Abstract
Ligation of death receptors or formation of the Apaf-1 apoptosome results in the activation of caspases and execution of apoptosis. We recently demonstrated that X-linked inhibitor-of-apoptosis protein (XIAP) associates with the apoptosome in vitro. By utilizing XIAP mutants, we now report that XIAP binds to the ‘native’ apoptosome complex via a specific interaction with the small p12 subunit of processed caspase-9. Indeed, we provide the first direct evidence that XIAP can simultaneously bind active caspases-9 and -3 within the same complex and that inhibition of caspase-3 by the Linker-BIR2 domain prevents disruption of BIR3-caspase-9 interactions. Recent studies suggest that inhibition of caspase-3 is dispensable for its anti-apoptotic effects. However, we clearly demonstrate that inhibition of caspase-3 is required to inhibit CD95 (Fas/Apo-1)-mediated apoptosis, whereas inhibition of either caspase-9 or caspase-3 prevents Bax-induced cell death. Finally, we illustrate for the first time that XIAP mutants, which are incapable of binding to caspases-9 and -3 are completely devoid of anti-apoptotic activity. Thus, XIAP's capacity to maintain inhibition of caspase-9 within the Apaf-1 apoptosome is influenced by its ability to simultaneously inhibit active caspase-3, and depending upon the apoptotic stimulus, inhibition of caspase-9 or 3 is essential for XIAP's anti-apoptotic activity.
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Abbreviations
- Apaf-1:
-
apoptotic protease-activating factor 1
- Smac:
-
second mitochondrial-activator of caspases
- CARD:
-
caspase recruitment domain
- DEVD.CHO:
-
acetyl-Asp-Glu-Val-Asp aldehyde
- IAP:
-
inhibitor-of-apoptosis protein
- BIR:
-
baculovirus IAP repeat
- XIAP:
-
X-linked inhibitor-of-apoptosis protein
- GST:
-
glutathione-S-transferase
References
Cohen GM . 1997 Caspases: the executioners of apoptosis Biochem. J. 326: 1–16
Earnshaw WC, Martins LM, Kaufmann SH . 1999 Mammalian caspases: structure, activation, substrates, and functions during apoptosis Annu. Rev. Biochem. 68: 383–424
Thornberry NA, Lazebnik Y . 1998 Caspases: enemies within Science 281: 1312–1316
Bratton SB, MacFarlane M, Cain K, Cohen GM . 2000 Protein complexes activate distinct caspase cascades in death receptor and stress-induced apoptosis Exp. Cell Res. 256: 27–33
Walczak H, Krammer PH . 2000 The CD95 (APO-1/Fas) and the TRAIL (APO-2L) apoptosis systems Exp. Cell Res. 256: 58–66
Bossy-Wetzel E, Newmeyer DD, Green DR . 1998 Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization EMBO J. 17: 37–49
Liu X, Kim CN, Yang J, Jemmerson R, Wang X . 1996 Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c Cell 86: 147–157
Harris MH, Thompson CB . 2000 The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability Cell Death Differ. 7: 1182–1191
Cain K, Brown DG, Langlais C, Cohen GM . 1999 Caspase activation involves the formation of the aposome, a large (∼700 kDa) caspase-activating complex J. Biol. Chem. 274: 22686–22692
Cain K, Bratton SB, Langlais C, Walker G, Brown DG, Sun XM, Cohen GM . 2000 Apaf-1 oligomerizes into biologically active ∼700 kDa and inactive ∼1.4 MDa apoptosome complexes J. Biol. Chem. 275: 6067–6070
Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X . 1997 Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade Cell 91: 479–489
Saleh A, Srinivasula SM, Acharya S, Fishel R, Alnemri ES . 1999 Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation J. Biol. Chem. 274: 17941–17945
Zou H, Li Y, Liu X, Wang X . 1999 An Apaf-1·cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9 J. Biol. Chem. 274: 11549–11556
Acehan D, Jiang X, Morgon DG, Heuser JE, Wang X, Akey CW . 2002 Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation Mol. Cell 9: 423–432
Rodriguez J, Lazebnik Y . 1999 Caspase-9 and APAF-1 form an active holoenzyme Genes Dev. 13: 3179–3184
Bratton SB, Walker G, Srinivasula S, Sun X-M, Butterworth M, Alnemri ES, Cohen GM . 2001 Recruitment, activation and retention of caspases-9 and -3 by Apaf-1 apoptosome and associated XIAP complexes EMBO J. 20: 998–2001
Stennicke HR, Deveraux QL, Humke EW, Reed JC, Dixit VM, Salvesen GS . 1999 Caspase-9 can be activated without proteolytic processing J. Biol. Chem. 274: 8359–8362
Srinivasula SM, Hegde R, Saleh A, Datta P, Shiozaki E, Chai J, Lee RA, Robbins PD, Fernandes-Alnemri T, Shi Y, Alnemri ES . 2001 A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis Nature 410: 112–116
