The proapoptotic BH3-only protein BAD transduces cell death signals independently of its interaction with Bcl-2.
The BH3-only protein BAD binds to Bcl-2 family proteins through its BH3 domain. Recent studies suggest that BAD binds to both Bcl-2 and Bcl-X(L), however mediates its pro-apoptotic functions through inhibition of Bcl-X(L), but not Bcl-2. In this paper we addressed this issue using a BAD mutant within the BH3 domain, by substitution of Asp 119 with Gly (BAD(D119G)), which selectively abrogates an ability to interact with Bcl-2. Confocal microscopy revealed that mutation of BAD at D119 does not affect BAD targeting to the mitochondrial membrane in serum-starved COS-7 cells. However, co-precipitation assays indicated that, whereas wild-type BAD (BADwt) directly interacts with Bcl-2 and Bcl-X(L), BAD(D119G) interacts only with Bcl-X(L). Nevertheless both BADwt and BAD(D119G) could introduce apoptosis and diminish the anti-apoptotic effect of Bcl-2 and Bcl-X(L) in a similar manner in a co-transfection assay. These data thus suggest that Asp119 is a crucial site within the BH3 domain of BAD for interaction of BAD with Bcl-2, but is dispensable for the interaction of BAD with Bcl-X(L), for its targeting to mitochondria, and most importantly, for its pro-apoptotic functions. Thus, we confirm that neutralization of Bcl-2 function is marginal for BAD-mediated apoptosis.
Adachi
M
M
Division of Molecular Oncology and Molecular Diagnosis, Graduate School of Medicine, Sapporo Medical University, Sapporo, Japan. adachi@sapmed.ac.jp
Imai
K
K
D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein
C494819:BAD protein, human; C494811:BCL2L1 protein, human; D002352:Carrier Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein