Regulation of caspase-9 activity by differential binding to the apoptosome complex.
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.
Saikumar
Pothana
P
Department of Pathology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA. saikumar@uthscsa.edu
Mikhailova
Margarita
M
Pandeswara
Sri Lakshmi
SL
D000818:Animals; D053478:Apoptosomes / Q000378:metabolism*; D053453:Caspase 9 / Q000302:isolation & purification / Q000378:metabolism*; D061945:Caspase Inhibitors; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D011499:Protein Processing, Post-Translational; D051381:Rats
D053478:Apoptosomes; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D053453:Caspase 9