Our findings therefore establish a new mechanism through which a small molecule, by targeting a single site on a dynamic protein interface, can lead to opposite biological effects depending on the regulatory context. Online Methods Cell Culture and Synchronization HeLa, HCT116, hTERT-RPE1, A549 TW-37 and U2OS cells (American Type Culture Collection, Manassas, VA) were cultured in a humidified incubator at 37 C in the presence of 5% CO2. and MCC-dependent APC/C inhibition. Furthermore, we found that apcin cooperates with p31comet to relieve MCC-dependent inhibition of APC/C. Apcin therefore causes either net APC/C inhibition, prolonging mitosis when SAC activity is low, or net APC/C activation, shortening mitosis when SAC activity is high, demonstrating that a small molecule can produce opposing biological effects depending on regulatory context. Introduction The Anaphase Promoting Complex/Cyclosome (APC/C) is a multi-subunit ubiquitin ligase (E3) that catalyzes ubiquitin transfer from associated E2s (Ube2C and Ube2S) to substrates, targeting them for degradation via the 26S proteasome1-3. The APC/C initiates TW-37 anaphase by targeting securin for degradation and triggers mitotic exit by inducing degradation of the Cdk1 activator cyclin B1. APC/C activity in mitosis depends on binding of a co-activator, Cdc20, which recruits substrates and stimulates catalysis. Distinct surfaces on Cdc20 recognize specific sequence motifs in substrates, including the destruction box (D-box), KEN box, and ABBA motif1-3. The D-box receptor (DBR) of Cdc20 binds to the RxxL sequence of the D-box, using an acidic patch to recognize the basic arginine side chain TW-37 and an adjacent hydrophobic pocket to accommodate the leucine side chain1-3. Accurate chromosome segregation requires that APC/C not be activated until all chromosomes have become properly attached to the mitotic spindle. The mitotic checkpoint complex (MCC) is the effector of the spindle assembly checkpoint (SAC), which is triggered by insufficient kinetochore-microtubule attachments that arise during early stages of mitosis. MCC binds and inhibits APC/CCdc20 to ensure sufficient time for proper chromosome attachment prior to anaphase onset4,5. The MCC consists of BubR1, Mad2, Bub3, and Cdc20 itself, and thus the inhibited APC/CCdc20-MCC complex contains of two molecules of Cdc206, designated Cdc20-A (the co-activator) and Cdc20-M (in MCC). The MCC makes multiple contacts with APC/CCdc20 to inhibit its activity7,8, including the binding of D-box sequences in BubR1 to the Cdc20 DBR6,9-11. In addition, KEN-box and ABBA motifs in BubR1 interact with other sites on Cdc20 to efficiently inhibit APC/CCdc20 6,9. The formation of MCC is dynamic and regulated by a network of protein kinases and phosphatases, including the kinase Mps14,5. SAC inactivation and mitotic exit are promoted by disassembly of free MCC, mediated by p31comet and TRIP13, as well as dissociation of MCC from APC/CCdc20, which requires ubiquitination of Cdc20-M5. How these dynamic processes are integrated to determine the overall level of APC/CCdc20 activity in mitosis is not fully understood. Through an unbiased screen in extract, we previously identified two small molecule inhibitors of APC/C : TAME (tosyl-L-arginine methyl ester) and apcin (APC Inhibitor)12. Subsequent studies revealed that these compounds TW-37 also inhibit human APC/C, and work by distinct mechanisms13-15. TAME binds Cdc27 and Apc8, subunits of APC/C, to block Cdc20 binding13,14,16. Apcin binds the leucine pocket of the TW-37 Rabbit Polyclonal to IkappaB-alpha Cdc20 DBR, interfering with association, ubiquitination and proteolysis of D-box-containing substrates15. TAME and apcin synergize to inhibit APC/CCdc20-dependent ubiquitination and proteolysis in mitotic extract and block mitotic exit in human cells15. Mitotic exit can also be inhibited by microtubule-targeting agents (MTAs), which cause defects in microtubule-kinetochore attachment, triggering MCC production, MCC-dependent APC/CCdc20 inhibition and a SAC-induced mitotic arrest. However, cells can prematurely exit from mitosis through a process known as mitotic slippage17-19. The rate of slippage varies across cell lines20 and blocking slippage by inhibiting APC/CCdc20 may potentiate the apoptotic effect of MTA-based cancer therapies 21,22. It has been shown that proTAME, the cell permeable form of TAME13, in combination with MTAs stabilizes cyclin B123, increases apoptosis23,24 and reduces mitotic slippage in cancer cells23. We hypothesized that apcin might also cooperate with MCC to inhibit APC/CCdc20 more robustly, preventing mitotic slippage. However, we found that apcin paradoxically induces mitotic slippage during a SAC-induced mitotic arrest. Using a reconstituted biochemical system and experiments in engineered cells, we provide a mechanism by which apcin causes this paradoxical effect. Results Apcin promotes slippage.