MAP-1, a Novel Proapoptotic Protein Containing a BH3-like Motif That Associates with Bax through Its Bcl-2 Homology Domains

A novel Bax-associating protein, named MAP-1 (Modulator of Apoptosis), has been identified in a yeast two-hybrid screen. MAP-1 contains a BH3-like (BH:Bcl-2 homology) motif and mediates caspase-dependent apoptosis in mammalian cells when overexpressed. MAP-1 homodimerizes and associates with the proapoptotic Bax and the prosurvival Bcl-2 and Bcl-XL of the Bcl-2 family in vitro and in vivo in mammalian cells. Mutagenesis analyses revealed that the BH3-like domain in MAP-1 is not required for its association with Bcl-XL but is required for association with Bax and for mediating apoptosis. Interestingly, in contrast to other Bax-associating proteins such as Bcl-XLand Bid, which require the BH3 and BH1 domains of Bax, respectively, for binding, the binding of MAP-1 to Bax appears to require all three BH domains (BH1, BH2, and BH3) of Bax, because point mutation of the critical amino acid in any one of these domains is sufficient to abolish its binding to MAP-1. These data suggest that MAP-1 mediates apoptosis through a mechanism that involves binding to Bax. A novel Bax-associating protein, named MAP-1 (Modulator of Apoptosis), has been identified in a yeast two-hybrid screen. MAP-1 contains a BH3-like (BH:Bcl-2 homology) motif and mediates caspase-dependent apoptosis in mammalian cells when overexpressed. MAP-1 homodimerizes and associates with the proapoptotic Bax and the prosurvival Bcl-2 and Bcl-XL of the Bcl-2 family in vitro and in vivo in mammalian cells. Mutagenesis analyses revealed that the BH3-like domain in MAP-1 is not required for its association with Bcl-XL but is required for association with Bax and for mediating apoptosis. Interestingly, in contrast to other Bax-associating proteins such as Bcl-XLand Bid, which require the BH3 and BH1 domains of Bax, respectively, for binding, the binding of MAP-1 to Bax appears to require all three BH domains (BH1, BH2, and BH3) of Bax, because point mutation of the critical amino acid in any one of these domains is sufficient to abolish its binding to MAP-1. These data suggest that MAP-1 mediates apoptosis through a mechanism that involves binding to Bax. Bcl-2 homology glutathione S-transferase polyacrylamide gel electrophoresis polymerase chain reaction 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid poly(ADP-ribose) polymerase green fluorescence protein hemagglutinin cytomegalovirus phenylmethylsulfonyl fluoride kilobase(s) Members of the Bcl-2 family of proteins are regulators of cell death that can be grouped into subfamilies of prosurvival and proapoptotic molecules (1Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4751) Google Scholar, 2Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3220) Google Scholar). They are characterized by the presence of several conserved motifs, known as the Bcl-2 homology (BH)1 domains, designated BH1, BH2, BH3, and BH4 (1Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4751) Google Scholar). Although the N-terminal BH4 domain is restricted to some prosurvival members, BH1, BH2, and BH3 can generally be found among members of both the prosurvival and proapoptotic subfamilies (1Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4751) Google Scholar, 2Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3220) Google Scholar). Mutagenesis and structural studies revealed that the BH1, BH2, and BH3 domains are important for dimerization function (1Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4751) Google Scholar, 2Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3220) Google Scholar). Association of the prosurvival member Bcl-XL with the proapoptotic member Bax requires the BH1, BH2, and BH3 domains of the former but only the BH3 domain of the latter (3Zha H. Aime-Sempe C. Sato T. Reed J.C. J. Biol. Chem. 1996; 271: 7440-7444Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 4Chittenden 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 (436) Google Scholar, 5Sattler 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 (1277) Google Scholar). In addition to its role as a protein-protein interaction domain, the BH3 domain of proapoptotic members appears to be important for mediating their proapoptotic function (3Zha H. Aime-Sempe C. Sato T. Reed J.C. J. Biol. Chem. 1996; 271: 7440-7444Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 4Chittenden 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 (436) Google Scholar). This notion is further supported by the recent discovery of a new group of cell death agonists containing only the BH3 domain (6Kelekar A. Thompson C.B. Trends Cell Biol. 1998; 8: 324-330Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar). Similar to other proapoptotic members of the Bcl-2 family, the BH3 domain in these molecules