Li H, Zhu H, Xu CJ, Yuan J . 1998 Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis Cell 94: 491–501
Luo X, Budihardjo I, Zou H, Slaughter C, Wang X . 1998 Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors Cell 94: 481–490
Nicholson DW . 1999 Caspase structure, proteolytic substrates, and function during apoptotic cell death Cell Death Differ. 6: 1028–1042
Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC, Nicholson DW, Alnemri ES, Green DR, Martin SJ . 1999 Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9- dependent manner J. Cell. Biol. 144: 281–292
Clem RJ, Miller LK . 1994 Control of programmed cell death by the baculovirus genes p35 and iap Mol. Cell Biol. 14: 5212–5222
Deveraux QL, Reed JC . 1999 IAP family proteins-suppressors of apoptosis Genes Dev. 13: 239–252
Richter BW, Mir SS, Eiben LJ, Lewis J, Reffey SB, Frattini A, Tian L, Frank S, Youle RJ, Nelson DL, Notarangelo LD, Vezzoni P, Fearnhead HO, Duckett CS . 2001 Molecular cloning of ILP-2, a novel member of the inhibitor of apoptosis protein family Mol. Cell Biol. 21: 4292–4301
Silke J, Vaux DL . 2001 Two kinds of BIR-containing protein-inhibitors of apoptosis, or required for mitosis J. Cell Sci. 114: 1821–1827
Chai J, Shiozaki E, Srinivasula SM, Wu Q, Datta P, Alnemri ES, Shi Y . 2001 Structural basis of caspase-7 inhibition by XIAP Cell 104: 769–780
Huang Y, Park YC, Rich RL, Segal D, Myszka DG, Wu H . 2001 Structural basis of caspase inhibition by XIAP: differential roles of the linker versus the BIR domain Cell 104: 781–790
Riedl SJ, Renatus M, Schwarzenbacher R, Zhou Q, Sun C, Fesik SW, Liddington RC, Salvesen GS . 2001 Structural basis for the inhibition of caspase-3 by XIAP Cell 104: 791–800
Sun C, Cai M, Gunasekera AH, Meadows RP, Wang H, Chen J, Zhang H, Wu W, Xu N, Ng SC, Fesik SW . 1999 NMR structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP Nature 401: 818–822
Sun C, Cai M, Meadows RP, Xu N, Gunasekera AH, Herrmann J, Wu JC, Fesik SW . 2000 NMR structure and mutagenesis of the third Bir domain of the inhibitor of apoptosis protein XIAP J. Biol. Chem. 275: 33777–33781
Suzuki Y, Nakabayashi Y, Nakata K, Reed JC, Takahashi R . 2001 X-linked inhibitor of apoptosis protein (XIAP) inhibits caspase-3 and -7 in distinct modes J. Biol. Chem. 276: 27058–27063
Takahashi R, Deveraux Q, Tamm I, Welsh K, Assa-Munt N, Salvesen GS, Reed JC . 1998 A single BIR domain of XIAP sufficient for inhibiting caspases J. Biol. Chem. 273: 7787–7790
Suzuki Y, Nakabayashi Y, Takahashi R . 2001 Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death Proc. Natl. Acad. Sci. USA 98: 8662–8667
Yang Y, Fang S, Jensen JP, Weissman AM, Ashwell JD . 2000 Ubiquitin protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli Science 288: 874–877
Du C, Fang M, Li Y, Li L, Wang X . 2000 Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition Cell 102: 33–42
Ekert PG, Silke J, Hawkins CJ, Verhagen AM, Vaux DL . 2001 DIABLO promotes apoptosis by removing MIHA/XIAP from processed caspase 9 J. Cell Biol. 152: 483–490
Srinivasula SM, Datta P, Fan XJ, Fernandes-Alnemri T, Huang Z, Alnemri ES . 2000 Molecular Determinants of the Caspase-promoting Activity of Smac/DIABLO and its Role in the Death Receptor Pathway J. Biol. Chem. 275: 36152–36157
Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM, Reid GE, Moritz RL, Simpson RJ, Vaux DL . 2000 Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins Cell 102: 43–53
Sun XM, Bratton SB, Butterworth M, MacFarlane M, Cohen GM . 2002 Bcl-2 and Bcl-xL inhibit CD95-mediated apoptosis by preventing mitochondrial release of Smac/DIABLO and subsequent inactivation of XIAP J. Biol. Chem. 277: 11345–11351
Birkey Reffey S, Wurthner JU, Parks WT, Roberts AB, Duckett CS . 2001 X-linked inhibitor of apoptosis protein functions as a cofactor in transforming growth factor-β signaling J. Biol. Chem. 28: 26542–26549
Hofer-Warbinek R, Schmid JA, Stehlik C, Binder BR, Lipp J, de Martin R . 2000 Activation of NF-kappa B by XIAP, the X chromosome-linked inhibitor of apoptosis, in endothelial cells involves TAK1 J. Biol. Chem. 275: 22064–22068
Sanna MG, Duckett CS, Richter BW, Thompson CB, Ulevitch RJ . 1998 Selective activation of JNK1 is necessary for the anti-apoptotic activity of hILP Proc. Natl. Acad. Sci. USA 95: 6015–6020
