Characterization of the anti-apoptotic mechanism of Bcl-B.
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.
Zhai
Dayong
D
The Burnham Institute, La Jolla, CA 92037, USA.
Ke
Ning
N
Zhang
Haichao
H
Ladror
Uri
U
Joseph
Mary
M
Eichinger
Andreas
A
Godzik
Adam
A
Ng
Shi-Chung
SC
Reed
John C
JC
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
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