Zwint-1 is a novel Aurora B substrate required for the assembly of a dynein-binding platform on kinetochores.
Zwint-1 is a novel Aurora B substrate required for the assembly of a dynein-binding platform on kinetochores.
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DOI：
10.1091/mbc.e11-03-0213
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发表时间：
2011-09
影响因子：
3.3
通讯作者：
Vaughan KT
中科院分区：
文献类型：
--
作者：
Kasuboski JM;Bader JR;Vaughan PS;Tauhata SB;Winding M;Morrissey MA;Joyce MV;Boggess W;Vos L;Chan GK;Hinchcliffe EH;Vaughan KT
这项研究确定zwint-1作为一种新的底物AURB在有丝分裂。磷酸化是前中期外部动粒组装所必需的。然而，zwint-1去磷酸化需要在中期检查点沉默。Aurora B（AurB）是一种有丝分裂激酶，负责有丝分裂进程的多个方面，包括外动粒的组装。细胞质动力蛋白是一种丰富的动粒蛋白，其募集到动粒需要磷酸化。为了评估AurB是否调节动力蛋白向着丝粒的募集，我们使用ZM 447439或激酶死亡的AurB构建体抑制AurB。抑制AurB大大减少了动力蛋白在着丝粒的积累，然而，这反映了动力蛋白相关蛋白的损失，而不是动力蛋白磷酸化的缺陷。我们确定AurB抑制影响ROD、ZW 10、zwilch（RZZ）复合物向动粒的募集，但不影响zwint-1或更近端的动粒蛋白。AurB在体外磷酸化zwint-1而非ZW 10，并且通过串联质谱分析鉴定了三个新的磷酸化位点。三重Ala zwint-1突变体的表达阻断了RZZ依赖性蛋白的动粒组装，并诱导了前中期染色体运动的缺陷。三重Glu zwint-1突变体的表达使细胞在前中期对AurB抑制具有抗性。然而，细胞表达的三重Glu突变体未能满足纺锤体组装检查点（SAC）在中期，因为极流的动力蛋白/dynactin/RZZ被抑制。这些研究确定zwint-1作为一种新的AurB基板所需的动粒组装和适当的SAC沉默在中期。
This study identifies zwint-1 as a novel substrate for AurB during mitosis. Phosphorylation is required for outer kinetochore assembly during prometaphase. However, zwint-1 dephosphorylation is required at metaphase for checkpoint silencing. Aurora B (AurB) is a mitotic kinase responsible for multiple aspects of mitotic progression, including assembly of the outer kinetochore. Cytoplasmic dynein is an abundant kinetochore protein whose recruitment to kinetochores requires phosphorylation. To assess whether AurB regulates recruitment of dynein to kinetochores, we inhibited AurB using ZM447439 or a kinase-dead AurB construct. Inhibition of AurB reduced accumulation of dynein at kinetochores substantially; however, this reflected a loss of dynein-associated proteins rather than a defect in dynein phosphorylation. We determined that AurB inhibition affected recruitment of the ROD, ZW10, zwilch (RZZ) complex to kinetochores but not zwint-1 or more-proximal kinetochore proteins. AurB phosphorylated zwint-1 but not ZW10 in vitro, and three novel phosphorylation sites were identified by tandem mass spectrometry analysis. Expression of a triple-Ala zwint-1 mutant blocked kinetochore assembly of RZZ-dependent proteins and induced defects in chromosome movement during prometaphase. Expression of a triple-Glu zwint-1 mutant rendered cells resistant to AurB inhibition during prometaphase. However, cells expressing the triple-Glu mutant failed to satisfy the spindle assembly checkpoint (SAC) at metaphase because poleward streaming of dynein/dynactin/RZZ was inhibited. These studies identify zwint-1 as a novel AurB substrate required for kinetochore assembly and for proper SAC silencing at metaphase.