Taylor S S, Hussein D, Wang Y, Elderkin S, Morrow C J
School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK.
J Cell Sci. 2001 Dec;114(Pt 24):4385-95. doi: 10.1242/jcs.114.24.4385.
BUB1 is a budding yeast gene required to ensure that progression through mitosis is coupled to correct spindle assembly. Two related human protein kinases, Bub1 and BubR1, both localise to kinetochores during mitosis, suggesting that they play a role in delaying anaphase until all chromosomes achieve correct, bipolar attachment to the spindle. However, how the activities of Bub1 and BubR1 are regulated by spindle events and how their activities regulate downstream cell cycle events is not known. To investigate how spindle events regulate Bub1 and BubR1, we characterised their relative localisations during mitosis in the presence and absence of microtubule toxins. In prometaphase cells, both kinases colocalise to the same domain of the kinetochore. However, whereas the localisation of BubR1 at sister kinetochores is symmetrical, localisation of Bub1 is often asymmetrical. This asymmetry is dependent on microtubule attachment, and the kinetochore exhibiting weaker Bub1 staining is typically closer to the nearest spindle pole. In addition, a 30 minute nocodazole treatment dramatically increases the amount of Bub1 localising to kinetochores but has little effect on BubR1. Furthermore, Bub1 levels increase at metaphase kinetochores following loss of tension caused by taxol treatment. Thus, these observations suggest that Bub1 localisation is sensitive to changes in both tension and microtubule attachment. Consistent with this, we also show that Bub1 is rapidly phosphorylated following brief treatments with nocodazole or taxol. In contrast, BubR1 is phosphorylated in the absence of microtubule toxins, and spindle damage has little additional effect. Although these observations indicate that Bub1 and BubR1 respond differently to spindle dynamics, they are part of a common complex during mitosis. We suggest therefore that Bub1 and BubR1 may integrate different 'spindle assembly signals' into a single signal which can then be interpreted by downstream cell cycle regulators.
BUB1是一种出芽酵母基因，对于确保有丝分裂进程与正确的纺锤体组装相偶联是必需的。两种相关的人类蛋白激酶Bub1和BubR1在有丝分裂期间均定位于动粒，这表明它们在延迟后期直至所有染色体实现与纺锤体正确的双极附着方面发挥作用。然而，Bub1和BubR1的活性如何受纺锤体事件调控以及它们的活性如何调控下游细胞周期事件尚不清楚。为了研究纺锤体事件如何调控Bub1和BubR1，我们在有和没有微管毒素的情况下，对它们在有丝分裂期间的相对定位进行了表征。在前中期细胞中，两种激酶共定位于动粒的同一结构域。然而，虽然BubR1在姐妹动粒上的定位是对称的，但Bub1的定位通常是不对称的。这种不对称性取决于微管附着，并且显示较弱Bub1染色的动粒通常更靠近最近的纺锤体极。此外，30分钟的诺考达唑处理显著增加了定位于动粒的Bub1的量，但对BubR1影响很小。此外，紫杉醇处理导致张力丧失后，中期动粒处的Bub1水平升高。因此，这些观察结果表明Bub1的定位对张力和微管附着的变化均敏感。与此一致，我们还表明，用诺考达唑或紫杉醇短暂处理后，Bub1会迅速磷酸化。相比之下，BubR1在没有微管毒素的情况下被磷酸化，纺锤体损伤几乎没有额外影响。虽然这些观察结果表明Bub1和BubR1对纺锤体动力学的反应不同，但它们在有丝分裂期间是一个共同复合物的一部分。因此，我们认为Bub1和BubR1可能将不同的“纺锤体组装信号”整合为一个单一信号，然后该信号可被下游细胞周期调节因子解读。
