Oncogene (2003) 22, 6340–6346 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Caspase-3-mediated cleavage of Rad9 during apoptosis Michael W Lee1,2, Itaru Hirai1,3 and Hong-Gang Wang*,1,2,3 1Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, USA; 2Department of Pharmacology & Therapeutics, University of South Florida College of Medicine, 12902 Magnolia Drive, Tampa, FL 33612, USA; 3Department of Interdisciplinary Oncology, University of South Florida College of Medicine, 12902 Magnolia Drive, Tampa, FL 33612, USA The activation of caspases is a critical event for the distinct from necrosis.Grossly, apoptotic cells exhibit a execution phase of programmed cell death. Caspases are variety of identifiable characteristics such as membrane highly specific in their ability to activate or inhibit many blebbing, chromatin condensation, and typically, crucial proteins in the cell via cleavage. In this study, we fragmentation into membrane-enclosed vesicles report the identification of several caspase-3-like cleavage (Earnshaw et al., 1999). Biochemical changes include sites in the cell-cycle checkpoint protein Rad9. We the externalization of phosphatidylserine, degradation demonstrate that human Rad9 can be specifically cleaved of chromosomal DNA into high-molecular-weight in cells induced to enter apoptosis by both DNA damage oligonucleosomal fragments, and cleavage of specific and staurosporine treatment. Indeed, we show that human polypeptides such as PARP, ICAD, and antiapoptotic Rad9 can be effectively cleaved both in vitro and in vivo, proteins such as Bcl-2 and Bcl-XL. which can be inhibited by either a pan-caspase inhibitor or Many stimuli that trigger apoptosis without ligating a caspase-3-specific inhibitor. Additionally, no cleavage death receptors cause mitochondria to release the of Rad9 can be seen in the caspase-3-deficient cell line electron transport protein cytochrome c to the cytosol MCF-7. Site-directed mutagenesis of three of the most (Green and Reed, 1998).Apaf-1 binds the released conserved cleavage sites dramatically abrogates cleavage cytochrome c, and then undergoes a nucleotide-depen- of Rad9 by caspase-3 in vitro, and in intact cells after dent conformational change that allows the binding of DNA damage. Expression of the cleavage-resistant procaspase-9.As a consequence of this binding, mutant Rad9 DDD/AAA appears to protect the cell from procaspase-9 is cleaved to active caspase-9, which in DNA damage-induced apoptosis. Immunofluorescence turn leads to activation of the downstream effector studies of Rad9 localization before and after induction caspase-3, -6, and -7.These effector caspases then of apoptosis showa translocation of Rad9 from the directly cause the morphological changes associated nucleus to the cytosol, concomitant to the appearance of with apoptosis by specifically cleaving ‘death substrates’ apoptotic morphology. Furthermore, analysis of a trun- (Earnshaw et al., 1999). Caspases cleave a limited cated Rad9 mutant that corresponds to a putative N- number of cellular proteins, and the process is one of terminal cleavage fragment shows that the N-terminal limited proteolysis (Salvesen and Dixit, 1997).Owing to portion of Rad9 localizes in the cytosol, binds to Bcl-XL, their substrate specificity, caspase-mediated cleavage and induces apoptosis. These results support a dual role can result in activation or inactivation of their protein for cleavage of Rad9: (1) the liberation and translocation substrates, but never degradation (Salvesen and Dixit, of the BH3 domain-containing N-terminus of Rad9 to the 1997). cytosol, as a means of promoting apoptosis via antagon- The Bcl-2 family is comprised of well over a dozen ism of Bcl-XL, and (2) the disruption of the Rad9-Rad1- proteins, which have been classified into three functional Hus1 DNA damage checkpoint complex. groups (Wang and Reed, 1998).Members of the first Oncogene (2003) 22, 6340–6346. doi:10.1038/sj.onc.1206729 group such as Bcl-2 and Bcl-XL contain four conserved Bcl-2 homology (BH) domains (BH1–BH4), and possess Keywords: caspase-3; Rad9; DNA damage; cell-cycle antiapoptotic activity.In contrast, group II consists of checkpoint; apoptosis proapoptotic Bcl-2 family members including Bax and Bak, which have a similar overall structure to group I proteins but lack the N-terminal BH4 domain.Group III consists of a large and diverse collection of Introduction proapoptotic proteins whose only common feature is the presence of the BH3 domain.The Bcl-2 family Apoptosis is an evolutionarily conserved process of cell of proteins appears to control the ‘decision’ step of suicide that is both morphologically and biochemically apoptosis, determining whether certain caspases will or will not become