2651 Research Article Introduction Programmed cell death or apoptosis is an evolutionarily conserved process that is critically important in embryonic development, tissue homeostasis, in lymphocyte development and immune response. Apoptotic signalling is initiated either through extrinsic (e.g. through death receptors) or intrinsic (mitochondria-dependent) stimulation, resulting in the activation of caspases, the central players within the apoptotic program (Boatright and Salvesen, 2003; Wallach et al., 1999). Dysregulation of apoptosis has been implicated in cancer, neurodegenerative and autoimmune disorders, immunodeficiency and viral infections (Fadeel and Orrenius, 2005). The nuclear factor kappa B (NFκB)/Rel family of transcription factors are important regulators of cell death, survival and proliferation (Karin and Greten, 2005; Karin and Lin, 2002). Various stimuli that include treatment with pro-inflammatory cytokines such as tumor necrosis factor (TNF)α, or phorbol myristyl acetate (PMA), T cell receptor (TCR) crosslinking using either antigen presentation or anti-CD3 antibody treatment, are known to activate NFκB through signal-induced phosphorylation and degradation of the endogenous inhibitors of NFκB, the IκBs, thereby facilitating NFκB translocation to the nucleus and activation of specific gene expression programs (Karin and Lin, 2002). NFκB induces the transcription of anti-apoptotic genes including the cellular inhibitors of apoptosis (IAPs), XIAP, FLIP as well as Bcl2 family members A1, Bcl-xL and Bcl-2, hence dampening caspase activation. In addition to NFκB, the Jun-N-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases respond rapidly to environmental stress, pro-inflammatory cytokines and TCR ligation, and promote both cell survival and cell death pathways (Davis, 2000; Sabapathy et al., 2001). The mechanisms of proapoptotic signalling by JNK have not yet been clearly established; however, they are known to promote mitochondria dysfunction by increasing the production of reactive oxygen species (ROS) (Ventura et al., 2004). In addition, they are known to activate and regulate both pro- and anti-apoptotic Bcl2 protein family members. Importantly, NFκB inhibits both ROS production and JNK activity through activation of specific target genes including ferritin heavy chain, Mn 2+ superoxide dismutase, Gadd45β and XIAP (Bubici et al., 2006), resulting in a protective effect towards apoptotic noxae. The inhibitor of apoptosis (IAP) family is characterized by the presence of at least one BIR (baculovirus IAP repeat) domain that has been identified in IAP1, IAP2, NAIP, survivin and XIAP, and accounts for their ability to inhibit apoptosis (Liston et al., 2003). XIAP is known as the most potent inhibitor of the executor caspase 3 and in addition plays a role in various signalling pathways. For instance, XIAP acts as a cofactor in transforming growth factor-β (TGFβ) signalling (Yamaguchi et al., 1999) through its interaction with TGF-beta-activated kinase 1-binding protein 1 (TAB1; also known as MAP3K7IP1) and activates TGFβ-activated kinase 1 (TAK1), leading to activation of NFκB and JNK signalling pathways (Birkey Reffey et al., 2001; Sanna et al., 2002). Ectopically expressed XIAP is known to enhance phosphorylation of AKT and activate both JNK and NFκB pathways (Asselin et al., 2001; Hofer-Warbinek et al., 2000; Lewis et al., 2004). The C-terminal RING domain of XIAP has E3-ubiquitin-ligase activity, which has been shown to XIAP is known as a potent inhibitor of apoptosis, but in addition is involved in cellular signalling, including the NFκB, JNK and TGFβ pathways. Our search for XIAP-interacting partners led us to Siva1, a proapoptotic protein that is known to play a role in T-cell apoptosis through a caspase-dependent mitochondrial pathway. The interaction sites between XIAP and Siva1 were mapped to the RING domain of XIAP and the N-terminal, SAH- containing and death-homology-region-containing domains of Siva1. Co-immunoprecipitation experiments showed that XIAP, Siva1 and TAK1 form a ternary complex in Jurkat T cells. Reporter-gene analysis revealed that Siva1 inhibits XIAP- and TAK1-TAB1-mediated NFκB activation. By contrast, Siva1 increased XIAP- and TNFα-mediated AP1 activity and prolonged TNFα-induced JNK activation, whereas knock down of Siva1 resulted in reduced JNK activation. This suggests that Siva1 differentially modulates signalling by JNK and NFκB and shifts the balance between these pathways towards enhanced JNK activation, a situation that promotes apoptosis. Ectopically expressed Siva1 increased caspase-3 activity, which was inhibited by XIAP in a ubiquitin-ligase-dependent manner. In line with this, Siva1 was lysine-48-linked polyubiquitylated by XIAP. Our findings suggest that, via physical interaction with XIAP and TAK1, Siva1 diminishes NFκB and enhances JNK activity to favour apoptosis. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/122/15/2651/DC1 Key words: Siva1, XIAP, TAK1, JNK, Apoptosis Summary Siva1 is a XIAP-interacting protein that balances NFκB and JNK signalling to promote apoptosis Ulrike Resch 1, *, Yvonne M. Schichl 1 , Gabriele Winsauer 1 , Radhika Gudi 2 , Kanteti Prasad 2 and Rainer de Martin 1 1 Department of Vascular Biology and Thrombosis Research, Medical University of Vienna, Lazarettg. 19, A-1090, Vienna, Austria 2 Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612, USA *Author for correspondence (e-mail: Ulrike.resch@meduniwien.ac.at) Accepted 22 April 2009 Journal of Cell Science 122, 2651-2661 Published by The Company of Biologists 2009 doi:10.1242/jcs.049940 Journal of Cell Science