Distinct domains of Bcl-XL are involved in Bax and Bad antagonism and in apoptosis inhibition.
Pro-survival factor Bcl-X(L) can antagonize the pro-apoptotic functions of Bax and Bad via two distinct mechanisms. It can block Bax-mediated cell death by preventing Bax translocation from the cytosol to mitochondria. On the other hand, Bcl-X(L) can neutralize Bad by sequestering it to mitochondria. In order to map the domains of Bcl-X(L) involved in inhibiting Bax and Bad, we have carried out mutational analyses of this protein. This was done by deleting the key domains of Bcl-X(L), including its BH1-4 domains, the flexible loop, the C-terminal hydrophobic domain, and segments of the alpha5-alpha6 hairpin. The resulting Bcl-X(L) mutant constructs were then co-transfected with either GFP-Bax or GFP-Bad. We found that the BH1-4 domains and the C-terminal segment of Bcl-X(L) were essential for blocking Bax localization to mitochondria. On the other hand, only its BH1 and BH3 domains and the C-terminal hydrophobic segment were necessary for sequestering Bad to mitochondria. In addition, by immunoprecipitation analyses, we found that these deletions differentially affected the ability of the Bcl-X(L) mutant proteins to bind Bax and Bad. Finally, cell viability assays indicated that the BH1-4 domains of Bcl-X(L) were the primary domains required for inhibiting staurosporine-induced apoptosis, suggesting that distinct domains of Bcl-X(L) are involved in antagonizing Bax and Bad and in apoptosis inhibition.
Zhou
Hui
H
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, PO Box 250509, Charleston, SC 29425, USA.
Hou
Qi
Q
Chai
Yunfei
Y
Hsu
Yi-Te
YT
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D019556:COS Cells; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002522:Chlorocebus aethiops; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D019311:Staurosporine / Q000494:pharmacology; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein
C494819:BAD protein, human; C494826:BAX protein, human; C494811:BCL2L1 protein, human; D002352:Carrier Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein; D019311:Staurosporine