plays an important role in mediating proapoptotic function as well as association with the prosurvival members of the Bcl-2 family. The three-dimensional structure of Bcl-XL demonstrates that the BH1, BH2, and BH3 regions form an elongated hydrophobic cleft to which a BH3 amphipathic α helix can bind (5Sattler 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 (1277) Google Scholar, 7Muchmore 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 (1276) Google Scholar). The other members of the Bcl-2 family that have similar domains are predicted to have a similar tertiary structure. Hence, proapoptotic members of the Bcl-2 family that contain multiple BH domains such as Bax are proposed to exist in two different conformations: one that is similar to Bcl-XL and the other with the BH3 domain rotated outside to allow its insertion into the hydrophobic cleft of a prosurvival protein (1Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4751) Google Scholar). However, there is a lack of evidence to support the existence of a Bcl-XL-like conformation among the proapoptotic molecules that contain the BH1–3 domains. In our effort to gain further understanding of the functions of the BH domains of Bax, we used the yeast two-hybrid screen to identify Bax-associating proteins. A novel protein, termed MAP-1, was identified. MAP-1 contains a putative BH3-like domain and induces caspase-dependent apoptosis in mammalian cells when overexpressed. MAP-1 forms homodimers and associates with Bax, Bcl-2, and Bcl-XL in vitro and in vivo in mammalian cells. The BH3-like domain in MAP-1 appeared to be required for mediating apoptosis and its binding to Bax, but not Bcl-XL. Interestingly, in contrast to other Bax-associating proteins, the binding of MAP-1 to Bax requires all three BH (BH1, BH2, and BH3) domains of Bax. MAP-1 thus represents a novel Bax-associating protein that may mediate apoptosis through binding to Bax. Mono- and polyclonal antibodies against the Myc epitope (9E10, A14) and HA epitope (F7) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against PARP (C2–10) was from Dr. G. Poirier, Laval University Medical Research Center, Québec Canada. MCF-7 (gift from Dr. V. Dixit), 293T, and SH-SY5Y cells were grown and maintained as described (8Chan S.L. Tan K.O. Zhang L. Yee K.S. Ronca F. Chan M.Y. Yu V.C. J. Biol. Chem. 1999; 274: 32461-32468Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 9Chan S.L. Yee K.S. Tan K.M. Yu V.C. J. Biol. Chem. 2000; 275: 17925-17928Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). HeLa cells were obtained from the American Type Culture Collection (ATCC), and the cells were grown and maintained according to the directions provided by the suppliers. The peptide protease inhibitors YVAD-fmk, ZVAD-fmk, and DEVD-fmk were from Enzyme System Products. λ-Zap human and mouse libraries were purchased from Stratagene (La Jolla, CA). Expression plasmids for hMAP-1, hMAP-1 deletion mutants, mBax, hBcl-XL, hBcl-2, mBimL, and mBid were generated by polymerase chain reaction (PCR) amplification of cDNA clones using the Expand High Fidelity PCR system (Roche Molecular Biochemicals, Germany), with primers incorporating appropriate restriction sites, and subcloned into pXJ40HA or pXJ40Myc mammalian expression vectors (10Yee K.S. Yu V.C. J. Biol. Chem. 1998; 273: 5366-5374Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) driven by the CMV promoter. Full-length MAP-1 cDNA was subsequently released from pXJHA-MAP-1 and cloned in-frame into GST fusion protein vector pGEX-TK4E (8Chan S.L. Tan K.O. Zhang L. Yee K.S. Ronca F. Chan M.Y. Yu V.C. J. Biol. Chem. 1999; 274: 32461-32468Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). A coding region of each construct was sequenced to ensure that no error was introduced. All epitope tags were positioned at the N terminus. Substitution mutants of MAP-1 and Bax were created using the Transformer site-directed mutagenesis kit (CLONTECH). Deletion mutants of MAP-1 were generated by PCR with appropriate primers. Yeast two-hybrid screening was done essentially as described previously (11Bai C. Elledge S.J. Methods Enzymol. 