Kim Mijin, Murphy Katie, Liu Fang, Parker Sharon E, Dowling Melissa L, Baff Wesley, Kao Gary D
Department of Radiation Oncology, University of Pennsylvania School of Medicine, John Morgan Bldg. 180 H, 3620 Hamilton Walk, Philadelphia, PA 19104, USA.
Mol Cell Biol. 2005 Nov;25(21):9232-48. doi: 10.1128/MCB.25.21.9232-9248.2005.
The fidelity of chromosomal duplication is monitored by cell cycle checkpoints operational during mitosis. One such cell cycle delay is invoked by microtubule-targeting agents such as nocodazole or paclitaxel (Taxol) and is mediated by mitotic checkpoint proteins that include BubR1. Relatively little is known about the regulation of expression and stability of BubR1 (or other checkpoint proteins) and how these factors dictate the durability of the cell cycle delay. We report here that treatment of HeLa cells with spindle-disrupting agents resulted in caspase activation and precipitated the cleavage of BubR1. This mechanism ultimately leads to reduced levels of full-length protein, which are accompanied by abrogation of the mitotic block; the checkpoint abrogation is substantially accelerated by inhibition of de novo protein synthesis. In contrast, inhibition of caspase activity blocked BubR1 degradation and prolonged mitosis. To confirm a direct link between caspase activity and BubR1 protein expression, we identified by site-directed mutagenesis the specific caspase cleavage sites cleaved after exposure to paclitaxel. Surprisingly, BubR1 has two sites of cleavage: primarily at Asp607/Asp610 and secondarily at Asp576/Asp579. BubR1 mutated at both locations (BubR1Delta579Delta610) was resistant to paclitaxel-induced degradation. Expression of BubR1Delta579Delta610 augmented the mitotic delay induced by spindle disruption in transfected cells as well as in clones engineered to inducibly express the mutant protein upon exposure to doxycycline and ultimately led to increased aneuploidy. Underscoring the importance of these caspase cleavage sites, both tetrapeptide motifs are identified in the amino acid sequences of human, mouse, chicken, and Xenopus BubR1. These results are potentially the first to link the control of the stability of a key mitotic checkpoint protein to caspase activation, a regulatory pathway that may be involved in killing defective cells and that has been evolutionarily conserved.
染色体复制的保真度由有丝分裂期间运行的细胞周期检查点监控。微管靶向剂如诺考达唑或紫杉醇（泰素）可引发一种这样的细胞周期延迟，且该延迟由包括BubR1在内的有丝分裂检查点蛋白介导。关于BubR1（或其他检查点蛋白）的表达调控和稳定性以及这些因素如何决定细胞周期延迟的持续时间，人们了解得相对较少。我们在此报告，用纺锤体破坏剂处理HeLa细胞会导致半胱天冬酶激活并促使BubR1裂解。这种机制最终导致全长蛋白水平降低，同时伴随着有丝分裂阻滞的消除；通过抑制从头合成蛋白质，检查点的消除会大大加速。相反，抑制半胱天冬酶活性可阻止BubR1降解并延长有丝分裂。为了证实半胱天冬酶活性与BubR1蛋白表达之间的直接联系，我们通过定点诱变确定了紫杉醇处理后被裂解的特定半胱天冬酶切割位点。令人惊讶的是，BubR1有两个切割位点：主要在Asp607/Asp610，其次在Asp576/Asp579。在这两个位置发生突变的BubR1（BubR1Delta579Delta610）对紫杉醇诱导的降解具有抗性。BubR1Delta579Delta610的表达增强了转染细胞以及经工程改造在接触强力霉素时可诱导表达突变蛋白的克隆中由纺锤体破坏诱导的有丝分裂延迟，并最终导致非整倍体增加。强调这些半胱天冬酶切割位点的重要性的是，在人、小鼠、鸡和非洲爪蟾的BubR1氨基酸序列中都鉴定出了这两个四肽基序。这些结果可能首次将关键有丝分裂检查点蛋白稳定性的控制与半胱天冬酶激活联系起来，这是一条可能参与杀死缺陷细胞且在进化上保守的调控途径。
