Barr, F. | bioRxiv Science

bioRxiv2020

PP1 promotes cyclin B destruction and the metaphase-anaphase transition by dephosphorylating CDC20

Ubiquitin-dependent proteolysis of cyclin B and securin initiates sister chromatid segregation and anaphase. The anaphase promoting complex/cyclosome (APC/C) and its co-activator CDC20 form the main ubiquitin E3 ligase for these proteins. APC/CCDC20 is regulated by CDK1-cyclin B and counteracting PP1 and PP2A family phosphatases through modulation of both activating and inhibitory phosphorylations. Here we report that PP1 promotes cyclin B destruction at the onset of anaphase by removing specific inhibitory phosphorylation in the N-terminus of CDC20. Depletion or chemical inhibition of PP1 stabilises cyclin B and results in a pronounced delay at the metaphase-to-anaphase transition after chromosome alignment. This requirement for PP1 is lost in cells expressing CDK1-phosphorylation defective CDC206A mutants. These CDC206A cells show a normal spindle checkpoint response, but once all chromosomes have aligned rapidly degrade cyclin B and enter into anaphase in the absence of PP1 activity. PP1 therefore facilitates the metaphase-to-anaphase by promoting APC/CCDC20-dependent destruction of cyclin B in human cells.

cell biology↗

bioRxiv2020

Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit

APC/C-mediated proteolysis of cyclin B and securin promotes entry into anaphase, inactivating CDK1 and permitting chromosome segregation, respectively. Reduction of CDK1 activity relieves inhibition of the CDK1-opposing phosphatases PP1 and PP2A-B55 leading to dephosphorylation of substrates crucial for mitotic exit. Meanwhile, continued APC/C activity is required to target various proteins, including Aurora and Polo kinases, for degradation. Together, these activities orchestrate a complex series of events during mitotic exit. However, the relative importance of regulated proteolysis and dephosphorylation in dictating the order and timing of these events remains unclear. Using high temporal-resolution mass spectrometry, we compare the relative extent of proteolysis and protein dephosphorylation. This reveals highly-selective rapid ([~]5min half-life) proteolysis of cyclin B, securin and geminin at the metaphase to anaphase transition, followed by slow proteolysis (>60 min half-life) of other mitotic regulators. Protein dephosphorylation requires APC/C-dependent destruction of cyclin B and was resolved into PP1-dependent fast, intermediate and slow categories with unique sequence motifs. We conclude that dephosphorylation initiated by the selective proteolysis of cyclin B drives the bulk of changes observed during mitotic exit.

cell biology↗