1997; 283: 141-156Crossref PubMed Scopus (75) Google Scholar). Briefly, mBaxΔC21 was cloned in-frame with Gal-4 DNA binding domain in pAS2(HA) vector and used to screen a yeast two-hybrid library derived from adult human brain (CLONTECH). Screening and subsequent protein-protein interaction studies were carried out in yeast Y190 reporter strain. cDNA fragment of B26 was used to screen human brain (cerebellum) and mouse brain λ-ZAP cDNA libraries (Stratagene) using standard techniques as described (10Yee K.S. Yu V.C. J. Biol. Chem. 1998; 273: 5366-5374Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The human multiple tissue Northern blot (CLONTECH) was hybridized with a32P-labeled cDNA probe by using ExpressHyb hybridization solution (CLONTECH) according to the instructions of the manufacturer. Bacterial GST-MAP-1 fusion protein was prepared as described previously (8Chan S.L. Tan K.O. Zhang L. Yee K.S. Ronca F. Chan M.Y. Yu V.C. J. Biol. Chem. 1999; 274: 32461-32468Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 12Tan K.O. Tan K.M. Yu V.C. J. Biol. Chem. 1999; 274: 23687-23690Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), and the recombinant protein was immobilized onto glutathione-agarose beads. A GST binding assay was carried out according to a previously (8Chan S.L. Tan K.O. Zhang L. Yee K.S. Ronca F. Chan M.Y. Yu V.C. J. Biol. Chem. 1999; 274: 32461-32468Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 12Tan K.O. Tan K.M. Yu V.C. J. Biol. Chem. 1999; 274: 23687-23690Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Briefly, proteins were prepared vitro of using a system The of the proteins was by gel electrophoresis of proteins of acid were into of GST binding and for with GST-MAP-1 immobilized the were subsequently with binding and for in proteins were by cells a were with each of expression plasmids driven by the CMV (8Chan S.L. Tan K.O. Zhang L. Yee K.S. Ronca F. Chan M.Y. Yu V.C. J. Biol. Chem. 1999; 274: 32461-32468Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 12Tan K.O. Tan K.M. Yu V.C. J. Biol. Chem. 1999; 274: 23687-23690Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) the N-terminal and proteins using the cells were for assay and as described (8Chan S.L. Tan K.O. Zhang L. Yee K.S. Ronca F. Chan M.Y. Yu V.C. J. Biol. Chem. 1999; 274: 32461-32468Abstract Full Text Full Text PDF PubMed Scopus (33) Google K.O. Tan K.M. Yu V.C. J. Biol. Chem. 1999; 274: 23687-23690Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Briefly, the cells were and in of of the cell was for the expression of the proteins by blot using or The were to using of polyclonal for and with of a of protein and for at The were in of in of containing and in of by were carried out as described previously (8Chan S.L. Tan K.O. Zhang L. Yee K.S. Ronca F. Chan M.Y. Yu V.C. J. Biol. Chem. 1999; 274: 32461-32468Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Briefly, cells a at were with each of the expression plasmids or vector and of was to the of plasmids for each to were carried out with for by of and inhibitors were to the at point cells were and in to the cells. are as the of cells by the of cells the cells from MCF-7 cells were onto at and with of expression plasmids of MAP-1 or vector and of (CLONTECH) using the cells were with and for with to The cells were subsequently and using a as described previously S.L. Yee K.S. Tan K.M. Yu V.C. J. Biol. Chem. 2000; 275: 17925-17928Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). described previously F. Chan S.L. Yu V.C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), cells were with and from by of The cells were at and the was The was in of A and were by at and in of and The were by and the was at for and the was was by and of proteins were used for by was prepared from cell using according to the of from cell was with and using (Roche Molecular for the of of the cDNA was to the PCR at for of amplification for for and for with a of for each PCR for was to ensure that and of cDNA were containing of cDNA were to ensure that the PCR obtained was not to of The of the primers used are as and A yeast two-hybrid screen was to identify Bax-associating proteins. cDNA amino of Bax with the hydrophobic domain to in yeast H. B. Reed J.C. Biol. 1996; PubMed Scopus Google Scholar), was cloned in-frame with the DNA binding domain and used as to screen a yeast two-hybrid human brain library (CLONTECH). two clones from the screen were of B26 and were found to with but not with other Gal-4 fusion proteins these two clones were found to contain two DNA derived from the B26 cDNA fragment as we a human brain (cerebellum) cDNA library and obtained several cDNA The a cDNA with a with a and with an in-frame that a protein of amino with a predicted of named protein MAP-1 for of apoptosis. A cDNA was subsequently obtained by screening a mouse brain cDNA The amino acid from the cDNA is to the human revealed that the predicted human MAP-1 protein amino acid with two other human proteins J. T. C. T. J. J. 1999; PubMed Scopus Google Scholar) and J. J. J. 1999; PubMed Scopus Google Scholar), that MAP-1 is a member of a family. and were both identified as by in the of some with with for and for J. T. C. T. J. J. 1999; PubMed Scopus Google J. J. J. 1999; PubMed Scopus Google Scholar). no function has been described for the and proteins. of the amino acid of MAP-1, a region of amino was found to be similar to the BH3 domains among members of the Bcl-2 family The BH3-like motif identified appears to be similar to the identified domain K.O. Tan K.M. Yu V.C. J. Biol. Chem. 1999; 