Harris Loleta, Davenport James, Neale Geoffrey, Goorha Rakesh
Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794, USA.
Exp Cell Res. 2005 Aug 1;308(1):85-100. doi: 10.1016/j.yexcr.2005.03.036.
BubR1 is one of two putative vertebrate homologs of the yeast spindle checkpoint protein Bub1. We have used deletion and point mutants to elucidate the functions of BubR1 in mitosis. The nocodazole-activated spindle checkpoint of HeLa cells was disrupted by expression of a 39 amino acid fragment (residues 382-420) of BubR1 containing the Bub3-binding GLEBS motif. In contrast, we observed normal checkpoint function in a truncation mutant comprising residues 1-477, despite the lack of the C-terminal BubR1 kinase domain. In the absence of nocodazole, expression of the 477 amino acid fragment slowed progress through prometaphase of mitosis, causing accumulation of mitotic cells. This accumulation was also seen in a kinase dead mutant. The prolongation of mitosis required both kinetochore binding and an intact, functional spindle checkpoint. The prolongation of mitosis by kinase deficient BubR1 constructs indicates a crucial role for the BubR1 C-terminal kinase domain in chromosome movement, in addition to the role of the N-terminus in the checkpoint.
BubR1是酵母纺锤体检查点蛋白Bub1的两个假定脊椎动物同源物之一。我们使用缺失和点突变体来阐明BubR1在有丝分裂中的功能。含有Bub3结合GLEBS基序的BubR1的39个氨基酸片段（第382 - 420位残基）的表达破坏了HeLa细胞中诺考达唑激活的纺锤体检查点。相比之下，我们在包含第1 - 477位残基的截短突变体中观察到正常的检查点功能，尽管缺乏C末端的BubR1激酶结构域。在没有诺考达唑的情况下，477个氨基酸片段的表达减缓了有丝分裂前中期的进程，导致有丝分裂细胞的积累。在激酶失活突变体中也观察到了这种积累。有丝分裂的延长需要动粒结合以及完整、功能性的纺锤体检查点。激酶缺陷型BubR1构建体导致的有丝分裂延长表明，除了N末端在检查点中的作用外，BubR1 C末端激酶结构域在染色体移动中也起着关键作用。
