Dimerization properties of human BAD. Identification of a BH-3 domain and analysis of its binding to mutant BCL-2 and BCL-XL proteins.
Bad, an inducer of programmed cell death, was recently isolated from a mouse cDNA library by its ability to bind to the anti-apoptotic protein BCL-2. Sequence analysis suggested that Bad was a member of the BCL-2 gene family that encodes both inducers and inhibitors of programmed cell death. To further analyze the role of BAD in the network of homo- and heterodimers formed by the BCL-2 family, we have cloned the human homologue of BAD and assessed its biological activity and its interactions with wild type and mutant BCL-2 family proteins. Our results indicate that the human BAD protein, like its mouse homologue, is able to induce apoptosis when transfected into mammalian cells. Furthermore, in yeast two-hybrid assays as well as quantitative in vitro interaction assays, human Bad interacted with BCL-2 and BCL-XL. Sequence alignments of human BAD revealed the presence of a BH-3 homology domain as seen in other BCL-2 family proteins. Peptides derived from this domain were able to completely inhibit the dimerization of BAD with BCL-XL. Thus, as previously shown for BAX, BAK, BCL-2, and BCL-XL, the BH3 domain of BAD is required for its dimerization with other BCL-2 family proteins. BAD was further analyzed for its ability to bind to various mutants of BCL-2 and BCL-XL that have lost the ability to bind BAX and BAK, some of which retain biological activity and some of which do not. Surprisingly, all of the mutated BCL-2 and BCL-XL proteins analyzed strongly interacted with human BAD. Our data thus indicate that mutations in BCL-2 and BCL-XL can differentially affect the heterodimeric binding of different death-promoting proteins and have implications concerning the relationship between heterodimerization and biological activity.
Ottilie
S
S
IDUN Pharmaceuticals Inc., La Jolla, California 92037, USA.
Diaz
J L
JL
Horne
W
W
Chang
J
J
Wang
Y
Y
Wilson
G
G
Chang
S
S
Weeks
S
S
Fritz
L C
LC
Oltersdorf
T
T
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein
C494819:BAD protein, human; C494772:BAK1 protein, human; C494826:BAX protein, human; C494811:BCL2L1 protein, human; C494820:Bad protein, mouse; C494773:Bak1 protein, mouse; C494827:Bax protein, mouse; C494812:Bcl2l1 protein, mouse; D002352:Carrier Proteins; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein