Limits...
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

Related in: MedlinePlus

Cytochrome c initiates processing and activation of  caspase-3 but not caspase-1 in Jurkat cell extracts. (A) Processing  of caspase-3 (top) and caspase-1 (bottom) in Jurkat cell–free extracts incubated for the indicated times with or without cytochrome  c (50 μg/ml). Caspase processing was assessed by Western blot. (B)  Cell-free reactions were set up as in A except that [35S]methionine-labeled caspase-3, prepared by coupled in vitro transcription/translation, was added to the extracts. At the indicated times, portions of  the extract were removed and were separated by SDS-PAGE followed by direct autoradiography. (C) Schematic representation of  cytochrome c–initiated caspase-3 processing. The pattern of processing was deduced from the data shown in A and B. (D) At the  indicated time points, extracts were assessed for DEVD-pNA or  YVAD-pNA hydrolyzing activity, as described in Materials and  Methods. Cell-free reactions were set up as described above and  were incubated in the presence (closed circles) or absence (open  circles) of cytochrome c (50 μg/ml). At the indicated times, samples  of extract were removed and were assessed for their ability to  hydrolyze the peptides DEVD-pNA and YVAD-pNA. Results  shown are representative of three separate experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132895&req=5

Figure 2: Cytochrome c initiates processing and activation of caspase-3 but not caspase-1 in Jurkat cell extracts. (A) Processing of caspase-3 (top) and caspase-1 (bottom) in Jurkat cell–free extracts incubated for the indicated times with or without cytochrome c (50 μg/ml). Caspase processing was assessed by Western blot. (B) Cell-free reactions were set up as in A except that [35S]methionine-labeled caspase-3, prepared by coupled in vitro transcription/translation, was added to the extracts. At the indicated times, portions of the extract were removed and were separated by SDS-PAGE followed by direct autoradiography. (C) Schematic representation of cytochrome c–initiated caspase-3 processing. The pattern of processing was deduced from the data shown in A and B. (D) At the indicated time points, extracts were assessed for DEVD-pNA or YVAD-pNA hydrolyzing activity, as described in Materials and Methods. Cell-free reactions were set up as described above and were incubated in the presence (closed circles) or absence (open circles) of cytochrome c (50 μg/ml). At the indicated times, samples of extract were removed and were assessed for their ability to hydrolyze the peptides DEVD-pNA and YVAD-pNA. Results shown are representative of three separate experiments.

Mentions: Previous studies have shown that caspases-3 and -9 are activated in response to cytochrome c (Liu et al., 1996; Li et al., 1997; Kluck et al., 1997b; Zou et al., 1997; Pan et al., 1998a). We initially confirmed these observations before assessing the activation of other caspases in this context. Fig. 2 demonstrates that caspase-3 endogenous to Jurkat cell extracts was rapidly converted from the 36-kD proenzyme to the p17/p12 mature form in the presence of cytochrome c. Processing occurred in a two-step manner, with the initial appearance of a p24/p12 intermediate in the extracts, followed by accumulation of the mature p17/ p12 form of the enzyme (Fig. 2, A and C), reminiscent of the mechanism of activation of caspase-3 in response to granzyme B (Martin et al., 1996). This was further confirmed by addition of [35S]methionine-labeled caspase-3 to the extracts, which enabled detection of the caspase-3-p12 chain that was not recognized by the anti–caspase-3 polyclonal antibody used (Fig. 2 B). In direct contrast, conversion of caspase-1 (ICE) to its mature form was not detected in the same extracts over an identical time course (Fig. 2 A). To further confirm that processed caspases were active, we used synthetic tetrapeptide substrates that are preferentially cleaved by caspase-1–like (YVAD-pNA) or caspase-3–like (DEVD-pNA) proteases to assess the induction of caspase-1– or caspase-3–like proteolytic activity in response to cytochrome c. We observed a striking induction of DEVD-pNA cleaving activity within 15 min of addition of cytochrome c to the extracts, whereas YVAD-pNA cleaving activity did not rise above basal levels during the same time course (Fig. 2 D), in agreement with our observations on the absence of caspase-1 processing in this context (Fig. 2 A).


