The Cancer Institute, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao 266061, China.
Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen DK-2200, Denmark.
J Mol Cell Biol. 2023 Apr 6;14(11). doi: 10.1093/jmcb/mjac062.
The Bub1 and BubR1 kinetochore proteins support proper chromosome segregation and mitotic checkpoint activity. Bub1 and BubR1 are paralogs with Bub1 being a kinase, while BubR1 localizes the PP2A-B56 protein phosphatase to kinetochores in humans. Whether this spatial separation of kinase and phosphatase activity is important is unclear as some organisms integrate both activities into one Bub protein. Here, we engineer human Bub1 and BubR1 proteins integrating kinase and phosphatase activities into one protein and show that these do not support normal mitotic progression. A Bub1-PP2A-B56 complex can support chromosome alignment but results in impairment of the checkpoint due to dephosphorylation of the Mad1 binding site in Bub1. Furthermore, a chimeric BubR1 protein containing the Bub1 kinase domain induces delocalized H2ApT120 phosphorylation, resulting in the reduction of centromeric hSgo2 and chromosome segregation errors. Collectively, these results argue that the spatial separation of kinase and phosphatase activities within the Bub complex is required for balancing its functions in the checkpoint and chromosome alignment.
Bub1 和 BubR1 着丝粒蛋白支持染色体正确分离和有丝分裂检查点活性。Bub1 和 BubR1 是平行基因，Bub1 是一种激酶，而 BubR1 将 PP2A-B56 蛋白磷酸酶定位于人类的着丝粒。激酶和磷酸酶活性的这种空间分离是否重要尚不清楚，因为一些生物将这两种活性整合到一个 Bub 蛋白中。在这里，我们设计了将激酶和磷酸酶活性整合到一个蛋白中的人 Bub1 和 BubR1 蛋白，并表明这些蛋白不能支持正常的有丝分裂进程。Bub1-PP2A-B56 复合物可以支持染色体排列，但由于 Bub1 中 Mad1 结合位点的去磷酸化，会导致检查点受损。此外，含有 Bub1 激酶结构域的嵌合 BubR1 蛋白诱导 H2ApT120 的去定位磷酸化，导致着丝粒 hSgo2 的减少和染色体分离错误。总之，这些结果表明，Bub 复合物中激酶和磷酸酶活性的空间分离对于平衡其在检查点和染色体排列中的功能是必需的。
