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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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Selective binding of caspase-9 to Apaf-1. Glutathione  Sepharose–immobilized GST-Apaf-11-97 fusion protein (CED-3  homology domain) was assessed for its ability to precipitate the  indicated [35S]methionine-labeled caspases, as described in Materials and Methods. (A) The signal generated by 10% of the total  input amount of each caspase is shown. (B) The total amount of  each caspase precipitated by 6 μg of GST or GST-Apaf-11-97 fusion protein. Identical exposures of each gel are shown to enable  direct comparison.
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Figure 6: Selective binding of caspase-9 to Apaf-1. Glutathione Sepharose–immobilized GST-Apaf-11-97 fusion protein (CED-3 homology domain) was assessed for its ability to precipitate the indicated [35S]methionine-labeled caspases, as described in Materials and Methods. (A) The signal generated by 10% of the total input amount of each caspase is shown. (B) The total amount of each caspase precipitated by 6 μg of GST or GST-Apaf-11-97 fusion protein. Identical exposures of each gel are shown to enable direct comparison.

Mentions: The observation that multiple caspases were activated in response to cytochrome c suggested either that all of these caspase activation events occurred downstream of caspase-9 (the only caspase known to directly associate with Apaf-1/ cytochrome c), or that a number of distinct caspase activation pathways could be instigated by cytochrome c, independent of caspase-9. To discriminate between these possibilities, we first explored whether Apaf-1 could directly bind caspases other than caspase-9. Fig. 6 demonstrates that a GST fusion protein comprising the CED-3 homology region of Apaf-1 (amino acid [aa] residues 1–97) selectively bound caspase-9 but did not bind any of the other caspases (-1, -2, -3, -6, -7, -8, and -10) tested. Similar results were obtained using a different GST fusion that spanned the CED-3 as well as the CED-4 homology regions of Apaf-1 (aa 1–412; Slee, E.A., and S.J. Martin, data not shown). These data demonstrate that Apaf-1 is highly selective for caspase-9, although they do not rule out the possibility that other caspases may become recruited to Apaf-1 via suitable adaptor molecules.


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)

Selective binding of caspase-9 to Apaf-1. Glutathione  Sepharose–immobilized GST-Apaf-11-97 fusion protein (CED-3  homology domain) was assessed for its ability to precipitate the  indicated [35S]methionine-labeled caspases, as described in Materials and Methods. (A) The signal generated by 10% of the total  input amount of each caspase is shown. (B) The total amount of  each caspase precipitated by 6 μg of GST or GST-Apaf-11-97 fusion protein. Identical exposures of each gel are shown to enable  direct comparison.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Selective binding of caspase-9 to Apaf-1. Glutathione Sepharose–immobilized GST-Apaf-11-97 fusion protein (CED-3 homology domain) was assessed for its ability to precipitate the indicated [35S]methionine-labeled caspases, as described in Materials and Methods. (A) The signal generated by 10% of the total input amount of each caspase is shown. (B) The total amount of each caspase precipitated by 6 μg of GST or GST-Apaf-11-97 fusion protein. Identical exposures of each gel are shown to enable direct comparison.
Mentions: The observation that multiple caspases were activated in response to cytochrome c suggested either that all of these caspase activation events occurred downstream of caspase-9 (the only caspase known to directly associate with Apaf-1/ cytochrome c), or that a number of distinct caspase activation pathways could be instigated by cytochrome c, independent of caspase-9. To discriminate between these possibilities, we first explored whether Apaf-1 could directly bind caspases other than caspase-9. Fig. 6 demonstrates that a GST fusion protein comprising the CED-3 homology region of Apaf-1 (amino acid [aa] residues 1–97) selectively bound caspase-9 but did not bind any of the other caspases (-1, -2, -3, -6, -7, -8, and -10) tested. Similar results were obtained using a different GST fusion that spanned the CED-3 as well as the CED-4 homology regions of Apaf-1 (aa 1–412; Slee, E.A., and S.J. Martin, data not shown). These data demonstrate that Apaf-1 is highly selective for caspase-9, although they do not rule out the possibility that other caspases may become recruited to Apaf-1 via suitable adaptor molecules.

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