Chen Rey-Huei
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
J Cell Biol. 2002 Aug 5;158(3):487-96. doi: 10.1083/jcb.200204048.
The spindle checkpoint delays anaphase onset until all chromosomes have attached properly to the mitotic spindle. Checkpoint signal is generated at kinetochores that are not bound with spindle microtubules or not under tension. Unattached kinetochores associate with several checkpoint proteins, including BubR1, Bub1, Bub3, Mad1, Mad2, and CENP-E. I herein show that BubR1 is important for the spindle checkpoint in Xenopus egg extracts. The protein accumulates and becomes hyperphosphorylated at unattached kinetochores. Immunodepletion of BubR1 greatly reduces kinetochore binding of Bub1, Bub3, Mad1, Mad2, and CENP-E. Loss of BubR1 also impairs the interaction between Mad2, Bub3, and Cdc20, an anaphase activator. These defects are rescued by wild-type, kinase-dead, or a truncated BubR1 that lacks its kinase domain, indicating that the kinase activity of BubR1 is not essential for the spindle checkpoint in egg extracts. Furthermore, localization and hyperphosphorylation of BubR1 at kinetochores are dependent on Bub1 and Mad1, but not Mad2. This paper demonstrates that BubR1 plays an important role in kinetochore association of other spindle checkpoint proteins and that Mad1 facilitates BubR1 hyperphosphorylation at kinetochores.
纺锤体检查点会延迟后期开始，直到所有染色体都正确附着到有丝分裂纺锤体上。检查点信号在未与纺锤体微管结合或未处于张力下的动粒处产生。未附着的动粒与几种检查点蛋白相关联，包括BubR1、Bub1、Bub3、Mad1、Mad2和CENP-E。我在此表明，BubR1对非洲爪蟾卵提取物中的纺锤体检查点很重要。该蛋白在未附着的动粒处积累并发生超磷酸化。免疫去除BubR1会大大降低Bub1、Bub3、Mad1、Mad2和CENP-E在动粒处的结合。BubR1的缺失也会损害后期激活因子Mad2、Bub3和Cdc20之间的相互作用。野生型、激酶失活型或缺少激酶结构域的截短型BubR1可挽救这些缺陷，这表明BubR1的激酶活性对卵提取物中的纺锤体检查点并非必不可少。此外，BubR1在动粒处的定位和超磷酸化依赖于Bub1和Mad1，而不是Mad2。本文证明BubR1在其他纺锤体检查点蛋白的动粒关联中起重要作用，并且Mad1促进BubR1在动粒处的超磷酸化。
