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Caspase-3-mediated cleavage of Rad9 during apoptosis

Oncogene volume 22, pages 6340–6346 (2003)Cite this article

Abstract

The activation of caspases is a critical event for the execution phase of programmed cell death. Caspases are highly specific in their ability to activate or inhibit many crucial proteins in the cell via cleavage. In this study, we report the identification of several caspase-3-like cleavage sites in the cell-cycle checkpoint protein Rad9. We demonstrate that human Rad9 can be specifically cleaved in cells induced to enter apoptosis by both DNA damage and staurosporine treatment. Indeed, we show that human Rad9 can be effectively cleaved both in vitro and in vivo, which can be inhibited by either a pan-caspase inhibitor or a caspase-3-specific inhibitor. Additionally, no cleavage of Rad9 can be seen in the caspase-3-deficient cell line MCF-7. Site-directed mutagenesis of three of the most conserved cleavage sites dramatically abrogates cleavage of Rad9 by caspase-3 in vitro, and in intact cells after DNA damage. Expression of the cleavage-resistant mutant Rad9 DDD/AAA appears to protect the cell from DNA damage-induced apoptosis. Immunofluorescence studies of Rad9 localization before and after induction of apoptosis show a translocation of Rad9 from the nucleus to the cytosol, concomitant to the appearance of apoptotic morphology. Furthermore, analysis of a truncated Rad9 mutant that corresponds to a putative N-terminal cleavage fragment shows that the N-terminal portion of Rad9 localizes in the cytosol, binds to Bcl-XL, and induces apoptosis. These results support a dual role for cleavage of Rad9: (1) the liberation and translocation of the BH3 domain-containing N-terminus of Rad9 to the cytosol, as a means of promoting apoptosis via antagonism of Bcl-XL, and (2) the disruption of the Rad9-Rad1-Hus1 DNA damage checkpoint complex.

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Acknowledgements

We thank Dr Guy S Salvesen (The Burnham Institute) for kindly providing caspase-3 and -6 enzymes, and Dr Hirohito Yamaguchi for helpful discussion. This work was supported by grants CA82197 and CA90315 from the National Institutes of Health. Michael W Lee is a fellow of the American Heart Association.

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Authors and Affiliations

  1. Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, USA

    Michael W Lee, Itaru Hirai & Hong-Gang Wang

  2. Department of Pharmacology & Therapeutics, University of South Florida College of Medicine, 12902 Magnolia Drive, Tampa, 33612, FL, USA

    Michael W Lee & Hong-Gang Wang

  3. Department of Interdisciplinary Oncology, University of South Florida College of Medicine, 12902 Magnolia Drive, Tampa, 33612, FL, USA

    Itaru Hirai & Hong-Gang Wang

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  1. Michael W Lee
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Correspondence to Hong-Gang Wang.

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Lee, M., Hirai, I. & Wang, HG. Caspase-3-mediated cleavage of Rad9 during apoptosis. Oncogene 22, 6340–6346 (2003). https://doi.org/10.1038/sj.onc.1206729

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