Inhibitor of Apoptosis Proteins (IAPs) are commonly over-expressed in cancers, suppressing apoptosis through diverse mechanisms. Conversely, certain endogenous inhibitors of IAPs, particularly the gene encoding ARTS (Septin4), appear to operate as tumor suppressors whose expression becomes lost in certain malignancies. IAP family members XIAP, c-IAP1, and c-IAP2 are E3 ligases, catalyzing formation of polyubiquitin chains on substrates involving either lysine 48 (K48) or K63 linkages, where K48 targets proteins for proteasome- mediated degradation while K63 is a post-translational modification associated with formation of signaling complexes. The biological consequences of partnering of IAPs with K48- versus K63-specific UBCs are profound, having important implications for understanding mechanisms of apoptosis resistance of cancers. For example, c-IAP1 and c-IAP2 associate with Tumor Necrosis Factor (TNF) Receptor complexes to promote K63-linked ubiquitinylation of Rip1, a post-translational modification that suppresses Rip1-mediated cytotoxicity and that stimulates NF-?B activation, thereby aiding cell survival, cell proliferation and cell migration. This proposal addresses the mechanisms that control the IAPs and their roles in apoptosis suppression and signal transduction in cancer. The Central hypothesis to be tested is that mechanisms controlling the diverse E3 ligase activities of IAPs play a critical role in regulating both the levels of these proteins and their cellular activities. Specific Aims include: (1) Determining how chemical antagonists of IAPs (based on mimicking SMAC) stimulate their self-directed K48-linked polyubiquitination and proteasome-dependent degradation; (2) Exploring the influence of IAP interactions with the kinase Rip1 on their K63-directed E3 ligase activity and testing the hypothesis that Rip1 binding is key to conversion from K48 to K63-specific ligase activity; (3) Addressing the mechanisms by which c-IAP1 and c-IAP2 operate antagonistically with Deubiquitinase CYLD to modulate K63 ubiquitination of Rip1, thereby dictating the assembly of TNFR1 signaling complexes that either promote or suppress TNF?-induced apoptosis; and (4) Investigating the mechanisms by which ARTS, an endogenous antagonist of XIAP, exerts its tumor suppressor effect. Altogether, the proposed studies will reveal the biochemical and cellular mechanisms underlying the multi-functional nature of IAPs as regulators of cell death and signal transduction, suggesting novel strategies for therapeutic applications to cancer treatment.