Small molecule inhibitors reveal novel functions of the APC/C through proteomic and chemical genetic screening

Abstract

The Anaphase Promoting Complex/Cyclosome (APC/C) is a ~1.2 MDa ubiquitin ligase that controls the timing of the eukaryotic cell cycle by targeting key proteins for destruction by the 26S proteasome. During M-phase, the APC/C works with a co-activator protein called Cdc20 to recognize and ubiquitylate substrates. By regulating the timed destruction of cyclin B1 and securin, APC/CCdc20 drives mitotic exit. Prior to sister chromatid alignment at the metaphase plate, APC/CCdc20 activity is restrained by the Mitotic Checkpoint Complex (MCC). This prevents mitotic exit until DNA is poised to be equally distributed between daughter cells, thereby preventing the abnormal distribution of genetic material between daughter cells. The APC/C is also active during G1, where it works in conjunction with Cdh1, a closely related homolog of Cdc20. Some APC/CCdh1 substrates are important for controlling the G1/S-phase transition, whereas others are involved in diverse processes outside of cell cycle control. Our lab has previously reported two small molecule inhibitors of APC/CCdc20 and APC/CCdh1: proTAME and apcin. These inhibitors block APC/C function via distinct molecular mechanisms: proTAME disrupts interactions between Cdc20/Cdh1 and the APC3 subunit of the APC/C, whereas apcin blocks a critical substrate recognition site on Cdc20/Cdh1. When used together in cells, proTAME and apcin robustly stabilize APC/C substrates and consequently cause mitotic arrest. These small molecules have served as valuable tools for probing the biochemical mechanism of the APC/C. In this dissertation, I describe the use of small molecule APC/C inhibitors in investigating the APC/C regulatory landscape on a systems level. In Chapter 2, I describe a chemical proteomic screen that used small molecule APC/C inhibition in M-phase cells to identify new APC/CCdc20 substrates and to investigate the relative timing of APC/CCdc20 substrate degradation during mitosis. Based on this dataset, I identify several novel putative APC/CCdc20 substrates and distinguish substrates that are degraded during prometaphase versus late metaphase. Moreover, this dataset reveals that treating mitotic cells with proTAME causes an accumulation of Cdc20. Further investigation demonstrated that both free and APC/C:MCC-bound Cdc20 levels increase upon proTAME treatment, and that this correlates with an increase in MCC components observed bound to APC/C. Taken together, these data support a model in which proTAME causes MCC accumulation on APC/C, possibly by antagonizing MCC turnover. In Chapter 3, I describe another chemical proteomic screen that was designed to identify APC/CCdh1 substrates in G1 cells. Based on this experiment, I identify and characterize the insulin receptor adaptor IRS2 as a new APC/CCdh1 substrate. Using IRS2 CRISPR knockout cell lines, I show that cells lacking IRS2 display depressed expression of several cell cycle related proteins and have defects in cell cycle timing. Together, these results establish IRS2 as a new component of the cell cycle control system. In Chapter 4, I describe a chemical genetic screen that was conducted to identify gene deletions that sensitize cells to small molecule APC/C inhibitors. Based on this screen, I identify and characterize a synthetic lethal relationship between APC/C inhibition and deletion of the APC/C’s associated ubiquitin conjugating enzymes, Ube2C and Ube2S. I also describe a deubiquitylating enzyme (DUB) whose deletion sensitizes cells to APC/C inhibition. Further investigation revealed that depletion of this DUB causes mitotic timing defects, possibly due to decreased abundance of the MCC component Mad2.