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

Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC, Nicholson DW, Alnemri ES, Green DR, Martin SJ - J. Cell Biol. (1999)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

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.

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Depletion of caspase-9 from Jurkat extracts abolishes cytochrome c–initiated processing  of all caspases. (A) Jurkat extracts were depleted of  caspase-9 using glutathione Sepharose–immobilized  GST-Apaf-11-97 fusion protein, or mock-depleted  with GST, and were then assessed for their ability to  support caspase processing, as indicated. Caspase  processing was assessed in the presence of 50 μg/ml  cytochrome c and 1 mM dATP. (B) Extracts were  depleted of caspase-9 using protein A/G agarose– immobilized anti–caspase-9 antibody, or mock- depleted using a control antibody (anti–Rel A), as  described in Materials and Methods. The same concentrations of cytochrome c/dATP were used as in  A. Results are representative of three independent  experiments.
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Figure 7: Depletion of caspase-9 from Jurkat extracts abolishes cytochrome c–initiated processing of all caspases. (A) Jurkat extracts were depleted of caspase-9 using glutathione Sepharose–immobilized GST-Apaf-11-97 fusion protein, or mock-depleted with GST, and were then assessed for their ability to support caspase processing, as indicated. Caspase processing was assessed in the presence of 50 μg/ml cytochrome c and 1 mM dATP. (B) Extracts were depleted of caspase-9 using protein A/G agarose– immobilized anti–caspase-9 antibody, or mock- depleted using a control antibody (anti–Rel A), as described in Materials and Methods. The same concentrations of cytochrome c/dATP were used as in A. Results are representative of three independent experiments.

Mentions: To determine whether caspase-9 was required for activation of all other caspases in this context, we depleted this protease from Jurkat extracts before the addition of cytochrome c. Depletion of caspase-9 using Sepharose- immobilized GST-Apaf-11-97 (Fig. 7 A), or anti–caspase-9 polyclonal antibody (Fig. 7 B), rendered all caspases unresponsive to cytochrome c. In contrast, mock depletions performed using Sepharose-GST or control polyclonal antibody did not interfere with cytochrome c–induced activation of any of the caspases examined (Fig. 7, A and B). These data suggest that caspase-9 is critical for cytochrome c–initiated caspase activation events and cannot be substituted for by the other caspases present in the extracts. They also provide further support for the idea that Apaf-1 is selective for caspase-9 (Fig. 6) and fails to initiate the apoptotic program in its absence.


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

Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC, Nicholson DW, Alnemri ES, Green DR, Martin SJ - J. Cell Biol. (1999)

Depletion of caspase-9 from Jurkat extracts abolishes cytochrome c–initiated processing  of all caspases. (A) Jurkat extracts were depleted of  caspase-9 using glutathione Sepharose–immobilized  GST-Apaf-11-97 fusion protein, or mock-depleted  with GST, and were then assessed for their ability to  support caspase processing, as indicated. Caspase  processing was assessed in the presence of 50 μg/ml  cytochrome c and 1 mM dATP. (B) Extracts were  depleted of caspase-9 using protein A/G agarose– immobilized anti–caspase-9 antibody, or mock- depleted using a control antibody (anti–Rel A), as  described in Materials and Methods. The same concentrations of cytochrome c/dATP were used as in  A. Results are representative of three independent  experiments.
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Related In: Results  -  Collection

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Figure 7: Depletion of caspase-9 from Jurkat extracts abolishes cytochrome c–initiated processing of all caspases. (A) Jurkat extracts were depleted of caspase-9 using glutathione Sepharose–immobilized GST-Apaf-11-97 fusion protein, or mock-depleted with GST, and were then assessed for their ability to support caspase processing, as indicated. Caspase processing was assessed in the presence of 50 μg/ml cytochrome c and 1 mM dATP. (B) Extracts were depleted of caspase-9 using protein A/G agarose– immobilized anti–caspase-9 antibody, or mock- depleted using a control antibody (anti–Rel A), as described in Materials and Methods. The same concentrations of cytochrome c/dATP were used as in A. Results are representative of three independent experiments.
Mentions: To determine whether caspase-9 was required for activation of all other caspases in this context, we depleted this protease from Jurkat extracts before the addition of cytochrome c. Depletion of caspase-9 using Sepharose- immobilized GST-Apaf-11-97 (Fig. 7 A), or anti–caspase-9 polyclonal antibody (Fig. 7 B), rendered all caspases unresponsive to cytochrome c. In contrast, mock depletions performed using Sepharose-GST or control polyclonal antibody did not interfere with cytochrome c–induced activation of any of the caspases examined (Fig. 7, A and B). These data suggest that caspase-9 is critical for cytochrome c–initiated caspase activation events and cannot be substituted for by the other caspases present in the extracts. They also provide further support for the idea that Apaf-1 is selective for caspase-9 (Fig. 6) and fails to initiate the apoptotic program in its absence.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

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.

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