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.

中文翻译：

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PP2A-B56 与 Apc1 结合并促进 Cdc20 与有丝分裂中的 APC/C 泛素连接酶结合。

细胞周期进程和基因组稳定性受泛素连接酶、后期促进复合物/环体 (APC/C) 调节。长期以来，细胞周期蛋白依赖性激酶 1 (Cdk1) 与 APC/C 激活有关。然而，在体内控制这一过程的分子机制在很大程度上是未知的。最近，已显示 Apc3-Apc1 亚基内的 Cdk1 依赖性磷酸化中继可减轻 Apc1 介导的自抑制，有丝分裂 APC/C 共激活因子 Cdc20 通过这种自抑制结合并激活 APC/C。然而，Cdc20 和 APC/C 去磷酸化的潜在机制仍然难以捉摸。在这里，我们展示了 Apc1 (Apc1-loop500) 的无序环结构域直接结合蛋白磷酸酶 2A (PP2A) 的 B56 调节亚基并刺激 Cdc20 加载到 APC/C。在非洲爪蟾卵提取物中使用 APC/C 重组系统，我们证明了消除 B56 结合的 Apc1-loop500 突变降低了 Cdc20 负载和 APC/C 依赖性泛素化。相反，即使 PP2A-B56 结合受到阻碍，非磷酸化突变 Cdc20 也可以有效地结合 APC/C。此外，PP2A-B56 优先于 Apc1 抑制域使 Cdc20 去磷酸化。这些结果表明 Apc1-loop500 在 Cdc20 去磷酸化中发挥作用，促进有丝分裂中 APC/C-Cdc20 复合物的形成。