Dephosphorylation of Cdc20 is required for its C-box-dependent activation of the APC/C

Abstract

The anaphase‐promoting complex/cyclosome (APC/C) ubiquitin ligase is tightly regulated to ensure programmed proteolysis in cells. The activity of the APC/C is positively controlled by cyclin‐dependent kinase (CDK), but a second level of control must also exist because phosphorylation inactivates Cdc20, a mitotic APC/C co‐activator. How Cdc20 is dephosphorylated specifically, when CDK is high, has remained unexplained. Here, we show that phosphatases are crucial to activate the APC/C. Cdc20 is phosphorylated at six conserved residues (S50/T64/T68/T79/S114/S165) by CDK in Xenopus egg extracts. When all the threonine residues are phosphorylated, Cdc20 binding to and activation of the APC/C are inhibited. Their dephosphorylation is regulated depending on the sites and protein phosphatase 2A, active in mitosis, is essential to dephosphorylate the threonine residues and activate the APC/C. Consistently, most of the Cdc20 bound to the APC/C in anaphase evades phosphorylation at T79. Furthermore, we show that the ‘activation domain’ of Cdc20 associates with the Apc6 and Apc8 core subunits. Our data suggest that dephosphorylation of Cdc20 is required for its loading and activation of the APC/C ubiquitin ligase.

Role for regulated phosphatase activity in generating mitotic oscillations in Xenopus cell-free extracts

“…S2). This aspect of the model is consistent with observations [ figure 5E of Labit et al (32)] that removal of CDK-phosphorylation sites from Cdc20 causes little change to the dynamics of MPF oscillations. So the double-negative feedback between MPF and Cdc20, which plays a prominent role in Ciliberto's model (24), does not seem to be as important as we once thought.…”

“…Unlike PP2A:B55δ, the PP2A that dephosphorylates Cdc20 does not show reduced activity in mitosis. Also, the PP2A:B55δ-targeted site (S50) of Cdc20 does not influence APC/C binding (32). These findings provide strong support for Ciliberto's assumption of constant, rapid turnover of Cdc20 phosphorylation; therefore, we keep this feature of the model.…”

“…For the case of Cdc20 dephosphorylation, this assumption has recently received experimental support. Using Xenopus cell-free extracts, Yamano and co-workers (32) showed that phosphorylation of three out of five Cdk1-sites on Cdc20 significantly reduces its binding to APC/C. They also showed that okadaic acid, an inhibitor of type 2A protein phosphatases, blocks APC/C:Cdc20 activity, suggesting that a PP2A dephosphorylates Cdc20.…”

The Biochemistry of Mitosis

“…Cyclin B -Cdk also phosphorylates Cdc20, but here the effect is more complicated. Some Cdk sites may activate Cdc20, whereas others may inhibit Cdc20 (Listovsky et al 2000;Kraft et al 2003;Labit et al 2012); therefore, it is likely that there is an important interplay between cyclin B-Cdk phosphorylation and Cdc20 or APC/C-associated phosphatase(s), which control APC/C activation.…”

The end of mitosis from a phosphatase perspective

“…Cdc20 phosphorylation, as well, has been shown to help sustain the SAC, while Cdc20 dephosphorylation has been recently shown to stimulate interaction with APC/C and ubiquitin ligase activity of the complex. 5,32 In addition, the observation that Fcp1 is required to dephosphorylate Wee1-T-239, to control cdk1 inhibitory phosphorylation, and that this dephosphorylation begins very early during SAC resolution (before significant cyclin degradation), 30 suggests that also in somatic cells, reversal of inhibitory phosphorylation of cdk1 is an integral pathway of the mitosis exit program, much like in meiosis and early embryo. 33,34 Indeed, blocking cdk1 inhibitory phosphorylation during mitosis exit in somatic cells, albeit it does not indefinitely arrests cells in mitosis, affects the Repo-Man:PP1 that is required for rearrangement of chromosome structure at the end of mitosis.…”