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‐loop500) 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 Apc1‐loop500 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‐loop500 plays a role in dephosphorylating Cdc20, promoting APC/C‐Cdc20 complex formation in mitosis.
Highlights
The cell cycle is a biological process by which genetic information is accurately duplicated and segregated into daughter cells
Cyclin-dependent kinases (Cdks) activity is vital for DNA replication and driving mitosis, while the anaphase-promoting complex/cyclosome (APC/C)-dependent ubiquitylation and proteolysis of several key regulatory proteins at specific times are essential for mitotic progression
Using egg extract of Xenopus laevis and reconstitution of apo-APC/Cs in the MultiBac system, we show here that Apc1-loop500 has a role in phosphatase 2A (PP2A)-B56 recruitment in mitosis, which in turn dephosphorylates Cdc20 and controls its loading for APC/C activation
Summary
The cell cycle is a biological process by which genetic information is accurately duplicated and segregated into daughter cells. Cdk and APC/C mutually regulate each other in mitosis, forming a negative feedback loop by which a periodicity of cell division cycle is ensured; there can be no mitotic APC/C activation without Cdk, and there can be no cyclin destruction and no cell division without APC/C. Cdk1-dependent Apc3-loop phosphorylation acts as a scaffold and recruits a Cdk regulatory subunit p9/Cks protein via its anion binding domain. This engages a ternary complex Cks-Cdk1-cyclin and phosphorylates an auto-inhibitory domain in a distant subunit, Apc, in an intramolecular phosphorylation relay [12]. Using egg extract of Xenopus laevis and reconstitution of apo-APC/Cs in the MultiBac system, we show here that Apc1-loop500 has a role in PP2A-B56 recruitment in mitosis, which in turn dephosphorylates Cdc and controls its loading for APC/C activation. Our work reveals a mechanism explaining how a mitotic co-activator Cdc can bind to and activate the APC/C in anaphase and initiate sister chromatid separation and mitotic exit
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