Unit of Biochemistry, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.
Proc Natl Acad Sci U S A. 2012 May 22;109(21):8056-60. doi: 10.1073/pnas.1204081109. Epub 2012 May 7.
The mitotic checkpoint system delays anaphase until all chromosomes are correctly attached to the mitotic spindle. When the checkpoint is turned on, it promotes the formation of the mitotic checkpoint complex (MCC), which inhibits the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2 bound to the APC/C activator Cdc20. When the checkpoint is satisfied, MCC is disassembled and APC/C becomes active. Previous studies have shown that the Mad2-binding protein p31(comet) promotes the dissociation of Cdc20 from BubR1 in MCC in a process that requires ATP. We now show that a part of MCC dissociation is blocked by inhibitors of cyclin-dependent kinases (Cdks) and that purified Cdk1-cyclin B stimulates this process. The mutation of all eight potential Cdk phosphorylation sites of Cdc20 partially prevented its release from BubR1. Furthermore, p31(comet) stimulated Cdk-catalyzed phosphorylation of Cdc20 in MCC. It is suggested that the binding of p31(comet) to Mad2 in MCC may trigger a conformational change in Cdc20 that facilitates its phosphorylation by Cdk, and that the latter process may promote its dissociation from BubR1.
有丝分裂检查点系统会延迟后期，直到所有染色体都正确连接到有丝分裂纺锤体上。当检查点开启时，它会促进有丝分裂检查点复合物（MCC）的形成，从而抑制泛素连接酶后期促进复合物/环体（APC/C）。MCC 由与 APC/C 激活剂 Cdc20 结合的检查点蛋白 BubR1、Bub3 和 Mad2 组成。当检查点得到满足时，MCC 被分解，APC/C 变得活跃。以前的研究表明，Mad2 结合蛋白 p31（彗星）促进了在需要 ATP 的过程中 Cdc20 从 BubR1 上的解离。我们现在表明，MCC 部分解离被细胞周期蛋白依赖性激酶（Cdks）抑制剂阻断，并且纯化的 Cdk1-细胞周期蛋白 B 可刺激该过程。Cdc20 的所有八个潜在 Cdk 磷酸化位点的突变部分阻止了其从 BubR1 的释放。此外，p31（彗星）在 MCC 中刺激 Cdk 催化的 Cdc20 磷酸化。这表明，MCC 中 p31（彗星）与 Mad2 的结合可能触发 Cdc20 的构象变化，从而促进其被 Cdk 磷酸化，并且后一过程可能促进其与 BubR1 的解离。
