BCL-X-L INTERACTS WITH APAF-1 AND INHIBITS APAF-1-DEPENDENT CASPASE-9ACTIVATION

Recent studies indicate that Caenorhabditis elegans CED-4 interacts wi th and promotes the activation of the death protease CED-3, and that t his activation is inhibited by CED-9, Here we show that a mammalian ho molog of CED-4, Apaf-1, can associate with several death proteases, in cluding caspase-4, caspase-8, caspase-9, and nematode CED-3 in mammali an cells. The interaction with caspase-9 was mediated by the N-termina l CED-4-like domain of Apaf-1. Expression of Apaf-1 enhanced the killi ng activity of caspase-9 that required the CED-4-like domain of Apaf-1 . Furthermore, Apaf-1 promoted the processing and activation of caspas e-9 in vivo. Bcl-X-L, an antiapoptotic member of the Bcl-2 family, was shown to physically interact with Apaf-1 and caspase-9 in mammalian c ells. The association of Apaf-1 with Bcl-X-L was mediated through both its CED-4-like domain and the C-terminal domain containing WD-40 repe ats. Expression of Bcl-X-L inhibited the association of Apaf-1 with ca spase-9 in mammalian cells. Significantly, recombinant Bcl-X-L purifie d from Escherichia coli or insect cells inhibited Apaf-1-dependent pro cessing of caspase-9, Furthermore, Bcl-X-L failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggest ing that Bcl-X-L regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-X-L associates with caspase-9 and Apaf-1, and sh ow that Bcl-X-L inhibits the maturation of caspase-9 mediated by Apaf- 1, a process that is evolutionarily conserved from nematodes to humans .