Mps1 Phosphorylation by MAP Kinase Is Required for Kinetochore Localization of Spindle-Checkpoint Proteins
Mps1 Phosphorylation by MAP Kinase Is Required for Kinetochore Localization of Spindle-Checkpoint Proteins
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DOI：
10.1016/j.cub.2006.07.058
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发表时间：
2006-09
期刊：
影响因子：
9.2
通讯作者：
Yong Zhao;Rey-Huei Chen
中科院分区：
文献类型：
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作者：
Yong Zhao;Rey-Huei Chen
纺锤体检查点延迟后期的开始，直到所有染色体都实现与纺锤体微管的双极附着。未附着的动粒通过招募几种纺锤体检查点蛋白来激活纺锤体检查点，包括 Mps1、Mad1、Mad2、Bub1、Bub3 和 BubR1（酵母中的 Mad3）[1]。在脊椎动物细胞中，活性 MAP 激酶 (MAPK) 也在未附着的动粒处富集，并且是纺锤体检查点所必需的 [2-5]。研究表明，Mps1 的激酶活性是纺锤体检查点 [6] 以及 Bub1、Bub3、Mad1 和 Mad2 着丝粒定位所必需的 [6, 7]。我们在此证明 MAPK 磷酸化爪蟾提取物中 S844 处的 Mps1。有趣的是，将S844改为不可磷酸化的丙氨酸（S844A）对Mps1的激酶活性没有影响，尽管它废除了Mps1的检查点功能。生化和免疫荧光研究表明，S844A 突变扰乱了 Mps1 和其他纺锤体检查点蛋白的动粒定位，而模拟磷酸化的 S844D 突变体则恢复了它们的功能。我们的研究表明，MAPK 在 S844 处磷酸化 Mps1 可能会产生一个磷酸表位，使 Mps1 能够与着丝粒相互作用。此外，我们的结果表明，活跃的 Mps1 必须定位到动粒才能执行其检查点功能。
The spindle checkpoint delays anaphase onset until all chromosomes have achieved bipolar attachment to the spindle microtubules. Unattached kinetochores activate the spindle checkpoint by recruiting several spindle-checkpoint proteins, including Mps1, Mad1, Mad2, Bub1, Bub3, and BubR1 (Mad3 in yeast) [1]. In vertebrate cells, active MAP kinase (MAPK) is also enriched at unattached kinetochores and is required for the spindle checkpoint [2–5]. It has been shown that the kinase activity of Mps1 is required for the spindle checkpoint [6] and for kinetochore localization of Bub1, Bub3, Mad1, and Mad2 [6, 7]. We herein demonstrate that MAPK phosphorylates Mps1 at S844 inXenopusegg extracts. Interestingly, changing S844 to unphosphorylatable alanine (S844A) has no effect on the kinase activity of Mps1, although it abolishes the checkpoint function of Mps1. Biochemical and immunofluorescence studies show that S844A mutation perturbs kinetochore localization of Mps1 and other spindle-checkpoint proteins, whereas the phosphorylation-mimicking S844D mutant restores their functions. Our studies suggest that Mps1 phosphorylation by MAPK at S844 might create a phosphoepitope that allows Mps1 to interact with kinetochores. In addition, our results indicate that active Mps1 must localize to kinetochores in order to execute its checkpoint function.