Thesis Type:
PhD Thesis
Abstract:
The Anaphase Promoting Complex/Cyclosome (APC/C) is a mega-dalton ubiquitin ligase that regulates chromosome segregation and mitotic exit by targeting destruction box (D-box)-containing substrates for degradation by the proteasome. Because accurate chromosome segregation requires precise timing of APC/C activation, APC/C is subject to highly complex regulation through binding of essential activators and inhibitory protein complexes. The APC/C is activated by Cdc20, which acts as a substrate receptor and is inhibited by the mitotic checkpoint complex (MCC), which delays mitotic exit when the spindle assembly checkpoint (SAC) is activated in response to improper kinetochore-microtubule attachments. We previously identified apcin, a small molecule ligand of the D-box receptor of Cdc20, as an inhibitor of APC/C that competitively inhibits substrate binding in vitro. We show that although apcin inhibits substrate degradation and delays mitotic exit in human cells, high endogenous levels of Cdc20 limit apcin’s effectiveness. Surprisingly, we found that apcin paradoxically accelerates substrate degradation and promotes mitotic exit in cells with high SAC activity. Biochemical studies indicate that apcin cooperates with p31comet to relieve MCC-dependent inhibition of APC/C. Apcin’s behavior as an antagonist of Cdc20 can thus result in either net inhibition of APC/C, delaying mitotic exit when SAC activity is low, or net activation of APC/C, promoting mitotic exit when SAC activity is high. Genetic experiments suggest that the dual behaviors of apcin arise from targeting a common binding site in Cdc20, the D-box receptor, which is required for both substrate ubiquitination as well as efficient APC/C inhibition by MCC. Therefore, we establish a new mechanism through which a small molecule, by targeting a single site on a dynamic protein interface, can lead to opposing biological effects depending on the regulatory context.
