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, C.L. A. R.J. J. Biol. 1997; PubMed Scopus Google and to form homodimers A. J. Korsmeyer S.J. EMBO J. 1998; 17: PubMed Scopus Google may to its ability to form B. F. A. S. S. L. A. G. K. F. R. J.C. Science. 1997; PubMed Scopus Google Scholar, Gross A. X.M. K. M. Waksman G. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google and to caspase activation, and cell death (11Wang K. Gross A. Waksman G. Korsmeyer S.J. Mol. Cell. Biol. 1998; 18: 6083-6089Crossref PubMed Scopus (189) Google Scholar). The of Bax and Bcl-2 that to in and heterodimerization are their amino acid analysis studies that BH3 is a critical domain of Bax for both and heterodimerization (9Chittenden T. Flemington C. Houghton A.B. Ebb R.G. Gallo G.J. Elangovan B. Chinnadurai G. Lutz R.J. EMBO J. 1995; 14: 5589-5596Crossref PubMed Scopus (438) Google Scholar, 10Zha H. Aime-Sempe C. Sato T. Reed J.C. J. Biol. Chem. 1996; 271: 7440-7444Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). is also with the of BH3 peptides to and heterodimerization with Bcl-2 family members T. M. G. S. T. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Bax molecules also showed activity T.W. Oltvai Z.N. Yang E. Wang K. Boise L.H. Thompson C.B. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7834-7838Crossref PubMed Scopus (784) Google Scholar, T. Flemington C. Houghton A.B. Ebb R.G. Gallo G.J. Elangovan B. Chinnadurai G. Lutz R.J. EMBO J. 1995; 14: 5589-5596Crossref PubMed Scopus (438) Google Scholar, J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). cell death in Bcl-2 family only the BH3 domain not BH1, BH2, or BH4 B. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, within the BH3 domain not the anti-apoptotic of with a with and also that Bax function of Bcl-2 Korsmeyer S.J. Nat. Genet. 1997; PubMed Scopus Google Scholar). Bax to apoptosis by its activity by the anti-apoptotic activity of Bcl-2 or Bcl-XL, Bcl-2 and inhibit apoptosis through heterodimers with Bax or of proteins protein and to proteins that (15Wang H.G. Rapp U.R. Reed J.C. Cell. 1996; 87: 629-638Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 16Shibasaki F. Kondo E. Akagi T. McKeon F. Nature. 1997; 386: 728-731Crossref PubMed Scopus (333) Google Scholar, 17Huang D.C. Adams J.M. Cory S. EMBO J. 1998; 17: 1029-1039Crossref PubMed Scopus (200) Google Scholar, B. Boise L.H. Thompson C.B. J.M. Nature. 1996; PubMed Scopus Google Scholar). Previous results and the report showed that the of Bcl-2 in human and mouse by R.J. R. A. K. J.M. Science. 1997; PubMed Scopus Google Scholar, D. E. L. T. C. EMBO J. 1998; 17: PubMed Scopus Google the NH2-terminal region of Bcl-2 acid which contains the BH4 domain that is conserved the anti-apoptotic Bcl-2 family members. Our revealed that the NH2-terminal deletion mutant which the cleaved Bcl-2 fragment, not suppress Bax- and STS-induced apoptosis in transfected 293T cells and HT1080 respectively 2 and the NH2-terminal region is critical for anti-apoptotic Bcl-2 family proteins to cells apoptosis, the by which loss of NH2-terminal region the activity of Bcl-2 not been Our showed that deletion of the NH2-terminal region of Bcl-2 and their heterodimerization with Bax 3 in cells in and lost their that the heterodimerization of Bcl-2 with Bax on not only the also BH4 is with the showed a for NH2-terminal region of Bcl-2 for to Bax (6Sato T. Hanada M. Bodrug S. Irie S. Iwama N. Boise L.H. Thompson C.B. Golemis E. Fong L. Wang H.-G. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9238-9242Crossref PubMed Scopus (592) Google and A. A.J. 1997; PubMed Google in a The analysis of the Bcl-XL monomer a of the BH4 domain within the NH2-terminal region of Bcl-2, which revealed an on the that hydrophobic interactions with and (13Muchmore S.W. Sattler M. Liang H. Meadows R.P. Harlan J.E. Yoon H.S. Nettesheim D. Chang B.S. Thompson C.B. Wong S.L. Ng S.L. Fesik S.W. Nature. 1996; 381: 335-341Crossref PubMed Scopus (1286) Google Scholar). mutagenesis analysis of the BH4 domain in Bcl-2, we revealed critical amino acids for the of BH4 domain and its function. The V15E which a hydrophobic with a instructive lost the ability to heterodimerize with Bax as well as its anti-apoptotic activity with the that the ability of mutant of Bcl-2 to inhibit staurosporine-induced apoptosis in was A. C. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). and mutants also to heterodimerize with Bax, V15E In contrast, of the phosphorylation site a of heterodimerization with Bax may the that the Bax ability of the is to a close to the phosphorylation results suggest that the the of the in BH4 domain is critical for Bcl-2 to form a heterodimer with Bax and to cells hydrophobic of the on the of the Bcl-2 protein and the protein its that the hydrophobic which the BH3 domain of Bax, is a on hydrophobic to be to the anti-apoptotic function of may that hydrophobic interactions at the of the pocket of Bcl-2 are important results that the BH4 the BH1 and BH2 of Bcl-2 is also essential for heterodimerization with Bax. Our that the of Bcl-2 with Bax is critical for exhibiting its anti-apoptotic we not that proteins may be also involved in BH4 domain function. Our provide an to new agents to cell apoptosis by the function of the BH4 domain. We and for