activated (Reed, 1998).Antiapoptotic *Correspondence: H-G Wang, Drug Discovery Program, H.Lee members of the Bcl-2 family tend to prevent activation Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, of these terminal effector proteases by blocking the Tampa FL 33612, USA; E-mail: wanghg@moffitt.usf.edu Received 13 February 2003; revised 17 April 2003; accepted 22 April release of apoptogenic molecules such as cytochrome c 2003 from the mitochondria.Conversely, proapoptotic Caspase-3 cleavage of Rad9 MW Lee et al 6341 Bcl-2 family members facilitate caspase activation by promoting the release of cytochrome c from the mitochondria. In response to DNA damage, which can arise spontaneously from replication errors or from exogen- ous agents, a rapid-surveillance signaling cascade called the DNA damage checkpoint is initiated (Caspari and Carr, 2002).Activation of this cascade results in cell- cycle arrest, which provides time for the damage to be repaired.Deregulation of this checkpoint can lead to genomic instability that is associated with genetic diseases such as cancer (Hartwell and Kastan, 1994). The Rad family of checkpoint proteins have emerged not only as critical mediators of this damage response pathway but also as putative apical sensors in the DNA damage checkpoint.Indeed, three members of the Rad Figure 1 Human Rad9 is degraded after treatment with high-dose IR.U937 cells were treated with various doses of IR and cultured family Rad9, Rad1, and Hus1 have been shown to for 5 h after treatment.Lysate was prepared and subjected to interact with each other in a manner similar to the SDS—PAGE, and transferred to nitrocellulose filters for analysis of proliferating cell nuclear antigen (PCNA) (O’Connell Rad9 expression using a polyclonal anti-Rad9 antibody (top).The et al., 2000). Furthermore, a fourth Rad family member, same blot was then stripped and reprobed with anti-caspase-3 Rad17, bears structural similarity to replication factor C antibody (bottom) (RFC), the clamp loader which places PCNA onto DNA (Tsurimoto, 1999; Venclovas and Thelen, 2000).It et al., 2000b; St Onge et al., 2001), when resolved on is believed that, in times of damage, these four proteins SDS–polyacrylamide gel (SDS–PAGE), Rad9 migrates form a putative DNA damage sensor, which encircles as several distinct species ranging from 42 to 60 kDa damaged DNA and relays the damage signal to down- even in the absence of genotoxic stress.These different stream effectors (Zhou and Elledge, 2000; Wahl and bands are likely the result of phosphorylation, and can Carr, 2001).In addition, it was shown that Rad9 could be seen in both treated and untreated cells (St.Onge interact with Bcl-2 and Bcl-XL via a conserved BH3 et al., 1999, 2001; Volkmer and Karnitz, 1999; Komatsu domain located in the N-terminus of the Rad9 protein et al., 2000b; Chen et al., 2001). As shown in Figure 1, to antagonize the protective function of these proteins, for example, Rad9 appeared as three major species, promoting apoptosis (Komatsu et al., 2000a, b; Yoshida designated Rad9a, Rad9b, and Rad9g, on Western et al., 2002). It has also been demonstrated that Rad9 blots.Interestingly, after treating U937 cells with can be phosphorylated on Tyr-28 within the BH3 incrementally increasing doses of ionizing radiation domain by c-Abl, and that this phosphorylation (IR), we observed that the lesser modified forms (b, g) promotes the interaction of Rad9 with Bcl-XL (Yoshida of Rad9 decreased in a dose-dependent manner et al., 2002). (Figure 1).This decrease does not directly translate into In this study, we demonstrate that Rad9 possess the increases in the highly modified form (Rad9a), several potential caspase-3 cleavage sites and can be suggesting that Rad9, especially the lesser modified cleaved by caspase-3 both in vitro and in vivo.After forms (Rad9b and Rad9g), might undergo proteolysis exposure to DNA-damaging agents or staurosporine after DNA damage.Reprobing the same blot with anti- (STS), Rad9 undergoes proteolysis and cytosolic trans- caspase-3 antibody revealed that the decrease in Rad9 location, which can be blocked by caspase inhibitors. protein level was concomitant to the activation of Mutating the caspase-3 cleavage sites in Rad9 blocks caspase-3 (Figure 1).Upon analysis of the Rad9 protein cleavage in vivo, and also appears to protect the cell sequence, we noticed that human Rad9 contains several from death induced by DNA-damaging agents.Further- potential caspase-3 cleavage sites (Figure 2a).For more, Rad9DC, a mutant Rad9 that lacks the C- example, the sequence DDID (amino acids 301–304) is terminus and thus mimics a potential N-terminal quite similar to the consensus motif DXXD for caspase- cleavage fragment of Rad9, retains the