Apoptosis Induced by Drosophila Reaper and Grim in a Human System
ATTENUATION BY INHIBITOR OF APOPTOSIS PROTEINS (cIAPs)*
Abstract
Previous genetic studies have established Reaper and Grim as central regulators of apoptosis in Drosophila melanogaster. Reaper and Grim induce extensive apoptosis inDrosophila, yet share no homology to known vertebrate proteins. In this study, we show for the first time that ectopic expression of Reaper or Grim induced substantial apoptosis in mammalian cells. Reaper- or Grim-induced apoptosis was inhibited by a broad range of caspase inhibitors and by human inhibitor of apoptosis proteins cIAP1 and cIAP2. Additionally, in vivo binding studies demonstrated that both Reaper and Grim physically interacted with human IAPs through a homologous 15-amino acid N-terminal segment. Deletion of this segment from either Reaper or Grim abolished binding to cIAPs.In vitro binding experiments indicated that Reaper and Grim bound specifically to the BIR domain-containing region of cIAPs as deletion of this region resulted in loss of binding. The physical interaction was further confirmed by immunolocalization. When co-expressed, Reaper or Grim co-localized with cIAP1. However, deletion of the N-terminal 15 amino acids of Reaper or Grim abolished co-localization with cIAP1, suggesting that this homologous region can serve as a protein-protein interacting domain in regulating cell death. Moreover, by virtue of this interaction, we demonstrate that cIAPs can regulate Reaper and Grim by abrogating their ability to activate caspases and thereby inhibit apoptosis. This is the first function attributed to this 15-amino acid N-terminal domain that is the only region having significant homology between these Drosophiladeath inducers.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. of Molecular Oncology, Genentech Inc., 1 DNA Way, Bldg. 10, Room 290, South San Francisco, CA 94080. Tel.: 650-225-1312; Fax: 650-225-6443; E-mail:dixit{at}gene.com.
- Abbreviations:
- IAP
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inhibitor of apoptosis proteins
- rpr
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reaper
- SMA
-
spinal muscular atrophy
- TNFR2
-
tumor necrosis factor receptor type 2
- BIR
-
baculovirus IAP repeat
- X-gal
-
5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside
- DAPI
-
4,6-diamidino-2-phenylindole
- GST
-
glutathione S- transferase
- PBS
-
phosphate-buffered saline
- mAb
-
monoclonal antibody
- FADD
-
Fas/APO-1-associated death domain protein.
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- Received June 18, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
