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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.

Show MeSH
Kinetics of activation of caspases-2, -3, -6, -7, -8, -9, and  -10 in response to cytochrome c. Cell-free reactions were assembled containing the indicated [35S]methionine-labeled caspases  and were incubated with or without 50 μg/ml cytochrome c and  1 mM dATP, as indicated. Reactions were incubated at 37°C and  samples were removed at the indicated times, followed by analysis of caspase processing by SDS-PAGE/fluorography. Cleavage  products are denoted by arrows. To allow direct comparison, all  reactions were set up in an identical manner. Results are representative of a minimum of three independent experiments.
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Figure 5: Kinetics of activation of caspases-2, -3, -6, -7, -8, -9, and -10 in response to cytochrome c. Cell-free reactions were assembled containing the indicated [35S]methionine-labeled caspases and were incubated with or without 50 μg/ml cytochrome c and 1 mM dATP, as indicated. Reactions were incubated at 37°C and samples were removed at the indicated times, followed by analysis of caspase processing by SDS-PAGE/fluorography. Cleavage products are denoted by arrows. To allow direct comparison, all reactions were set up in an identical manner. Results are representative of a minimum of three independent experiments.

Mentions: To explore the range of cytochrome c–inducible caspase activation events in more detail, we monitored the kinetics of activation of all caspases relative to each other in this system. Fig. 5 shows that detectable activation of most caspases, with the exceptions of caspases-8 and -10, appeared to occur contemporaneously, typically within 30 min of addition of cytochrome c to the extracts. In contrast, processing of caspases-8 and -10 were noticeably delayed relative to the other caspases, suggesting that these caspases might be activated late in this pathway.


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)

Kinetics of activation of caspases-2, -3, -6, -7, -8, -9, and  -10 in response to cytochrome c. Cell-free reactions were assembled containing the indicated [35S]methionine-labeled caspases  and were incubated with or without 50 μg/ml cytochrome c and  1 mM dATP, as indicated. Reactions were incubated at 37°C and  samples were removed at the indicated times, followed by analysis of caspase processing by SDS-PAGE/fluorography. Cleavage  products are denoted by arrows. To allow direct comparison, all  reactions were set up in an identical manner. Results are representative of a minimum of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132895&req=5

Figure 5: Kinetics of activation of caspases-2, -3, -6, -7, -8, -9, and -10 in response to cytochrome c. Cell-free reactions were assembled containing the indicated [35S]methionine-labeled caspases and were incubated with or without 50 μg/ml cytochrome c and 1 mM dATP, as indicated. Reactions were incubated at 37°C and samples were removed at the indicated times, followed by analysis of caspase processing by SDS-PAGE/fluorography. Cleavage products are denoted by arrows. To allow direct comparison, all reactions were set up in an identical manner. Results are representative of a minimum of three independent experiments.
Mentions: To explore the range of cytochrome c–inducible caspase activation events in more detail, we monitored the kinetics of activation of all caspases relative to each other in this system. Fig. 5 shows that detectable activation of most caspases, with the exceptions of caspases-8 and -10, appeared to occur contemporaneously, typically within 30 min of addition of cytochrome c to the extracts. In contrast, processing of caspases-8 and -10 were noticeably delayed relative to the other caspases, suggesting that these caspases might be activated late in this pathway.

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.

Show MeSH