Role of cytochrome c and dATP/ATP hydrolysis in Apaf‐1‐mediated caspase‐9 activation and apoptosis

Role of cytochrome c and dATP/ATP hydrolysis in Apaf‐1‐mediated caspase‐9 activation and... Apaf‐1 plays a critical role in apoptosis by binding to and activating procaspase‐9. We have identified a novel Apaf‐1 cDNA encoding a protein of 1248 amino acids containing an insertion of 11 residues between the CARD and ATPase domains, and another 43 amino acid insertion creating an additional WD‐40 repeat. The product of this Apaf‐1 cDNA activated procaspase‐9 in a cytochrome c and dATP/ATP‐dependent manner. We used this Apaf‐1 to show that Apaf‐1 requires dATP/ATP hydrolysis to interact with cytochrome c, self‐associate and bind to procaspase‐9. A P‐loop mutant (Apaf‐1K160R) was unable to associate with Apaf‐1 or bind to procaspase‐9. Mutation of Met368 to Leu enabled Apaf‐1 to self‐associate and bind procaspase‐9 independent of cytochrome c, though still requiring dATP/ATP for these activities. The Apaf‐1M368L mutant exhibited greater ability to induce apoptosis compared with the wild‐type Apaf‐1. We also show that procaspase‐9 can recruit procaspase‐3 to the Apaf‐1–procaspase‐9 complex. Apaf‐1(1–570), a mutant lacking the WD‐40 repeats, associated with and activated procaspase‐9, but failed to recruit procaspase‐3 and induce apoptosis. These results suggest that the WD‐40 repeats may be involved in procaspase‐9‐mediated procaspase‐3 recruitment. These studies elucidate biochemical steps required for Apaf‐1 to activate procaspase‐9 and induce apoptosis. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Wiley

Role of cytochrome c and dATP/ATP hydrolysis in Apaf‐1‐mediated caspase‐9 activation and apoptosis

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Publisher
Wiley
Copyright
Copyright © 2013 Wiley Periodicals, Inc
ISSN
0261-4189
eISSN
1460-2075
D.O.I.
10.1093/emboj/18.13.3586
Publisher site
See Article on Publisher Site

Abstract

Apaf‐1 plays a critical role in apoptosis by binding to and activating procaspase‐9. We have identified a novel Apaf‐1 cDNA encoding a protein of 1248 amino acids containing an insertion of 11 residues between the CARD and ATPase domains, and another 43 amino acid insertion creating an additional WD‐40 repeat. The product of this Apaf‐1 cDNA activated procaspase‐9 in a cytochrome c and dATP/ATP‐dependent manner. We used this Apaf‐1 to show that Apaf‐1 requires dATP/ATP hydrolysis to interact with cytochrome c, self‐associate and bind to procaspase‐9. A P‐loop mutant (Apaf‐1K160R) was unable to associate with Apaf‐1 or bind to procaspase‐9. Mutation of Met368 to Leu enabled Apaf‐1 to self‐associate and bind procaspase‐9 independent of cytochrome c, though still requiring dATP/ATP for these activities. The Apaf‐1M368L mutant exhibited greater ability to induce apoptosis compared with the wild‐type Apaf‐1. We also show that procaspase‐9 can recruit procaspase‐3 to the Apaf‐1–procaspase‐9 complex. Apaf‐1(1–570), a mutant lacking the WD‐40 repeats, associated with and activated procaspase‐9, but failed to recruit procaspase‐3 and induce apoptosis. These results suggest that the WD‐40 repeats may be involved in procaspase‐9‐mediated procaspase‐3 recruitment. These studies elucidate biochemical steps required for Apaf‐1 to activate procaspase‐9 and induce apoptosis.

Journal

The EMBO JournalWiley

Published: Jan 1, 1999

Keywords: ; ; ;

References

  • Mammalian cell death proteases: a family of highly conserved aspartate specific cysteine proteases
    Alnemri, ES
  • CED‐4 induces chromatin condensation in Schizosaccharomyces pombe and is inhibited by direct physical association with CED‐9
    James, C; Gschmeissner, S; Fraser, A; Evan, GI
  • Evolutionary conservation of a genetic pathway of programmed cell death
    Yuan, J

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