274: 23687-23690Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) in the N-terminal region of The amino acid in and to the region that contains the BH3-like domain, to be similar to motif Northern blot revealed a of MAP-1 to be but at a in and brain The of MAP-1 in from MCF-7 cells not be in Northern blot However, MAP-1 were in MCF-7 and other human cell HeLa and SH-SY5Y with Interestingly, and have a restricted expression protein is only in brain and J. T. C. T. J. J. 1999; PubMed Scopus Google Scholar), protein is found in brain J. J. J. 1999; PubMed Scopus Google Scholar). The of MAP-1 to with Bax was further vivo in MAP-1, Bax, Bid, Bcl-2, and Bcl-XL were with MAP-1 into and were prepared for MAP-1 with Bax, Bcl-2, and Bcl-XL but not members of the Bcl-2 family, and to with MAP-1 in the assay the not that MAP-1 not bind to all members of the Bcl-2 family. In MAP-1 was to in vivo has been that the conformation of Bax can be by such as in the in interaction of with some members of the Bcl-2 family R.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). was not to the N-terminal conformation of Bax R.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), we the as described in A in by the cells in containing of Similar to the obtained with MAP-1 and with Bax, Bcl-2, and but not not These in the were further in vitro by using the The in vitro of MAP-1 from the in MAP-1 which as two when The to the predicted of MAP-1 and appears to have the as the MAP-1 in cells not was only to the in vitro of MAP-1 by the not that the protein by the not contain the Myc and is to be generated from of In with the in vivo GST-MAP-1 was to in vitro MAP-1, Bax, Bcl-2, and Bcl-XL but not the role of MAP-1 in cell death by MCF-7 cells were with MAP-1 expression or vector with or as a for cells. a of cells with MAP-1 as by the of and cells of was in a of green cells with MAP-1 of poly(ADP-ribose) polymerase which as a for the of in cells apoptosis (8Chan S.L. Tan K.O. Zhang L. Yee K.S. Ronca F. Chan M.Y. Yu V.C. J. Biol. Chem. 1999; 274: 32461-32468Abstract Full Text Full Text PDF PubMed Scopus (33) Google S. Nature. PubMed Scopus Google Scholar), was in cells with MAP-1 the of PARP to the fragment in MCF-7 cells with MAP-1 was by with A of inhibitors were for their to apoptosis by MAP-1 a of and DEVD-fmk an of no and respectively, against However, a was to the of MAP-1 that MAP-1 mediates apoptosis through a caspase-dependent Similar to of Bcl-XL the of MAP-1 The of MAP-1 in mediating apoptosis is not restricted to MCF-7 because of MAP-1 was to apoptosis by cell and in both HeLa and SH-SY5Y cells not Similar to human MAP-1, mouse MAP-1 apoptosis in MCF-7 cells and with Bax and Bcl-XL in assay in not to the of the BH3-like domain in MAP-1 for mediating protein-protein interaction and proapoptotic A of MAP-1 mutants was created Deletion of the BH3-like domain or of several conserved amino in the putative BH3-like domain of MAP-1 its interaction with Bax The conserved in the BH3 domains of Bax and was to be critical for mediating interaction with Bcl-2 or J. H. J. G. Korsmeyer S.J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, A. G. Korsmeyer S.J. Biol. 1998; PubMed Scopus Google Scholar) as well as for the N-terminal domain of to with its K.O. Tan K.M. Yu V.C. J. Biol. Chem. 1999; 274: 23687-23690Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Substitution of conserved of the BH3 domain, but not the to acid in MAP-1 was found to its interaction with Bax Interestingly, in contrast to proapoptotic members of the Bcl-2 family, the BH3-like domain in MAP-1 not to be required for interaction with deletion of the BH3-like domain of MAP-1 not its binding to Bcl-XL In deletion containing the BH3-like domain to with the and regions of MAP-1 were to with Similar to containing molecules (1Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4751) Google Scholar, 2Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3220) Google Scholar), the BH3-like domain in MAP-1 appeared to be required for its cell death function as deletion of domain or of the conserved amino in domain in protein The of MAP-1 that maintained binding to Bax proapoptotic at a similar to the MAP-1. the presence of the BH3-like domain, the to apoptosis that the BH3-like domain is not sufficient for mediating apoptosis. Although has been that essentially the BH3 domain are of apoptosis V. S. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Lutz R.J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), the BH3 domain has been to be for apoptosis B. Chinnadurai G. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, G. V.C. J. Reed J.