Sub-Cellular Localization Determines the Delicate Balance between the Anti and Proapoptotic Activity of Livin.

Inhibitor of Apoptosis proteins (IAP) comprise a family of intracellular proteins that play an essential role in the regulation of apoptosis. IAP family members are defined by one or more repeats of a highly conserved anti-apoptotic domain termed the baculovirus IAP repeat (BIR), located at the amino-terminus. In addition, some IAPs contain a conserved domain termed RING finger at the carboxy-terminus. In a previous study, we identified an IAP family member termed Livin, which contains a single BIR domain and a carobxy-terminal RING finger motif. Livin inhibits apoptosis induced by a variety of stimuli. We further demonstrated that following apoptotic stimuli, Livin is cleaved by effector caspases 3 and 7, to produce a truncated form with paradoxical proapoptotic activity. In our current study, we reveal that while the full-length Livin shows a cytoplasmic distribution, the truncated form is readily detected in the nucleus. Using mutation analysis, we identified two domains outside the BIR sequence that are strongly engaged in determining the subcellular localization as well as the proapoptotic activity of Livin. These two domains are the N-terminus post-cleavage exposed area and the C-terminus RING domain. Remarkably, a single substitution of the first exposed amino acid Glycine, is sufficient to significantly diminish nuclear localization as well as the proapoptotic activity, thus linking the nuclear accumulation of the cleavage fragment with the proapoptotic activity. We demonstrate that the cytoplasmic localization of Livin is mediated through the RING domain, as a mutation that disables Livin from forming the zinc finger results in both a cytoplasmic and nuclear localization. Moreover, RING-mutated Livin subunit also loses its proapoptotic activity. Thus we suggest a model in which the exposed N-terminal region counteracts the cytoplasmic localization signal of the RING domain, promoting nuclear localization of the truncated form. The change in sub-cellular localization allows the RING domain to mediate the proapoptotic effect. To the best of our knowledge, this is the first example of such a regulatory mechanism in IAP family members.