Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner

E A Slee, M T Harte, R M Kluck, B B Wolf, C A Casiano, D D Newmeyer, H G Wang, J C Reed, D W Nicholson, E S Alnemri, D R Green, S J Martin

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Abstract

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 languageEnglish
Pages (from-to)281-92
Number of pages12
JournalThe Journal of cell biology
Volume144
Issue number2
Publication statusPublished - 25 Jan 1999

Keywords

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

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    Slee, E. A., Harte, M. T., Kluck, R. M., Wolf, B. B., Casiano, C. A., Newmeyer, D. D., Wang, H. G., Reed, J. C., Nicholson, D. W., Alnemri, E. S., Green, D. R., & Martin, S. J. (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. The Journal of cell biology, 144(2), 281-92.