Bcl-X(L) interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation

Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. Here we show that a mammalian homolog of CED-4, Apaf-1, can associate with several death proteases, including caspase-4, caspase-8, caspase-9, and nematode CED-3 in mammalian cells. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhanced the killing activity of caspase-9 that required the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promoted the processing and activation of caspase-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 cells. 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 repeats. Expression of Bcl-X(L) inhibited the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-X(L) purified from Escherichia coli or insect cells inhibited Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-X(L) failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting 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 show that Bcl-X(L) inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans.