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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.

Original publication

DOI

10.1083/jcb.144.2.281

Type

Journal article

Journal

The Journal of cell biology

Publication Date

01/1999

Volume

144

Pages

281 - 292

Addresses

Molecular Cell Biology Laboratory, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland.

Keywords

Jurkat Cells, Animals, Rabbits, Humans, Cytochrome c Group, Caspases, Proteins, Cell Extracts, Apoptosis, Protein Processing, Post-Translational, Enzyme Activation, Caspase 8, Caspase 9, Caspase 3, Apoptotic Protease-Activating Factor 1, Caspase 2, Caspase 6, Caspase 7, Caspase 10