Abstract
BackgroundA 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.ResultsData 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.ConclusionsThe 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.
Highlights
A functioning ubiquitin proteasome system (UPS) is essential for a number of diverse cellular processes and maintenance of overall cellular homeostasis
Altering this balance in favor of apoptosis resistance is a common feature in cancer cells [1], and overcoming cell death barriers is a primary goal of many chemotherapeutic treatments used in the clinic today
A ubiquitin ligase screen identifies Siah2 and POSH as regulators of caspase-8 activity We previously demonstrated the ability of proteasome inhibition to sensitize cancer cells to death receptormediated apoptosis both in vitro and in vivo [10,11]
Summary
A functioning ubiquitin proteasome system (UPS) is essential for a number of diverse cellular processes and maintenance of overall cellular homeostasis. Tight regulation of apoptosis is essential for maintaining tissue homeostasis. The ubiquitin proteasome system is the major cellular pathway for regulated protein turnover. Ubiquitination and protein degradation allows the cell to respond quickly to extra- or intracellular signals, thereby maintaining cellular homeostasis. The addition of multiple ubiquitin moieties to a targeted protein “tags” the protein for degradation by the 26S proteasome. The ubiquitin activating enzyme (E1) catalyzes the formation of a C-terminal thiol ester in an ATP dependent reaction. The ubiquitin ligase enzymes (E3) catalyze the transfer of ubiquitin from the E2 to a lysine residue on the targeted protein via an isopeptide linkage. A polyubiquitinated protein is recognized by the 19S regulatory subunit of the 26S proteasome and degraded by the 20S core particle
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