B-cyclin/CDKs regulate mitotic spindle assembly by phosphorylating kinesins-5 in budding yeast.
B-cyclin/CDKs regulate mitotic spindle assembly by phosphorylating kinesins-5 in budding yeast.
复制标题
DOI：
10.1371/journal.pgen.1000935
复制
发表时间：
2010-05-06
期刊：
影响因子：
4.5
通讯作者：
Haase SB
中科院分区：
文献类型：
--
作者：
Chee MK;Haase SB
尽管多年来人们已经知道B-细胞周期蛋白/CDK复合物调节有丝分裂纺锤体的组装和进入有丝分裂，但相关CDK靶点的完整补体尚未确定。以前已经在各种模型系统中显示，B型细胞周期蛋白/CDK复合物、驱动蛋白-5马达和SCFCdc 4泛素连接酶是分离纺锤体极和组装双极纺锤体所需的。有人认为，在芽殖酵母中，B型细胞周期蛋白/CDK（Clb/Cdc28）复合物通过后期促进复合物（APCCdh 1）抑制驱动蛋白-5 Kip 1和Cin8的降解来促进纺锤体极分离。然而，我们已经确定，Kip1和Cin8蛋白在Clb/Cdc28激酶活性的情况下以野生型水平存在。在这里，我们表明，Kip1和Cin8的Clb2/Cdc28的体外目标和Kip1上的保守的CDK磷酸化位点的突变抑制纺锤体极分离，而不影响蛋白质的体内定位或丰度。质谱分析证实，在Kip1的尾部结构域中的两个CDK位点在体内被磷酸化。此外，我们已经确定，Sic1，Clb/Cdc28特异性抑制剂，是SCFCdc4的目标，抑制纺锤体极分离缺乏功能Cdc4的细胞。基于这些发现，我们提出，Clb/Cdc28驱动纺锤体极分离驱动蛋白-5马达的直接磷酸化。双极有丝分裂纺锤体的组装对于有丝分裂期间姐妹染色单体的准确分离以及因此对于成功的细胞分裂至关重要。纺锤体组装取决于纺锤体极的成功复制，然后将它们分离到细胞的相对端。尽管多年来人们已经知道B-细胞周期蛋白/CDK复合物调节有丝分裂纺锤体的组装，但相关的CDK靶点尚未确定。驱动蛋白-5家族的马达蛋白在构成纺锤体的微管上产生运动，并且被认为是驱动纺锤体极分离的动力。通过采用芽殖酵母酿酒酵母作为模型，我们已经发现的证据表明，细胞周期蛋白/CDKs控制纺锤体组装磷酸化的驱动蛋白-5 Kip1和Cin8。当Kip1的马达结构域中保守的CDK位点的磷酸化被阻断时，纺锤体极分离大大减少，但蛋白质丰度和定位都不受影响。我们还通过质谱法获得了直接证据，表明Kip1和Cin8在其尾部结构域的共有CDK位点处被体内磷酸化。我们的研究结果表明，B-cyclin/CDKs通过调节驱动蛋白-5运动活性来调节纺锤体组装。
Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCFCdc4 ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that, in budding yeast, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APCCdh1). We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity. Here, we show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28 and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, we have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCFCdc4 target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors. The assembly of a bipolar mitotic spindle is essential for the accurate segregation of sister chromatids during mitosis and, hence, for successful cell division. Spindle assembly depends on the successful duplication of the spindle poles, followed by their separation to opposing ends of the cell. Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle, the relevant CDK targets have not been identified. Motor proteins of the kinesin-5 family generate movement on the microtubules that make up the spindle and are believed to power spindle pole separation. By employing the budding yeast Saccharomyces cerevisiae as a model, we have found evidence that cyclin/CDKs control spindle assembly by phosphorylating the kinesins-5 Kip1 and Cin8. When phosphorylation at a conserved CDK site in the motor domain of Kip1 is blocked, spindle pole separation is greatly diminished but neither protein abundance nor localization is affected. We have also obtained direct evidence by mass spectrometry that Kip1 and Cin8 are phosphorylated in vivo at consensus CDK sites in their tail domains. Our findings suggest that B-cyclin/CDKs regulate spindle assembly by regulating kinesin-5 motor activity.
登录
查看更多内容
影响因子：
64.5
作者：
Feldman, RMR;Correll, CC;Deshaies, RJ
通讯作者：
Deshaies, RJ
影响因子：
3.7
作者：
Cahu, Julie;Olichon, Aurelien;Hentrich, Christian;Schek, Henry;Drinjakovic, Jovana;Zhang, Cunjie;Doherty-Kirby, Amanda;Lajoie, Gilles;Surrey, Thomas
通讯作者：
Surrey, Thomas
影响因子：
3.2
作者：
BYERS, B;GOETSCH, L
通讯作者：
GOETSCH, L
影响因子：
5.8
作者：
Arnold, K;Bordoli, L;Schwede, T
通讯作者：
Schwede, T
影响因子：
64.8
作者：
Collins, Sean R.;Miller, Kyle M.;Krogan, Nevan J.
通讯作者：
Krogan, Nevan J.