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 initiates processing and activation of  caspase-3 but not caspase-1 in Jurkat cell extracts. (A) Processing  of caspase-3 (top) and caspase-1 (bottom) in Jurkat cell–free extracts incubated for the indicated times with or without cytochrome  c (50 μg/ml). Caspase processing was assessed by Western blot. (B)  Cell-free reactions were set up as in A except that [35S]methionine-labeled caspase-3, prepared by coupled in vitro transcription/translation, was added to the extracts. At the indicated times, portions of  the extract were removed and were separated by SDS-PAGE followed by direct autoradiography. (C) Schematic representation of  cytochrome c–initiated caspase-3 processing. The pattern of processing was deduced from the data shown in A and B. (D) At the  indicated time points, extracts were assessed for DEVD-pNA or  YVAD-pNA hydrolyzing activity, as described in Materials and  Methods. Cell-free reactions were set up as described above and  were incubated in the presence (closed circles) or absence (open  circles) of cytochrome c (50 μg/ml). At the indicated times, samples  of extract were removed and were assessed for their ability to  hydrolyze the peptides DEVD-pNA and YVAD-pNA. Results  shown are representative of three separate experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Cytochrome c initiates processing and activation of caspase-3 but not caspase-1 in Jurkat cell extracts. (A) Processing of caspase-3 (top) and caspase-1 (bottom) in Jurkat cell–free extracts incubated for the indicated times with or without cytochrome c (50 μg/ml). Caspase processing was assessed by Western blot. (B) Cell-free reactions were set up as in A except that [35S]methionine-labeled caspase-3, prepared by coupled in vitro transcription/translation, was added to the extracts. At the indicated times, portions of the extract were removed and were separated by SDS-PAGE followed by direct autoradiography. (C) Schematic representation of cytochrome c–initiated caspase-3 processing. The pattern of processing was deduced from the data shown in A and B. (D) At the indicated time points, extracts were assessed for DEVD-pNA or YVAD-pNA hydrolyzing activity, as described in Materials and Methods. Cell-free reactions were set up as described above and were incubated in the presence (closed circles) or absence (open circles) of cytochrome c (50 μg/ml). At the indicated times, samples of extract were removed and were assessed for their ability to hydrolyze the peptides DEVD-pNA and YVAD-pNA. Results shown are representative of three separate experiments.
Mentions: Previous studies have shown that caspases-3 and -9 are activated in response to cytochrome c (Liu et al., 1996; Li et al., 1997; Kluck et al., 1997b; Zou et al., 1997; Pan et al., 1998a). We initially confirmed these observations before assessing the activation of other caspases in this context. Fig. 2 demonstrates that caspase-3 endogenous to Jurkat cell extracts was rapidly converted from the 36-kD proenzyme to the p17/p12 mature form in the presence of cytochrome c. Processing occurred in a two-step manner, with the initial appearance of a p24/p12 intermediate in the extracts, followed by accumulation of the mature p17/ p12 form of the enzyme (Fig. 2, A and C), reminiscent of the mechanism of activation of caspase-3 in response to granzyme B (Martin et al., 1996). This was further confirmed by addition of [35S]methionine-labeled caspase-3 to the extracts, which enabled detection of the caspase-3-p12 chain that was not recognized by the anti–caspase-3 polyclonal antibody used (Fig. 2 B). In direct contrast, conversion of caspase-1 (ICE) to its mature form was not detected in the same extracts over an identical time course (Fig. 2 A). To further confirm that processed caspases were active, we used synthetic tetrapeptide substrates that are preferentially cleaved by caspase-1–like (YVAD-pNA) or caspase-3–like (DEVD-pNA) proteases to assess the induction of caspase-1– or caspase-3–like proteolytic activity in response to cytochrome c. We observed a striking induction of DEVD-pNA cleaving activity within 15 min of addition of cytochrome c to the extracts, whereas YVAD-pNA cleaving activity did not rise above basal levels during the same time course (Fig. 2 D), in agreement with our observations on the absence of caspase-1 processing in this context (Fig. 2 A).

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