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

Pothana Saikumar, Margarita Mikhailova, Srilakshmi Pandeswara

Research output: Contribution to journalArticle

21 Scopus citations

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 coelution 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.

Original languageEnglish (US)
Pages (from-to)3343-3354
Number of pages12
JournalFrontiers in Bioscience
Volume12
Issue number9
DOIs
StatePublished - May 1 2007

Keywords

  • Apaf-1
  • Apoptosis
  • Apoptosome
  • Caspase-3
  • Caspase-9
  • Cytochrome c
  • Inhibitor
  • Research article

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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