Caspase-mediated specific cleavage of BubR1 is a determinant of mitotic progression
Caspase-mediated specific cleavage of BubR1 is a determinant of mitotic progression
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DOI：
10.1128/mcb.25.21.9232-9248.2005
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发表时间：
2005-11-01
影响因子：
5.3
通讯作者：
Kao, GD
中科院分区：
文献类型：
--
作者：
Kim, M;Murphy, K;Kao, GD
染色体复制的保真度是由有丝分裂期间操作的细胞周期检查点监测的。一种这样的细胞周期延迟是由微管靶向药物如诺可唑或紫杉醇（Taxol）引起的，并由包括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久性，我们知之甚少。我们在这里报道，用纺锤体破坏剂处理HeLa细胞导致半胱天冬酶激活并促进BubR1的裂解。这一机制最终导致全长蛋白水平的降低，并伴随着有丝分裂阻滞的消除；抑制从头蛋白合成可大大加速检查点的废除。相反，抑制caspase活性可阻断BubR1降解并延长有丝分裂。为了证实caspase活性与BubR1蛋白表达之间的直接联系，我们通过位点定向诱变确定了暴露于紫杉醇后caspase切割的特定位点。令人惊讶的是，BubR1有两个切割位点：主要在Asp607/Asp610，其次在Asp576/Asp579。BubR1在两个位点突变（BubR1 Delta 579 Delta 610）对紫杉醇诱导的降解具有抗性。BubR1 Delta 579 Delta 610的表达增加了转染细胞以及暴露于强力霉素诱导表达突变蛋白的克隆中纺锤体断裂引起的有丝分裂延迟，并最终导致非整倍性增加。这两种四肽基序在人类、小鼠、鸡和非洲爪蟾（Xenopus BubR1）的氨基酸序列中都被鉴定出来，强调了这些半胱天冬酶裂解位点的重要性。这些结果可能是第一个将一种关键有丝分裂检查点蛋白的稳定性控制与半胱天甲酶激活联系起来的研究，半胱天甲酶激活是一种可能参与杀死缺陷细胞的调节途径，并且在进化上是保守的。
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 (BubR1 Delta 579 Delta 610) was resistant to paclitaxel-induced degradation. Expression of BubR1 Delta 579 Delta 610 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.