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). the other because no such as protein-protein interaction or apoptosis has been with the we out the that may not at which in a other BH3 protein or of MAP-1 are to the the BH3-like region in MAP-1 is sufficient for mediating protein-protein interaction apoptosis. these data suggest that the BH3-like domain in MAP-1 is required for interaction with Bax and mediating apoptosis. Although both the and of MAP-1 were to with of these mutants and were found to be proapoptotic that interaction with Bcl-XL is to the conserved amino acid of the BH1, BH2, and BH3 domains among Bcl-2 family members have been to be critical for functions A. G. Korsmeyer S.J. Biol. 1998; PubMed Scopus Google Scholar, Korsmeyer S.J. Nature. PubMed Scopus Google Scholar, Korsmeyer S.J. Genes Dev. 1996; PubMed Scopus Google Scholar). the of the BH domains of Bax in mediating interaction with MAP-1, point of these critical amino acid in the BH1, BH2, and BH3 domains were In with mutation of the BH3 but not the BH1 or domains of Bax, its binding to However, of the point mutants were to bind MAP-1 that the three BH domains (BH1, BH2, and BH3) of Bax are all required for mediating protein-protein interaction with MAP-1. Yeast two-hybrid with these Bax in the to their interaction with the B26 similar not In we the and of a Bax-associating protein, MAP-1. proapoptotic members of the Bcl-2 family have been identified by using prosurvival members of the Bcl-2 family as in the yeast two-hybrid system J. J. Thompson C.B. Korsmeyer S.J. 1995; Full Text PDF PubMed Scopus Google Scholar, L. S. G. EMBO J. 1997; PubMed Scopus Google Scholar, A. M. A.J. S. A. 1997; PubMed Scopus Google Scholar). However, there has been no in the for the of protein of proapoptotic members of the Bcl-2 family such as Bax using This in may be to the by some proapoptotic members of Bcl-2 family in yeast which its in system H. B. Reed J.C. Biol. 1996; PubMed Scopus Google Scholar, C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of the by the hydrophobic of Bax, the of Bax was in was of mediating apoptosis in mammalian cells H. B. Reed J.C. Biol. 1996; PubMed Scopus Google Scholar). as we to screen for protein of Bax. Interestingly, we to identify any prosurvival members of the Bcl-2 family, Bcl-XL in our screen. may for prosurvival members of the Bcl-2 family may be in in the Bax may only with the prosurvival members of the Bcl-2 family in we that interaction of Bax with Bcl-XL was in the yeast two-hybrid system not MAP-1 contains a putative BH3 domain and associates with Bax, and Bcl-2 in vitro and in vivo in mammalian cells. mediates caspase-dependent apoptosis when overexpressed. The BH3-like domain in MAP-1 is required for binding to Bax, but not and for mediating apoptosis. Interestingly, in contrast to other known Bax-associating proteins, the binding of Bax to MAP-1 requires all of the BH (BH1, BH2, and BH3) domains of Bax, because point any one of the BH domains its binding to MAP-1. MAP-1 thus represents the protein of Bax identified that requires all the BH domains of Bax for some proapoptotic members of the Bcl-2 family contain BH1, BH2, and BH3 domains of the prosurvival members of Bcl-2 The structure of Bcl-XL that to the domains of such as and and 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 (1276) Google Scholar). has been that regions of the BH1 and domains may have function A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3220) Google Scholar). In Bax was to form an in vitro A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3220) Google Scholar). However, to be the Bcl-2 family of proteins forms vivo and these proteins apoptosis the of S. 2000; PubMed Scopus Google Scholar). of the BH1, BH2, and BH3 domains a as in the of Bcl-XL (6Kelekar A. Thompson C.B. Trends Cell Biol. 1998; 8: 324-330Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar), the BH domains in Bax have been to The BH3 domain of Bax has been proposed to be in binding the and and from the S. 2000; PubMed Scopus Google Scholar, Reed J.C. Science. 1998; 281: PubMed Google Scholar). In has been proposed that the BH3 domain of Bax mediates its proapoptotic by binding to the prosurvival Bcl-XL the binding of from Bcl-XL to G. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, D. G. S. A. 1998; PubMed Scopus Google Scholar). However, the of was because a of interaction and Bcl-XL be Cory S. J.M. S. A. 1999; PubMed Scopus Google Scholar, Cell 2000; PubMed Scopus Google Scholar). the was found to with Bax through its BH1 domain Korsmeyer S.J. Genes Dev. 1996; PubMed Scopus Google Scholar). is the only proapoptotic member of the Bcl-2 family known to with Bax to MAP-1. This association appears to the conformation of Bax and to the of the S. A. A. S. S. B. J.C. J. Cell Biol. 1999; PubMed Scopus Google Scholar). Hence, multiple are to be for the by Bax. of MAP-1 as a novel Bax-associating protein may thus further for the by Bax. are to Cory and for with of the cDNA clones in

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