Park Chong J, Park Jung-Eun, Karpova Tatiana S, Soung Nak-Kyun, Yu Li-Rong, Song Sukgil, Lee Kyung H, Xia Xue, Kang Eugene, Dabanoglu Ilknur, Oh Doo-Yi, Zhang James Y, Kang Young Hwi, Wincovitch Stephen, Huffaker Tim C, Veenstra Timothy D, McNally James G, Lee Kyung S
Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Eukaryot Cell. 2008 Mar;7(3):444-53. doi: 10.1128/EC.00283-07. Epub 2008 Jan 4.
In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of the budding yeast polo kinase Cdc5, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal noncatalytic polo box domain, a region that is critical for proper subcellular localization. One of these mutants, cdc5-11, exhibited a temperature-sensitive growth defect with an abnormal spindle morphology. Strikingly, provision of a moderate level of benomyl, a microtubule-depolymerizing drug, permitted cdc5-11 cells to grow significantly better than the isogenic CDC5 wild type in a FEAR (cdc Fourteen Early Anaphase Release)-independent manner. In addition, cdc5-11 required MAD2 for both cell growth and the benomyl-remedial phenotype. These results suggest that cdc5-11 is defective in proper spindle function. Consistent with this view, cdc5-11 exhibited abnormal spindle morphology, shorter spindle length, and delayed microtubule regrowth at the nonpermissive temperature. Overexpression of CDC5 moderately rescued the spc98-2 growth defect. Interestingly, both Cdc28 and Cdc5 were required for the proper modification of the spindle pole body components Nud1, Slk19, and Stu2 in vivo. They also phosphorylated these three proteins in vitro. Taken together, these observations suggest that concerted action of Cdc28 and Cdc5 on Nud1, Slk19, and Stu2 is important for proper spindle functions.
在许多生物体中，polo激酶在M期进程中似乎发挥着多种作用。为了深入了解出芽酵母polo激酶Cdc5的功能，我们通过诱变C端非催化性polo盒结构域（该区域对正确的亚细胞定位至关重要），生成了新的温度敏感型cdc5突变体。其中一个突变体cdc5-11表现出温度敏感型生长缺陷，纺锤体形态异常。令人惊讶的是，添加中等水平的苯菌灵（一种微管解聚药物）能使cdc5-11细胞以不依赖FEAR（cdc十四早期后期释放）的方式生长得比同基因的CDC5野生型细胞明显更好。此外，cdc5-11细胞生长以及苯菌灵补救表型都需要MAD2。这些结果表明cdc5-11在正常纺锤体功能方面存在缺陷。与此观点一致，cdc5-11在非允许温度下表现出异常的纺锤体形态、较短的纺锤体长度以及延迟的微管再生。CDC5的过表达适度挽救了spc98-2的生长缺陷。有趣的是，在体内，纺锤极体成分Nud1、Slk19和Stu2的正确修饰需要Cdc28和Cdc5共同参与。它们在体外也能磷酸化这三种蛋白质。综上所述，这些观察结果表明Cdc28和Cdc5对Nud1、Slk19和Stu2的协同作用对于正常的纺锤体功能很重要。
