Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation

Abstract

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-XL, 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-XL was mediated through both its CED-4-like domain and the C-terminal domain containing WD-40 repeats. Expression of Bcl-XL inhibited the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-XL purified from Escherichia coli or insect cells inhibited Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-XL failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-XL regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-XL associates with caspase-9 and Apaf-1, and show that Bcl-XL inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans.

Figure 1

Apaf-1 interacts with several caspases with long prodomains in mammalian cells. (A) Schematic drawing of the Apaf-1 constructs used in this study. (B–D) Interaction of Apaf-1 with caspase-4, caspase-9, and CED-3. 293T cells were cotransfected with the indicated tagged caspases and control vector, or tagged Apaf-1, N-terminal, or C-terminal Apaf-1 constructs (Apaf-1-Myc, N-Apaf-1-Myc, and C-Apaf-1-Myc, respectively). (Upper) Western blot analysis of immunoprecipitated protein complexes with indicated antibodies. (Lower) The expression of caspase-3 and -4 (B), caspase-9 (C), and CED-3 and Apaf-1 proteins (D). Reduced level of pro-caspase-9 in lane 3 of C is due to efficient N-Apaf-1-mediated processing of pro-caspase-9. IP: immunoprecipitation, WB: Western blot analysis. Asterisks indicate nonspecific bands.

Figure 2

Apaf-1 interacts with the prodomain of caspase-8. (A) Interaction of Apaf-1 with caspase-8. (B) Apaf-1 interacts with the prodomain of caspase-8. Immunoprecipitation analysis was performed as described in Fig. 1. (Upper) Western blot analysis of immunoprecipitated protein complexes with indicated antibodies. (A and B, Lower) show the expression of caspase-8 (A) or Apaf-1 (B) in total lysates by Western blot analysis. IP: immunoprecipitation, WB: Western blot analysis. Asterisks indicate nonspecific bands.

Figure 3

Regulation of caspase-9 by full-length and N-terminal Apaf-1. (A) Apaf-1 enhances caspase-9-induced apoptosis. 293T cells were transiently transfected with a reporter pcDNA3-β-galactosidase plus the indicated plasmids. WT, full-length Apaf-1-Myc; N, N-terminal Apaf-1-Myc; C, C-terminal Apaf-1-Myc; UT, Untagged full-length Apaf-1. The results represent the percentage of blue cells that exhibit morphologic features of apoptosis and are given as the mean +/− SD of triplicate cultures. (B) Apaf-1 promotes caspase-9 activation in vivo. Lysates from cells transfected with the indicated plasmids were immunoblotted with anti-caspase-9 (Upper) or anti-PARP antibodies (Lower). Arrows indicate full-length and processed caspase-9 and PARP fragments. Asterisk indicates nonspecific band.

Figure 4

Bcl-X_L interacts with caspase-9 and Apaf-1. (A) Bcl-X_L interacts with caspase-9. (Upper) Western blot analysis of immunoprecipitated Bcl-X_L and coimmunoprecipitated pro-caspase-9 and processed form (p35). (Lower) The expression of caspase-9 in total lysate. (B and C) Bcl-X_L interacts with Apaf-1. 293T cells were transfected with indicated plasmids and the lysates immunoprecipitated with anti-Flag (B) or anti-Myc (C) antibody. WT, full-length Apaf-1-Myc; N, N-terminal Apaf-1-Myc; C, C-terminal Apaf-1-Myc. Panels show Western blot analysis of coimmunoprecipitated Apaf-1 and Bcl-X_L proteins. Asterisk indicates nonspecific band.

Figure 5

Bcl-X_L inhibits the association of Apaf-1 with caspase-9. 293T cells were transfected with indicated plasmids and the lysates immunoprecipitated with anti-HA antibody. (Upper) Western blot analysis of immunoprecipitated caspase-9 and coimmunoprecipitated Apaf-1. (Lower) Western blot analysis of total lysates with anti-Myc and anti-Flag antibody.

Figure 6

Purified Bcl-X_L inhibits Apaf-1-dependent maturation of caspase-9 in vitro. (A) Apaf-1-dependent processing of caspase-9 by Apaf-1 in vitro. In the last two lanes normal rabbit serum (control) or anti-Apaf-1 serum were added 30 min prior to the reaction. (B) N-terminal Apaf-1 mutant (residues 1–559) activates caspase-9 independently of cytochrome c and dATP. Extracts from 293T cells transfected with pcDNA3 (control extract) or pcDNA3-N-Apaf-1 (N-Apaf-1 extract) were used in the analysis. (C) Regulation of caspase-9 maturation by recombinant Bcl-X_L. Cellular extracts were incubated with indicated amounts of recombinant Bcl-X_L or Bad and then cytochrome c and dATP were added to the reaction. (Right) Extracts from cells expressing a N-terminal Apaf-1 mutant (residues 1–559) were incubated with indicated amounts of recombinant Bcl-X_L. Proform and processed caspase-9 are indicated by arrows.