Abstract
ABSTRACTThe spindle assembly checkpoint (SAC) inhibits the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores by generating a diffusible inhibitor termed the mitotic checkpoint complex (MCC). At metaphase, rapid activation of the APC/C requires removal of the MCC, a process that has been shown to depend on the APC/C E2 enzymes, UBE2C and UBE2S. Here we investigate the in vivo role of the APC/C E2 enzymes in SAC silencing using CRISPR/Cas9 genetically engineered HCT116 UBE2C or UBE2S null cell lines. Using live cell assays, we show that UBE2C and UBE2S make a minor contribution to SAC silencing in HCT116 cells. Strikingly, in cells specifically lacking UBE2C, we observe a strong synergistic inhibition of mitotic progression when we stabilize the MCC on the APC/C by depleting APC15, potentially reflecting increased competition between the MCC and the remaining initiating E2 enzyme UBE2D. In conclusion, we provide in vivo insight into the APC/C E2 module and its interplay with SAC silencing components.
Highlights
The anaphasepromoting complex/cyclosome (APC/C) is a large ubiquitin ligase regulating mitotic progression by targeting proteins for ubiquitin-mediated destruction (Pines, 2011)
Analysis of spindle assembly checkpoint (SAC) silencing in HCT116 cells lacking specific APC/C E2 enzymes We recently reported the generation of genetically engineered HCT116 cell lines where the UBE2C and UBE2S genes were
The time from nuclear envelope breakdown (NEBD) to anaphase onset is determined by the time it takes to bi-orient all chromosomes, how efficient the APC/C is in ubiquitinating its substrates (APC/C activity) and how efficiently the SAC is silenced
Summary
The APC/C is a large ubiquitin ligase regulating mitotic progression by targeting proteins for ubiquitin-mediated destruction (Pines, 2011). An important substrate of the APC/C is cyclin B1 since the destruction of this protein results in entry into anaphase. The poly-ubiquitination of cyclin B1 depends on APC/C-specific E2 enzymes and in collaboration with the Choudhary lab, we have recently showed that at least three E2 enzymes can work with the APC/C in vivo, namely UBE2C (UBCH10), UBE2S and UBE2D (UBCH5) (Wild et al, 2016). UBE2C and UBE2D work with the APC/C to add the initial ubiquitin molecules to a substrate that is subsequently extended by UBE2S that catalyzes the formation of Lys11-linked chains on substrates (Garnett et al, 2009; Williamson et al, 2009; Wu et al, 2010). UBE2C interacts with the APC11 RING finger and APC2 in
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