Interaction and Regulation of the Caenorhabditis elegans Death Protease CED-3 by CED-4 and CED-9*
- From the Department of Pathology and Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan 48109
Abstract
In the nematode Caenorhabditis elegans, three genes, ced-3, ced-4, andced-9, play critical roles in the induction and execution of the death pathway. Genetic studies have suggested thatced-9 controls programmed cell death by regulatingced-4 and ced-3. However, the mechanism by which CED-9 controls the activities of CED-4 and the cysteine protease CED-3, the effector arm of the cell-death pathway, remains poorly understood. Immunoprecipitation analysis demonstrates that CED-9 forms a multimeric protein complex with CED-4 and CED-3 in vivo. Expression of wild-type CED-4 promotes the ability of CED-3 to induce apoptosis in mammalian cells, which is inhibited by CED-9. The pro-apoptotic activity of CED-4 requires the expression of a functional CED-3 protease. Significantly, loss-of-function CED-4 mutants are impaired in their ability to promote CED-3-mediated apoptosis. Expression of CED-4 enhances the proteolytic activation of CED-3. We also show that CED-9 inhibits the formation of p13 and p15, two cleavage products of CED-3 associated with its proteolytic activationin vivo. Moreover, CED-9 inhibits the enzymatic activity of CED-3 promoted by CED-4. Thus, these results provide evidence that CED-4 and CED-9 regulate the activity of CED-3 through physical interactions, which may provide a molecular basis for the control of programmed cell death in C. elegans.
Footnotes
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↵* This work was supported by Grant CA-64556 from the National Institutes of Health.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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↵‡ Contributed equally to the work.
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↵§ Supported by National Institutes of Health Predoctoral Training Grant T32A107413–03.
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↵¶ Recipient of Research Career Development Award CA-64421 from the National Institutes of Health and to whom correspondence should be addressed: Dept. of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109. Tel.: 313-764-3190; Fax: 313-763-6476; E-mail:Gabriel.Nunez{at}umich.edu.
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↵1 The abbreviations used are: PCD, programmed cell death; Ac-DEVD-AMC, acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin; HA, hemagglutinin; PCR, polymerase chain reaction; wt, wild type; X-gal, 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside; ICE, interleukin-1β-converting enzyme.
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↵2 D. Wu, H. Wallen, N. Inohara, and G. Nuñez, unpublished observations.
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↵3 J. Yuan, personal communication.
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- Received March 13, 1997.
- Revision received May 30, 1997.
