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

Abstract

APC/C-mediated proteolysis of cyclin B and securin promotes anaphase entry, inactivating CDK1 and permitting chromosome segregation, respectively. Reduction of CDK1 activity relieves inhibition of the CDK1-counteracting phosphatases PP1 and PP2A-B55, allowing wide-spread dephosphorylation of substrates. Meanwhile, continued APC/C activity promotes proteolysis of other mitotic regulators. 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 proteomics, we compare the relative extent of proteolysis and protein dephosphorylation. This reveals highly-selective rapid proteolysis of cyclin B, securin and geminin at the metaphase-anaphase transition, followed by slow proteolysis of other substrates. Dephosphorylation requires APC/C-dependent destruction of cyclin B and was resolved into PP1-dependent categories with unique sequence motifs. We conclude that dephosphorylation initiated by selective proteolysis of cyclin B drives the bulk of changes observed during mitotic exit.

Selective dephosphorylation by PP2A-B55 directs the meiosis I-meiosis II transition in oocytes

“…For example, TP sites with a small non-polar (A or G) or basic (K or R) amino acid were significantly more dephosphorylated than TP with acidic amino acids (E or D) or proline (Figure 4E, figure 4-figure supplement 1A). Importantly, we note that the sites preferentially dephosphorylated at the MI / MII transition (Cluster 3, TP followed by basic or nonpolar amino acids) match the sequence preferences of the phosphatase PP2A-B55 that we and others have recently reported (Cundell et al, 2016;Holder et al, 2020;Kruse et al, 2020;McCloy et al, 2015;Touati et al, 2019). These observations raise the possibility that PP2A-B55 drives the striking difference in behavior between TP and SP sites at the MI/MII transition.…”

Novel CRK-Cyclin Complex Controls Spindle Assembly Checkpoint in Toxoplasma Endodyogeny

“…As predicted of a SAC arrest, the lack of mitotic cyclin TgCyc1 significantly changed global protein expression and intensity of phosphorylation ( Fig. 6A and B and Table S3 and S4 ; 20 to 50% of global tachyzoite proteome) ( 34 , 35 ). Comparative analysis of asynchronous (−auxin) and metaphase arrested populations (+auxin, 4 h) showed that more proteins had elevated rather than reduced expression and phosphomodification.…”

“…Spindle assembly checkpoint provides fidelity of the genetic inheritance, ensuring a proper attachment of the chromosomes to the spindle fibers during metaphase ( 32 , 33 ). In studied model eukaryotes, phosphorylation-directed proteolysis coordinates key SAC events ( 34 , 35 ). Upon checkpoint satisfaction, mitotic CDK1/cyclin B complex activates large ubiquitinating machinery, the anaphase promoting complex or cyclosome (APC/C), that releases the protease separase from its inhibitory complex with securin.…”

Dephosphorylation in nuclear reassembly after mitosis

“…Frontiers in Cell and Developmental Biology frontiersin.org phosphothreonine residues over phosphoserine residues (Hein et al, 2017;Touati et al, 2018). PP2A-B55 also favors substrates containing basic amino-acid residues near its dephosphorylation site, seemingly because of acidic residues on the B55 substrate binding site (Xu et al, 2008;Cundell et al, 2016;Touati et al, 2018;Holder et al, 2020). Thus, substrates whose target site contains a phosphothreonine and adjacent basic residues tend to be dephosphorylated first by PP2A-B55 during mitotic exit.…”

“…Phosphoproteomic studies have profiled changes in global protein phosphorylation as cells exit mitosis. Studies with HeLa cells found hundreds of sites that are dephosphorylated during mitotic exit, many of them in structural nuclear proteins ( Malik et al, 2009 ; McCloy et al, 2015 ; Holder et al, 2020 ). Fewer sites were found to increase in phosphorylation during mitotic exit.…”