PP2A‐B56 binds to Apc1 and promotes Cdc20 association with the APC/C ubiquitin ligase in mitosis

Abstract

Cell cycle progression and genome stability are regulated by a ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C). Cyclin-dependent kinase 1 (Cdk1) has long been implicated in APC/ C activation; however, the molecular mechanisms of governing this process in vivo are largely unknown. Recently, a Cdk1-dependent phosphorylation relay within Apc3-Apc1 subunits has been shown to alleviate Apc1-mediated auto-inhibition by which a mitotic APC/ C co-activator Cdc20 binds to and activates the APC/C. However, the underlying mechanism for dephosphorylation of Cdc20 and APC/C remains elusive. Here, we show that a disordered loop domain of Apc1 (Apc1-loop 500 ) directly binds the B56 regulatory subunit of protein phosphatase 2A (PP2A) and stimulates Cdc20 loading to the APC/C. Using the APC/C reconstitution system in Xenopus egg extracts, we demonstrate that mutations in Apc1loop 500 that abolish B56 binding decrease Cdc20 loading and APC/ C-dependent ubiquitylation. Conversely, a non-phosphorylatable mutant Cdc20 can efficiently bind the APC/C even when PP2A-B56 binding is impeded. Furthermore, PP2A-B56 preferentially dephosphorylates Cdc20 over the Apc1 inhibitory domain. These results indicate that Apc1-loop 500 plays a role in dephosphorylating Cdc20, promoting APC/C-Cdc20 complex formation in mitosis.

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

“…In different experimental systems PP2A-B55, PP2A-B56 and PP1 have all been reported to act on and regulate components of the APC/C and spindle checkpoint pathways. (Fujimitsu and Yamano, 2020;Hein et al, 2017;Kim et al, 2017). Like PP1, PP2A-B55 is inhibited by CDK1-cyclin B1 and has also been suggested to be an important CDC20 phosphatase in mammalian cells (Hein et al, 2017;Mochida and Hunt, 2012).…”

“…Unlike PP1 and PP2A-B55, PP2A-B56 is thought to retain full activity in mitosis, and CDK1-dependent phosphorylation of PP2A-B56 recognition sites may in fact increase its activity towards key substrates (Kruse et al, 2013;Smith et al, 2019). Interestingly, a specific pool of PP2A-B56, attached directly to the APC/C, has been implicated in vertebrate CDC20 dephosphorylation during mitosis (Fujimitsu and Yamano, 2020;Lee et al, 2017). Due to the unique properties of PP2A-B56, we propose that this may promote the dynamic turnover of CDC20 phosphorylation during early mitosis, to create a limited level of APC/C activity prior to anaphase ( Figure 9).…”

“…It has been suggested that the preference of the PP2A phosphatase for phosphorylated threonine compared to serine may explain differential dephosphorylation kinetics in mammalian cells. For CDC20 a number of the important phosphorylated regulatory sites are on threonine residues, whereas those on the APC/C and CDH1 are serine (Fujimitsu and Yamano, 2020;Hein et al, 2017).…”

Cyclin B3 activates the Anaphase-Promoting Complex/Cyclosome in meiosis and mitosis

“…The interdependence between CycB3 and Tws that we uncovered may reflect a role of PP2A-Tws in the recruitment of Cdc20 co-activators to the APC/C. Cdc20 must be dephosphorylated at CDK sites before binding the APC/C, and in human cells both PP2A-B55 and PP2A-B56 promote this event (Fujimitsu and Yamano, 2020;Hein et al, 2017;Labit et al, 2012).…”

“…Cdc20 binding to the APC/C is also inhibited by its phosphorylation at CDK sites (Alfieri et al, 2017). Phosphatase activity is then required to dephosphorylate Cdc20 and allow its binding of the APC/C for its activation of anaphase (Fujimitsu and Yamano, 2020;Hein et al, 2017;Labit et al, 2012). In addition, phosphorylation of the APC/C itself is required to allow Cdc20 binding (Kramer et al, 2000).…”