Yamaguchi K, Nagai S, Ninomiya-Tsuji J, Nishita M, Tamai K, Irie K, Ueno N, Nishida E, Shibuya H, Matsumoto K . 1999 XIAP, a cellular member of the inhibitor of apoptosis protein family, links the receptors to TAB1-TAK1 in the BMP signaling pathway EMBO J. 18: 179–187
Richter BWM, Duckett CS . 2000 The IAP proteins: caspase inhibitors and beyond www.stke.org/cgi/content/full/OC_sigtrans;2000/44/pe1 1–4
Silke J, Ekert PG, Day CL, Hawkins CJ, Baca M, Chew J, Pakusch M, Verhagen AM, Vaux DL . 2001 Direct inhibition of caspase 3 is dispensable for the anti-apoptotic activity of XIAP EMBO J. 20: 3114–3123
Bratton SB, Walker G, Roberts DL, Cain K, Cohen GM . 2001 Caspase-3 cleaves Apaf-1 into an approximately 30 kDa fragment that associates with an inappropriately oligomerized and biologically inactive ∼1.4 MDa apoptosome complex Cell Death Differ. 8: 425–433
Deveraux QL, Leo E, Stennicke HR, Welsh K, Salvesen GS, Reed JC . 1999 Cleavage of human inhibitor of apoptosis protein XIAP results in fragments with distinct specificities for caspases EMBO. J. 18: 5242–5251
Johnson DE, Gastman BR, Wieckowski E, Wang GQ, Amoscato A, Delach SM, Rabinowich H . 2000 Inhibitor of apoptosis protein hILP undergoes caspase-mediated cleavage during T lymphocyte apoptosis Cancer Res. 60: 1818–1823
Liu Z, Sun C, Olejniczak ET, Meadows RP, Betz SF, Oost T, Herrmann J, Wu JC, Fesik SW . 2000 Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain Nature 408: 1004–1008
Wu G, Chai J, Suber TL, Wu JW, Du C, Wang X, Shi Y . 2000 Structural basis of IAP recognition by Smac/DIABLO Nature 408: 1008–1012
Chai J, Du C, Wu JW, Kyin S, Wang X, Shi Y . 2000 Structural and biochemical basis of apoptotic activation by Smac/DIABLO Nature 406: 855–862
Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA, Munday NA, Raju SM, Smulson ME, Yamin T-T, Yu VL, Miller DK . 1995 Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis Nature 376: 37–43
Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME . 1998 Two CD95 (APO-1/Fas) signaling pathways EMBO J. 17: 1675–1687
Deng Y, Lin Y, Wu X . 2002 TRAIL-induced apoptosis requires Bax-dependent mitochondrial release of Smac/DIABLO Genes Dev. 16: 33–45
Zhang XD, Zhang XY, Gray CP, Nguyen T, Hersey P . 2001 Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of human melanoma is regulated by smac/DIABLO release from mitochondria Cancer Res. 61: 7339–7348
Wang SL, Hawkins CJ, Yoo SJ, Muller HA, Hay BA . 1999 The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID Cell 98: 453–463
Harlin H, Reffey SB, Duckett CS, Lindsten T, Thompson CB . 2001 Characterization of XIAP-deficient mice Mol. Cell Biol. 21: 3604–3608
Deveraux QL, Welsh K, Reed JC . 2000 Purification and use of recombinant inhibitor of apoptosis proteins as caspase inhibitors Methods Enzymol. 322: 154–161
Roberts DL, Merrison W, MacFarlane M, Cohen GM . 2001 The inhibitor of apoptosis protein-binding domain of Smac is not essential for its proapoptotic activity J. Cell Biol. 153: 221–228
Duckett CS, Li F, Wang Y, Tomaselli KJ, Thompson CB, Armstrong RC . 1998 Human IAP-like protein regulates programmed cell death downstream of Bcl-xL and cytochrome c. Mol Cell Biol. 18: 608–615
Acknowledgements
Antibodies to caspases-9 and -3 were generously provided by Drs D Green (La Jolla Institute for Allergy and Immunology, San Diego, CA, USA) and DW Nicholson (Merck Frosst, Canada). Bacterial expression plasmids for caspases-9 and -3 were kindly provided by Dr ES Alnemri (Kimmel Cancer Institute, Philadelphia, PA, USA). Mammalian expression plasmids for CD95 and Bax were kindly provided by Drs R Siegel (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA) and S Korsmeyer (Howard Hughes Medical Institute, Harvard Medical School, Dana-Farber Cancer Institute, Boston, MA, USA). This work was partly funded by a European Union grant (QLG1-1999-00739 to GM Cohen).
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Bratton, S., Lewis, J., Butterworth, M. et al. XIAP inhibition of caspase-3 preserves its association with the Apaf-1 apoptosome and prevents CD95- and Bax-induced apoptosis. Cell Death Differ 9, 881–892 (2002). https://doi.org/10.1038/sj.cdd.4401069
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DOI: https://doi.org/10.1038/sj.cdd.4401069
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