Cyclin dependent kinase cascade.

细胞周期依赖性激酶级联。

基本信息

批准号：
13043057
负责人：
INAGAKI Masaki
金额：
$ 38.27万
依托单位：
Aichi Cancer Center (Research Institute)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2005
项目状态：
已结题

项目摘要

Mitotic chromosomal dynamics is regulated by the coordinated activities of many mitotic kinases, such as Cdc2 (Cdkl), Aurora-B or Polo-like kinase 1 (P1k1), but mechanisms of its coordination remain unknown. We demonstrated two novel Cdc2 kinase cascades. 1) We demonstrated a direct interaction between Plkl and vimentin-Ser55 phosphorylated by Cdc2, an event which led to Plkl activation and further vimentin phosphorylation by Plkl. In vitro analyses revealed that Plkl phosphorylated vimentin at,・1 mol phosphate/mol substrate, which partly inhibited its filament forming ability. Plkl phosphorylated Ser82 on vimentin in vitro, and this phosphorylation was elevated from metaphase and was maintained until the end of mitosis. This elevation thus followed the Cdc2-induced vimentin-Ser55 phosphorylation. Depletion of Plkl by RNA interference impaired the elevation of vimentin-Ser82 phosphorylation in mitosis. These results indicated a novel mechanism that Cdc2 regulated mitotic vimentin phosphorylation via not only a direct enzyme reaction but also Plkl recruitment to vimentin. 2) We found that Cdc2 phosphorylates Thr59 and Thr388 on inner centromere protein (INCENP), which regulates localization and kinase activity of Aurora-B, from prophase to metaphase. INCENP depletion disrupts Plkl localization specifically at the kinetochore. This phenotype is rescued by the exogenous expression of INCENP wild type (WT) and INCENP mutated at Thr59 to Ala (T59A), but not at Thr388 to Ala (T388A). The replacement of endogenous INCENP with T388A resulted in the delay of progression from metaphase to anaphage. Our findings suggested that INCENP phosphorylation by Cdc2 is necessary for the recruitment of Plkl to the kinetochore, and the complex formation of Plkl and Aurora-B on INCENP may play critical roles in the regulation of chromosomal dynamics.

有丝分裂染色体动力学受许多有丝分裂激酶（例如 Cdc2 (Cdkl)、Aurora-B 或 Polo 样激酶 1 (P1k1)）的协调活动调节，但其协调机制仍然未知。 1)我们证明了 Plkl 和被 Cdc2 磷酸化的波形蛋白-Ser55 之间的直接相互作用，这一事件导致 Plkl 激活和体外分析表明，Plkl 以 1 mol 磷酸盐/mol 底物磷酸化波形蛋白，这在体外部分抑制了 Plkl 磷酸化波形蛋白上的 Ser82 的能力，并且这种磷酸化从中期开始升高并得以维持。直到有丝分裂结束，这种升高是在 Cdc2 诱导的波形蛋白-Ser55 磷酸化之后发生的。 RNA 干扰的 Plkl 损害了有丝分裂中波形蛋白-Ser82 磷酸化的升高。这些结果表明 Cdc2 不仅通过直接酶反应而且还通过 Plkl 招募波形蛋白来调节有丝分裂波形蛋白磷酸化的新机制 2) 我们发现 Cdc2 磷酸化 Thr59 和 Thr59。内部着丝粒蛋白 (INCENP) 上的 Thr388，调节 Aurora-B 的定位和激酶活性，来自INCENP 缺失会破坏 Plkl 在着丝粒上的定位，这种表型可以通过外源表达 INCENP 野生型 (WT) 和在 Thr59 处突变为 Ala (T59A) 的 INCENP 来恢复，但在 Thr388 处突变为 Ala (T388A) 时则不会。用 T388A 替代内源性 INCENP 导致从中期到后期的进展延迟。 Cdc2 对 INCENP 的磷酸化对于将 Plkl 招募到着丝粒是必需的，并且 Plkl 和 Aurora-B 在 INCENP 上形成的复合物可能在染色体动力学的调节中发挥关键作用。