title: Characterization of the anti-apoptotic mechanism of Bcl-B.
abstract: Bcl-B protein is an anti-apoptotic member of the Bcl-2 family protein that contains all the four BH (Bcl-2 homology) domains (BH1, BH2, BH3 and BH4) and a predicted C-terminal transmembrane domain. Our previous results showed that Bcl-B binds Bax and suppresses apoptosis induced by over-expression of Bax; however, Bcl-B does not bind or suppress Bak. To explore the molecular basis for the differential binding and suppression of Bax and Bak, we studied the BH3 dimerization domains of Bax and Bak. Chimeric mutants of Bax and Bak were generated that swapped the BH3 domains of these pro-apoptotic proteins. Bcl-B associated with and blocked apoptosis induced by mutant Bak containing the BH3 domain of Bax, but not mutant Bax containing the BH3 domain of Bak. In contrast, Bcl-X(L) protein bound and suppressed apoptosis induction by Bax, Bak and both BH3-domain chimeras. A strong correlation between binding and apoptosis suppression was also obtained using a series of alanine substitutions spanning the length of the Bax BH3 domain to identify critical residues for Bcl-B binding. Conversely, using structure-based modelling to design mutations in the BH3-binding pocket of Bcl-B, we produced two Bcl-B mutants (Leu86-->Ala and Arg96-->Gln) that failed to bind Bax and that also were unable to suppress apoptosis induced by Bax over-expression. In contrast, other Bcl-B mutants that still bound Bax retained protective activity against Bax-induced cell death, thus serving as a control. We conclude that, in contrast with some other anti-apoptotic Bcl-2-family proteins, a strong correlation exists for Bcl-B between binding to pro-apoptotic multidomain Bcl-2 family proteins and functional apoptosis suppression.
authors.affiliation: The Burnham Institute, La Jolla, CA 92037, USA.
authors.forename: Dayong
authors.initials: D
authors.lastname: Zhai
authors.forename: Ning
authors.initials: N
authors.lastname: Ke
authors.forename: Haichao
authors.initials: H
authors.lastname: Zhang
authors.forename: Uri
authors.initials: U
authors.lastname: Ladror
authors.forename: Mary
authors.initials: M
authors.lastname: Joseph
authors.forename: Andreas
authors.initials: A
authors.lastname: Eichinger
authors.forename: Adam
authors.initials: A
authors.lastname: Godzik
authors.forename: Shi-Chung
authors.initials: SC
authors.lastname: Ng
authors.forename: John C
authors.initials: JC
authors.lastname: Reed
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D019610:Cytoprotection; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein
chemical_list: C494772:BAK1 protein, human; C494826:BAX protein, human; C117144:BCL2-like 10 protein; 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