Caspase-dependent processing activates the proapoptotic activity of deleted in breast cancer-1 during tumor necrosis factor-alpha-mediated death signaling Ramya Sundararajan 1 , Guanghua Chen 2 , Chandreyee Mukherjee 2 and Eileen White* ,1,2,3,4,5 1 Rutgers University, 679 Hoes Lane, Room 140, Piscataway, NJ 08854, USA; 2 Howard Hughes Medical Institute, NJ, USA; 3 Center for Advanced Biotechnology and Medicine, Rutgers University, 679 Hoes Lane, Room 140, Piscataway, NJ 08854, USA; 4 Department of Molecular Biology and Biochemistry, Rutgers University, 679 Hoes Lane, Room 140, Piscataway, NJ 08854, USA; 5 Cancer Institute of New Jersey, NJ, USA Deleted in breast cancer-1 (DBC-1) was initially cloned from a homozygously deleted region in breast and other cancers on human chromosome 8p21, although no function is known for the protein product it encodes. We identified the generation of amino-terminally truncated versions of DBC-1 during tumor necrosis factor (TNF)-a-mediated apoptosis. Full-length 150kDa DBC-1 underwent caspase- dependent processing during TNF-a-mediated death signaling, to produce p120 DBC-1 and p66 DBC-1 carboxy-terminal fragments. Endogenous DBC-1 local- ized to the nucleus in healthy cells, but localized to the cytoplasm during TNF-a-mediated apoptosis, consistent with the loss of the amino-terminus containing the nuclear localization signal. Overexpression of an amino-terminal truncated DBC-1, resembling p120 DBC-1, caused mitochondrial clustering, mitochondrial matrix condensa- tion, and sensitized cells to TNF-a-mediated apoptosis. The carboxy-terminal coiled-coil domain of DBC-1 was responsible for the cytoplasmic and mitochondrial locali- zation, and for the death-promoting activity of DBC-1. Thus, caspase-dependent processing of DBC-1 may act as a feed-forward mechanism to promote apoptosis and possibly also tumor suppression. DBC-1, like its homolog cell cycle and apoptosis regulatory protein-1 (CARP-1), may function in the regulation of apoptosis. Oncogene (2005) 24, 4908–4920. doi:10.1038/sj.onc.1208681; published online 11 April 2005 Keywords: apoptosis; TNF-a; mitochondria; DBC-1; caspase; proapoptotic Introduction Apoptosis is an essential physiological process that plays a critical role in development, tissue homeostasis, and defense against pathogens (Adams, 2003; Cuconati and White, 2002; Danial and Korsmeyer, 2004). The dying cell undergoes several biochemical and morphological changes and is eventually engulfed by neighboring cells. Bcl-2 family members are either proapoptotic or antiapoptotic and are key regulators of apoptotic signaling through mitochondria. The mitochondrial intermembrane space is a storehouse of proapoptotic proteins that are released from mitochondria during apoptosis enabling activation of some caspases. Cas- pases, a family of cysteine proteases that are activated by cleavage, are the executioners of apoptosis that implement apoptosis by selective cleavage of cellular substrates that implement the orderly dismantling of the cell. Apoptosis can be triggered by a variety of physio- logical and pathological stimuli such as cytokines, hormones, viruses, and toxic insults, among many others. Tumor necrosis factor-a (TNF-a) is representa- tive of a family of trimeric cytokines such as Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL) (Nagata, 1999). Many cell types produce TNF-a, including macrophages, monocytes, lympho- cytes, keratinocytes, and fibroblasts, in response to inflammation, infection, injury, and other environmen- tal challenges (Baud and Karin, 2001). A broad spectrum of responses is elicited by TNF-a, in- cluding activation and migration of lymphocytes and leukocytes, cell proliferation, differentiation, survival, and apoptosis. Activation of apoptosis by TNF-a in vitro requires coordinate inhibition of survival signaling by addition of protein synthesis inhibitors (Perez and White, 2000) that inhibit the generation of prosurvival factors mediated by NF-kB (Kucharczak et al., 2003). Engagement of TNF-a with tumor necrosis factor-a receptor 1 (TNFR1) results in the recruitment of adaptor proteins that facilitate multi- merization, autocatalytic cleavage, and activation of caspase-8 in a death-inducing signaling complex (DISC) (Igney and Krammer, 2002). In certain cell types, mitochondria amplify death signaling by enabling activation of caspase-9, which then activates other downstream caspases. Although several steps in the activation and regulation of TNF-a-mediated death signaling have been determined, our understanding of the signaling pathway is far from complete. Received 3 January 2005; revised 2 March 2005; accepted 2 March 2005; published online 11 April 2005 *Correspondence: E White, CABM, Rutgers University, 679 Hoes Lane, Room 140, Piscataway, NJ 08854, USA; E-mail: ewhite@cabm.rutgers.edu Oncogene (2005) 24, 4908–4920 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc