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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-6 from Jurkat extracts  abolishes cytochrome c/dATP-initiated processing of caspases-8 and -10, whereas depletion of caspase-7 has no  effect. Jurkat extracts were  depleted of caspase-6 (A) or  caspase-7 (B) as described in  Materials and Methods and  were then assessed for their  ability to support processing  of the indicated 35S-labeled  caspases. Caspase processing was assessed after incubation of extracts for 2 h at  37°C in the presence of 50  μg/ml cytochrome c and 1 mM  dATP. (C) To confirm that  caspases were depleted, equal  amounts of either mock- depleted, caspase-6–depleted,  or caspase-7–depleted Jurkat  extracts were loaded, followed by probing for caspase-6 or caspase-7 by Western blot, as indicated.
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Figure 10: Depletion of caspase-6 from Jurkat extracts abolishes cytochrome c/dATP-initiated processing of caspases-8 and -10, whereas depletion of caspase-7 has no effect. Jurkat extracts were depleted of caspase-6 (A) or caspase-7 (B) as described in Materials and Methods and were then assessed for their ability to support processing of the indicated 35S-labeled caspases. Caspase processing was assessed after incubation of extracts for 2 h at 37°C in the presence of 50 μg/ml cytochrome c and 1 mM dATP. (C) To confirm that caspases were depleted, equal amounts of either mock- depleted, caspase-6–depleted, or caspase-7–depleted Jurkat extracts were loaded, followed by probing for caspase-6 or caspase-7 by Western blot, as indicated.

Mentions: We next depleted caspase-6 or caspase-7 from Jurkat extracts to ask whether either of these caspases was required for any of the other caspase activation events seen in the presence of cytochrome c (Fig. 10). Immunodepletion of caspase-6 failed to have any effect on the processing of caspases-3, -7, and -9 in response to cytochrome c, consistent with caspase-6 activation being downstream of the latter caspases (Fig. 10 A). Caspase-2 processing was also unaffected in the absence of caspase-6, suggesting that caspase-2 is directly processed, along with caspase-6, upon activation of caspase-3 in the extracts. However, processing of caspases-8 and -10 was largely abrogated in extracts devoid of caspase-6 (Fig. 10 A). These data suggest that, upon activation by caspase-3, caspase-6 in turn promotes the processing of caspases-8 and -10 further down the cascade.


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-6 from Jurkat extracts  abolishes cytochrome c/dATP-initiated processing of caspases-8 and -10, whereas depletion of caspase-7 has no  effect. Jurkat extracts were  depleted of caspase-6 (A) or  caspase-7 (B) as described in  Materials and Methods and  were then assessed for their  ability to support processing  of the indicated 35S-labeled  caspases. Caspase processing was assessed after incubation of extracts for 2 h at  37°C in the presence of 50  μg/ml cytochrome c and 1 mM  dATP. (C) To confirm that  caspases were depleted, equal  amounts of either mock- depleted, caspase-6–depleted,  or caspase-7–depleted Jurkat  extracts were loaded, followed by probing for caspase-6 or caspase-7 by Western blot, as indicated.
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Related In: Results  -  Collection

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Figure 10: Depletion of caspase-6 from Jurkat extracts abolishes cytochrome c/dATP-initiated processing of caspases-8 and -10, whereas depletion of caspase-7 has no effect. Jurkat extracts were depleted of caspase-6 (A) or caspase-7 (B) as described in Materials and Methods and were then assessed for their ability to support processing of the indicated 35S-labeled caspases. Caspase processing was assessed after incubation of extracts for 2 h at 37°C in the presence of 50 μg/ml cytochrome c and 1 mM dATP. (C) To confirm that caspases were depleted, equal amounts of either mock- depleted, caspase-6–depleted, or caspase-7–depleted Jurkat extracts were loaded, followed by probing for caspase-6 or caspase-7 by Western blot, as indicated.
Mentions: We next depleted caspase-6 or caspase-7 from Jurkat extracts to ask whether either of these caspases was required for any of the other caspase activation events seen in the presence of cytochrome c (Fig. 10). Immunodepletion of caspase-6 failed to have any effect on the processing of caspases-3, -7, and -9 in response to cytochrome c, consistent with caspase-6 activation being downstream of the latter caspases (Fig. 10 A). Caspase-2 processing was also unaffected in the absence of caspase-6, suggesting that caspase-2 is directly processed, along with caspase-6, upon activation of caspase-3 in the extracts. However, processing of caspases-8 and -10 was largely abrogated in extracts devoid of caspase-6 (Fig. 10 A). These data suggest that, upon activation by caspase-3, caspase-6 in turn promotes the processing of caspases-8 and -10 further down the cascade.

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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