Possible role of death receptor-mediated apoptosis by the E3 ubiquitin ligases Siah2 and POSH

A functioning ubiquitin proteasome system (UPS) is essential for a number of diverse cellular processes and maintenance of overall cellular homeostasis. The ability of proteasome inhibitors, such as Velcade, to promote extrinsic apoptotic effects illustrates the importance of the ubiquitin proteasome system in the regulation of death receptor signaling. Here, we set out to define the UPS machinery, particularly the E3 ubiquitin ligases, that repress apoptosis through the extrinsic pathway. A cell-based genome-wide E3 ligase siRNA screen was established to monitor caspase-8 activity following the addition of TRAIL.

The data presented here define POSH and Siah2 as important mediators of death receptor mediated apoptosis and suggest targeting the interaction of these two E3 ligases is a promising novel cancer therapeutic strategy.

Data from the high-throughput screen revealed that targeting the RING-finger containing E3 ligase Siah2 as well as the signaling platform molecule POSH (SH3RF1) conferred robust caspase-8 activation in response to TRAIL stimulus. Silencing Siah2 or POSH in prostate cancer cells led to increased caspase activity and apoptosis in response to both TRAIL and Fas ligand. The E3 activity of Siah2 was responsible for mediating apoptosis resistance; while POSH protein levels were critical for maintaining viability. Further characterization of Siah2 revealed it to function downstream of early death receptor events in the apoptotic pathway. The observed apoptosis resistance provides one biological explanation for the induction of Siah2 and POSH reported in lung and prostate cancer, respectively. Expanding on an initial yeast-two-hybrid screen we have confirmed a physical interaction between E3 ligases Siah2 and POSH. Utilizing a yeast-two-hybrid mapping approach we have defined the spacer region of POSH, more specifically the RPxAxVxP motif encompassing amino acids 601-607, to be the site of Siah2 binding. Conclusions: The data presented here define POSH and Siah2 as important mediators of death receptor mediated apoptosis and suggest targeting the interaction of these two E3 ligases is a promising novel cancer therapeutic strategy.