BUBR1 phosphorylation is regulated during mitotic checkpoint activation.
BUBR1 phosphorylation is regulated during mitotic checkpoint activation.
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发表时间：
1999-11
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通讯作者：
Wenqing Li;Zhengdao Lan;Huiyun Wu;Shechao Wu;J. Meadows;Jie Chen;Veronica Zhu;Wei Dai
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作者：
Wenqing Li;Zhengdao Lan;Huiyun Wu;Shechao Wu;J. Meadows;Jie Chen;Veronica Zhu;Wei Dai
真核细胞已经进化出一种延迟有丝分裂进程的机制，直到浓缩的染色体正确定位在有丝分裂纺锤体上。为了了解人类细胞中这种监测机制的分子基础，我们一直在研究调节有丝分裂检查点的基因。我们的早期研究已经克隆了编码MAD 3样蛋白（也称为BUBR 1/MAD 3/SSK 1）的全长cDNA。斑点杂交分析表明，BUBR 1 mRNA表达的组织有丝分裂指数高，但不分化的组织。Western印迹分析表明，在异步细胞中，BUBR 1蛋白主要表现出120 kDa的分子量，其表达在大多数细胞系检测。此外，BUBR 1存在于细胞周期的各个阶段。随着细胞进入后期S期和G2期，BUBR 1水平显著增加。通过机械振摇获得的诺考达唑阻滞的有丝分裂细胞含有在变性SDS凝胶上具有较慢迁移率的BUBR 1抗原。磷酸酶处理使缓慢迁移的条带恢复到间期状态，表明BUBR 1抗原的缓慢迁移可归因于磷酸化。此外，纯化的重组His 6-BUBR 1能够自磷酸化。我们的研究表明，BUBR 1的磷酸化状态调节纺锤体中断。考虑到其与BUB 1蛋白激酶的高度同源性，BUBR 1也可能通过有丝分裂检查点途径的关键细胞组分的磷酸化在有丝分裂检查点控制中发挥重要作用。
Eukaryotic cells have evolved a mechanism that delays the progression of mitosis until condensed chromosomes are properly positioned on the mitotic spindle. To understand the molecular basis of such monitoring mechanism in human cells, we have been studying genes that regulate the mitotic checkpoint. Our early studies have led to the cloning of a full-length cDNA encoding MAD3-like protein (also termed BUBR1/MAD3/SSK1). Dot blot analyses show that BUBR1 mRNA is expressed in tissues with a high mitotic index but not in differentiated tissues. Western blot analyses show that in asynchronous cells, BUBR1 protein primarily exhibits a molecular mass of 120 kDa, and its expression is detected in most cell lines examined. In addition, BUBR1 is present during various stages of the cell cycle. As cells enter later S and G2, BUBR1 levels are increased significantly. Nocodazole-arrested mitotic cells obtained by mechanical shake-off contain BUBR1 antigen with a slower mobility on denaturing SDS gels. Phosphatase treatment restores the slowly migrating band to the interphase state, indicating that the slow mobility of the BUBR1 antigen is attributable to phosphorylation. Furthermore, purified recombinant His6-BUBR1 is capable of autophosphorylation. Our studies indicate that BUBR1 phosphorylation status is regulated during spindle disruption. Considering its strong homology to BUB1 protein kinase, BUBR1 may also play an important role in mitotic checkpoint control by phosphorylation of a critical cellular component(s) of the mitotic checkpoint pathway.