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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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Cytochrome c–initiated activation of multiple  caspases. [35S]Methionine-labeled caspases, prepared  by coupled in vitro transcription/translation (∼0.25–0.5  μl of translation reaction in a total reaction volume of  10 μl), were incubated with cytochrome c (50 μg/ml) in  the presence or absence of Jurkat postnuclear extract,  as indicated. Where no cell extract was added to the reaction, an appropriate volume of extract dilution buffer  was substituted. Cell-free reactions were incubated for  3 h at 37°C and were then analyzed by SDS-PAGE followed by fluorography.
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Figure 4: Cytochrome c–initiated activation of multiple caspases. [35S]Methionine-labeled caspases, prepared by coupled in vitro transcription/translation (∼0.25–0.5 μl of translation reaction in a total reaction volume of 10 μl), were incubated with cytochrome c (50 μg/ml) in the presence or absence of Jurkat postnuclear extract, as indicated. Where no cell extract was added to the reaction, an appropriate volume of extract dilution buffer was substituted. Cell-free reactions were incubated for 3 h at 37°C and were then analyzed by SDS-PAGE followed by fluorography.

Mentions: Recent studies have shown that caspase-9 is activated in response to cytochrome c due to clustering of caspase-9 by Apaf-1 and that this results in activation of Caspases-3 (Li et al., 1997; Pan et al., 1998a; Srinivasula et al., 1998). To explore the full range of caspase activation events in the cytochrome c–initiated proteolytic cascade, we introduced [35S]methionine-labeled caspases-1, -2, -3, -4, -5, -6, -7, -8, -9, and -10 into Jurkat cell–free extracts and monitored processing of these proteases to their mature forms. Fig. 4 demonstrates that cytochrome c/dATP triggered maturation of caspases-2, -3, -6, -7, -8, -9, and -10, whereas none of the ICE subfamily proteases (caspases-1, -4, and -5) were processed under the same conditions. Significantly, cytochrome c failed to activate any of the caspases in the absence of cell extract, suggesting that a cytosolic factor such as Apaf-1 was required for all of these activation events. Caspases with long prodomains such as caspases-2, -8, and -10 are generally considered to be upstream or signaling caspases in the cell death pathway due to their ability to associate with cell surface death receptor molecules such as Fas/CD95 or TNFR1. Therefore, it was somewhat surprising that these caspases became activated in the presence of cytochrome c. However, it has been reported recently that thymocytes from APAF-1 mice are impaired with respect to caspase-2 and caspase-8 activation in response to several proapoptotic stimuli (Yoshida et al., 1998). Similarly, dexamethasone-induced processing of caspases-2 and -8 was found to be impaired in mice deficient for caspase-9 (Hakem et al., 1998). These data suggest that these caspases are indeed activated in the Apaf-1 pathway in vivo.


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)

Cytochrome c–initiated activation of multiple  caspases. [35S]Methionine-labeled caspases, prepared  by coupled in vitro transcription/translation (∼0.25–0.5  μl of translation reaction in a total reaction volume of  10 μl), were incubated with cytochrome c (50 μg/ml) in  the presence or absence of Jurkat postnuclear extract,  as indicated. Where no cell extract was added to the reaction, an appropriate volume of extract dilution buffer  was substituted. Cell-free reactions were incubated for  3 h at 37°C and were then analyzed by SDS-PAGE followed by fluorography.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Cytochrome c–initiated activation of multiple caspases. [35S]Methionine-labeled caspases, prepared by coupled in vitro transcription/translation (∼0.25–0.5 μl of translation reaction in a total reaction volume of 10 μl), were incubated with cytochrome c (50 μg/ml) in the presence or absence of Jurkat postnuclear extract, as indicated. Where no cell extract was added to the reaction, an appropriate volume of extract dilution buffer was substituted. Cell-free reactions were incubated for 3 h at 37°C and were then analyzed by SDS-PAGE followed by fluorography.
Mentions: Recent studies have shown that caspase-9 is activated in response to cytochrome c due to clustering of caspase-9 by Apaf-1 and that this results in activation of Caspases-3 (Li et al., 1997; Pan et al., 1998a; Srinivasula et al., 1998). To explore the full range of caspase activation events in the cytochrome c–initiated proteolytic cascade, we introduced [35S]methionine-labeled caspases-1, -2, -3, -4, -5, -6, -7, -8, -9, and -10 into Jurkat cell–free extracts and monitored processing of these proteases to their mature forms. Fig. 4 demonstrates that cytochrome c/dATP triggered maturation of caspases-2, -3, -6, -7, -8, -9, and -10, whereas none of the ICE subfamily proteases (caspases-1, -4, and -5) were processed under the same conditions. Significantly, cytochrome c failed to activate any of the caspases in the absence of cell extract, suggesting that a cytosolic factor such as Apaf-1 was required for all of these activation events. Caspases with long prodomains such as caspases-2, -8, and -10 are generally considered to be upstream or signaling caspases in the cell death pathway due to their ability to associate with cell surface death receptor molecules such as Fas/CD95 or TNFR1. Therefore, it was somewhat surprising that these caspases became activated in the presence of cytochrome c. However, it has been reported recently that thymocytes from APAF-1 mice are impaired with respect to caspase-2 and caspase-8 activation in response to several proapoptotic stimuli (Yoshida et al., 1998). Similarly, dexamethasone-induced processing of caspases-2 and -8 was found to be impaired in mice deficient for caspase-9 (Hakem et al., 1998). These data suggest that these caspases are indeed activated in the Apaf-1 pathway in vivo.

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