Regulation of caspase-9 activity by differential binding to the apoptosome complex

Abstract

Proteolytic processing of procaspase-9 is required for its activation, but processing in itself appears to be insufficient for its activity. We studied caspase activation in a cell-free system and found that incubation of cytosol from rat kidney proximal tubule cells with Cytochrome c (Cyt c) and dATP results in rapid autocatalytic processing of procaspase-9 from ~50 kD to ~38 kD size fragment. Moreover, Cyt c concentration influences the production of alternatively processed forms of caspase-9. At lower Cyt c concentration (0.01-0.05 mg/ml), two fragments of caspase-9 of the size 38 and 40 kD are produced. In contrast, at higher concentrations of Cyt c (>0.1 mg/ml) only 38 kD fragment will prevail. However, our failure to capture processed caspase-9 by affinity labeling or co-elution with Apaf-1 suggested that caspase-9 undergoes a conformational change during its enzymatic action on effector caspases, resulting in its release from the apoptosome complex and inactivation. In support of this hypothesis, catalytic inhibitors of caspase-9 prevented its release from the apoptosome complex without affecting its auto-processing and allowed successful capture of active caspase-9 (38 kD) and its complex by affinity labeling. These observations suggest that complex allosteric interactions with the apoptosome complex influence caspase-9 activity and function by controlling not only the induction of its enzymatic activity, but also its rapid termination.

Figure 1

Proteolytic processing of procaspase-9. A, Cytosolic extract (10mg/ml) from RPTC was incubated with dATP (0.4 mM) or Cyt c (0.2 mg/ml) or both for 30 min at 30°C and were assayed for caspase processing activity by measuring DEVDase activity and immunoblotting as described in Materials and Methods. B, Control (lines 1 and 2) and caspase-9 immunodepleted (lines 3 and 4) cytosolic extracts from RPTC were concentrated and activated in presence of dATP and Cyt c for 30 min at 30°C, then proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-caspase-9, -2, -7 and -3 antibodies. C, Cytosolic extract was incubated at 30°C in presence of dATP and Cyt c for 2, 5, 10, 15, 30 min, the proteins were separated by SDS-PAGE and analyzed by immunoblotting with a monoclonal anti-caspase-9 (left panels) and a polyclonal anti-caspase-3 (right panels) antibody; * indicates a non-specific band detected by polyclonal anti-caspase-3 antibody. Presence of a caspase inhibitors z-VAD or z-LEHD during activation did not prevent but only slowed caspase-9 processing (middle and bottom left panels). In contrast, z-VAD completely blocked caspase-3 processing (bottom right panel).

Figure 2

Effect of Cytochrome c on DEVDase activity and Caspase-9 processing. Cytosolic extracts (10mg/ml) from RPTC and HK-2 were incubated with dATP (0.4 mM) and varying concentrations of Cyt c at 30°C for 30 min and were assayed for caspase processing activity by measuring DEVDase activity (A), an average of 4 different experiments and immunoblotting for Caspase-9 (B), as described in Materials and Methods.

Figure 3

Capture of active caspase-9. Cytosolic extract from RPTC was activated, as described in Figure 1, without (lines 2, 5 and 8) or with bio-VAD (lines 3, 6 and 9). If no bio-VAD was added during activation it was added later and incubated for 30 min at RT. Biotinylated proteins were captured on strep-MP, eluted, separated by SDS-PAGE and immunoblotted with monoclonal anti-caspase-9 (top panel) or polyclonal anti-caspase-3 (bottom panel) antibodies as described in materials and methods. Lines 1, 4 and 7 correspond to untreated extract. Nonspecific band detected by anti-caspase-3 antibody is indicated with an asterisk (*).

Figure 4

Inhibition of caspase-9 activity blocks its dissociation from the apoptosome complex. RPTC cytosolic extract was incubated at 30°C with dATP (panel II) or Cyt c (panel III) or both for 2 min (panels IV, VI and VII) or 30 min (panel V) and then loaded on to Superose-6 FPLC column. Panel I corresponds to untreated extract. To demonstrate that inhibition of caspase-9 activity prevents its dissociation from the high molecular weight complex, z-LEHD (panel VI), z-VAD (panel VII) or z-DQMD (panel VIII) were included during activation. Equal volumes of the column fractions after gel filtration chromatography were separated by SDS-PAGE and immunoblotted with anti-Apaf-1 and anti-caspase-9 antibodies. Nonspecific bands are indicated with an asterisk (*). The peak eluted fractions of protein standards on Superose-6 FPLC column are indicated at the bottom.

Figure 5

Effect of various caspase Inhibitors on caspase processing A, Cytosolic extracts (10mg/ml) from RPTC were incubated with dATP (0.4 mM) and Cyt c in the presence of various caspase inhibitors at 10 microM concentration at 30°C for 30 min and were assayed for caspase-9 and -3 processing by immunoblotting as described in Materials and Methods. B, Cytosolic extract from RPTC was activated, as described in Figure 1, activated without (lanes 3 and 4), with bio-VAD (lane 5) or with bio-DEVD (lane 6). If no bio-VAD or bio-DEVD was added during activation either one was added later as indicated with an asterisk and incubated for 30 min at RT. Biotinylated proteins were captured on strep-MP, eluted, separated by SDS-PAGE and immunoblotted with monoclonal anticaspase-9 (top panel) or monoclonal anti-caspase-3 (bottom panel) antibodies as described in materials and methods. Lanes 1 and 2 correspond to untreated extract.

Figure 6

Affinity capture of caspase-9 and Apaf-1 complexes Elution profiles of cytosolic extracts from control (not shown) and activated without (panels I and IV), with bio-VAD (panels II and V) or with bio-DEVD (panels III and VI) subjected to gel filtration chromatography. Equal volumes of the column fractions were separated by SDS-PAGE and immunoblotted with anti-Apaf-1, anti-caspase-9 and anti-caspase-3 antibodies. Proteins from the same column fractions were captured on strep-MP (panels IV and VI), eluted, separated by SDS-PAGE and immunoblotted with anti -Apaf-1 (polyclonal), -caspase-9 (monoclonal) and -caspase-3 (monoclonal) antibodies. If no bio-VAD was included during initial incubation, 0.25mM bio-VAD was added to gel filtration fractions before capturing. The peak eluted fractions of protein standards on Superose-6 FPLC column are indicated at the bottom.

Figure 7

A hypothetical model for caspase-9 activation and inactivation Apaf-1 undergoes conformational change by binding to Cytochrome c into the cytosol and forms heptameric apoptosome complex and recruits procaspase-9 to this complex in the presence of dATP. Upon recruitment to this complex procaspase-9 undergoes conformational change and auto-catalytic processing. In this conformation processed caspase-9 (Casp-9) has stronger affinity to Apaf-1 and is enzymatically active to process effector caspases like procaspase-3. During catalytic action, Caspase-9 undergoes further conformational change and dissociates from the apoptosome complex resulting in its inactivation. Inhibition of catalytic activity prevents the release of caspase-9 from apoptosome complex.