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J. Cell Biol., Volume 144, Number 2, January 25, 1999 281-292

Ordering the Cytochrome c-initiated Caspase Cascade: Hierarchical Activation of Caspases-2, -3, -6, -7, -8, and -10 in a Caspase-9-dependent Manner

Elizabeth A. Slee,* Mary T. Harte,* Ruth M. Kluck,Dagger Beni B. Wolf,Dagger Carlos A. Casiano,§ Donald D. Newmeyer,Dagger Hong-Gang Wang,parallel John C. Reed,parallel Donald W. Nicholson, Emad S. Alnemri,** Douglas R. Green,Dagger and Seamus J. Martin*

* Molecular Cell Biology Laboratory, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland; Dagger  Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121; § Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037; parallel   The Burnham Institute, La Jolla, California 92037;  Departments of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, H9R 4P8, Canada; and ** Center for Apoptosis Research and The Kimmel Cancer Institute, Jefferson Medical College, Philadelphia, Pennsylvania 19107

Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.

Key words: Apaf-1;  apoptosis;  caspases;  cell-free;  cytochrome c


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