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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-3 from  Jurkat extracts abolishes cytochrome  c–initiated processing of caspases-2, -6,  -8, and -10 and reveals a feedback amplification loop involving caspase-9.  (A) Jurkat extracts were depleted of  caspase-3 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. (B) Caspase-9 processing is impaired in the absence of  caspase-3. (Top) Jurkat extracts were  depleted of caspase-3, or were mock  depleted using a control antibody, and  incubated under similar conditions to  A. (Bottom) Cell extracts were prepared from Jurkat or MCF-7 cells and  were compared for their ability to process caspase-9 to its p37 and p35 forms  in the presence of cytochrome c/dATP.  Caspase-9 was detected by Western blot. (C) Schematic representation of the sites at which caspase-9 and -3 are likely to cleave the  caspase-9 pro-form to produce the p35 and p37 processed forms of caspase-9. (D) Verification of caspase-3 depletion from Jurkat extracts. Equal amounts of mock-depleted or caspase-3–depleted extract (top) or antibody-coupled protein A/G agarose beads used in the  immunodepletions (bottom) were loaded, followed by probing for caspase-3.
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Figure 9: Depletion of caspase-3 from Jurkat extracts abolishes cytochrome c–initiated processing of caspases-2, -6, -8, and -10 and reveals a feedback amplification loop involving caspase-9. (A) Jurkat extracts were depleted of caspase-3 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. (B) Caspase-9 processing is impaired in the absence of caspase-3. (Top) Jurkat extracts were depleted of caspase-3, or were mock depleted using a control antibody, and incubated under similar conditions to A. (Bottom) Cell extracts were prepared from Jurkat or MCF-7 cells and were compared for their ability to process caspase-9 to its p37 and p35 forms in the presence of cytochrome c/dATP. Caspase-9 was detected by Western blot. (C) Schematic representation of the sites at which caspase-9 and -3 are likely to cleave the caspase-9 pro-form to produce the p35 and p37 processed forms of caspase-9. (D) Verification of caspase-3 depletion from Jurkat extracts. Equal amounts of mock-depleted or caspase-3–depleted extract (top) or antibody-coupled protein A/G agarose beads used in the immunodepletions (bottom) were loaded, followed by probing for caspase-3.

Mentions: To confirm that caspase-3 was activated upstream of caspases-2, -6, -8, and -10 in response to cytochrome c and to ask whether caspase-3 was required for processing of any of the other caspases in this context, we immunodepleted caspase-3 from cell extracts (Fig. 9). Caspase-3–depleted extracts were compared with mock-depleted extracts for their ability to support cytochrome c–induced processing of caspases-2, -3, -6, -7, -8, -9, and -10. Strikingly, removal of caspase-3 from the extracts abrogated processing of caspases-2, -6, -8, and -10 but had only a marginal effect on the processing of caspase-7 in the presence of cytochrome c (Fig. 9 A). These data suggest that caspases-3 and -7 are directly processed downstream of caspase-9 in the cytochrome c/Apaf-1–inducible caspase cascade and that caspase-3 is necessary for the activation (either directly or indirectly) of caspases-2, -6, -8, and -10.


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-3 from  Jurkat extracts abolishes cytochrome  c–initiated processing of caspases-2, -6,  -8, and -10 and reveals a feedback amplification loop involving caspase-9.  (A) Jurkat extracts were depleted of  caspase-3 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. (B) Caspase-9 processing is impaired in the absence of  caspase-3. (Top) Jurkat extracts were  depleted of caspase-3, or were mock  depleted using a control antibody, and  incubated under similar conditions to  A. (Bottom) Cell extracts were prepared from Jurkat or MCF-7 cells and  were compared for their ability to process caspase-9 to its p37 and p35 forms  in the presence of cytochrome c/dATP.  Caspase-9 was detected by Western blot. (C) Schematic representation of the sites at which caspase-9 and -3 are likely to cleave the  caspase-9 pro-form to produce the p35 and p37 processed forms of caspase-9. (D) Verification of caspase-3 depletion from Jurkat extracts. Equal amounts of mock-depleted or caspase-3–depleted extract (top) or antibody-coupled protein A/G agarose beads used in the  immunodepletions (bottom) were loaded, followed by probing for caspase-3.
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Figure 9: Depletion of caspase-3 from Jurkat extracts abolishes cytochrome c–initiated processing of caspases-2, -6, -8, and -10 and reveals a feedback amplification loop involving caspase-9. (A) Jurkat extracts were depleted of caspase-3 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. (B) Caspase-9 processing is impaired in the absence of caspase-3. (Top) Jurkat extracts were depleted of caspase-3, or were mock depleted using a control antibody, and incubated under similar conditions to A. (Bottom) Cell extracts were prepared from Jurkat or MCF-7 cells and were compared for their ability to process caspase-9 to its p37 and p35 forms in the presence of cytochrome c/dATP. Caspase-9 was detected by Western blot. (C) Schematic representation of the sites at which caspase-9 and -3 are likely to cleave the caspase-9 pro-form to produce the p35 and p37 processed forms of caspase-9. (D) Verification of caspase-3 depletion from Jurkat extracts. Equal amounts of mock-depleted or caspase-3–depleted extract (top) or antibody-coupled protein A/G agarose beads used in the immunodepletions (bottom) were loaded, followed by probing for caspase-3.
Mentions: To confirm that caspase-3 was activated upstream of caspases-2, -6, -8, and -10 in response to cytochrome c and to ask whether caspase-3 was required for processing of any of the other caspases in this context, we immunodepleted caspase-3 from cell extracts (Fig. 9). Caspase-3–depleted extracts were compared with mock-depleted extracts for their ability to support cytochrome c–induced processing of caspases-2, -3, -6, -7, -8, -9, and -10. Strikingly, removal of caspase-3 from the extracts abrogated processing of caspases-2, -6, -8, and -10 but had only a marginal effect on the processing of caspase-7 in the presence of cytochrome c (Fig. 9 A). These data suggest that caspases-3 and -7 are directly processed downstream of caspase-9 in the cytochrome c/Apaf-1–inducible caspase cascade and that caspase-3 is necessary for the activation (either directly or indirectly) of caspases-2, -6, -8, and -10.

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
Related in: MedlinePlus