title: Caspase-3-mediated cleavage of Rad9 during apoptosis.
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.
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000502:physiology; D006801:Humans; D009154:Mutation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D020298:U937 Cells; D051020:bcl-X Protein
chemical_list: C494811:BCL2L1 protein, human; D018797:Cell Cycle Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; C069930:rad9 protein; C505407:CASP3 protein, human; C505514:CASP6 protein, human; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases