Identification of a Bcl-XL binding region within the ATPase domain of Apaf-1.
CED-4, a pro-apoptotic factor in Caenorhabditis elegans, activates the cell death protease CED-3. CED-9 directly binds to CED-4 and represses this. However, it has remained unclear whether a mammalian CED-9 homologue, Bcl-XL, inhibits the function of the mammalian CED-4 homologue, Apaf-1, by direct binding. To analyze the interaction, we adopted a yeast two-hybrid system. Since Bcl-XL and the CED-4-like portion of Apaf-1 failed to exhibit a positive result in the assay, we prepared "fragment libraries" of bcl-XL or apaf-1 cDNA. By screening of the apaf-1 "fragment library," we obtained nine clones interacting with Bcl-XL, all containing the same region within the ATPase domain, designated BBR: the Bcl-XL binding region. Binding of BBR to Bcl-XL was also confirmed by immunoprecipitation assays. Bcl-2, Bcl-w, A1/Bfl-1, and Boo/Diva failed to show the same capacity for binding to BBR as Bcl-XL. These results indicate that Bcl-XL directly binds to a specific region in Apaf-1.
Yajima
Hirohiko
H
Department of Molecular Radiobiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan. yajima@hiroshima-u.ac.jp
Suzuki
Fumio
F
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000595:Amino Acid Sequence; D053477:Apoptotic Protease-Activating Factor 1; D001665:Binding Sites; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D016415:Sequence Alignment; D020798:Two-Hybrid System Techniques; D051020:bcl-X Protein
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; C494811:BCL2L1 protein, human; D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins; C089224:Ced-4 protein, C elegans; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D000251:Adenosine Triphosphatases