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的协调活性调节，例如CDC2（CDKL），Aurora-B或Polo样激酶1（P1K1），但其协调机制仍然未知。我们展示了两个新型的CDC2激酶级联反应。 1）我们证明了由Cdc2磷酸化的PLKL和波形蛋白 - 塞55之间的直接相互作用，这一事件导致PLKL激活和PLKL进一步的波形蛋白磷酸化。体外分析表明，PLKL磷酸化的波形蛋白在・1ol磷酸/mol底物，部分抑制其丝形成能力。 PLKL在体外在波形蛋白上的磷脂化Ser82，这种磷酸化从中期升高，并一直保持到有丝分裂结束。因此，该升高遵循CDC2诱导的波形蛋白 - 塞55磷酸化。 RNA干扰对PLKL的消耗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，一种新的机制，即CDC2不仅通过直接的酶反应，而且还针对波形蛋白募集了PLKL。 2）我们发现CDC2在内侧丝粒蛋白（Incenp）上磷酸化Thr59和Thr388，该蛋白（Incenp）调节了Aurora-B的定位和激酶活性，从前期到中期。 Incenp耗竭破坏了PLKL定位，这种表型是由Incenp野生型（WT）表达性表达（WT）的特异性进行的，并在THR59突变为ALA（T59A）的Incenp，但在THR388对ALA（T388A）则不强。用T388A替换内源性印加P，导致从中期到放置的进展延迟。我们的发现表明，Cdc2的磷酸化对于将PLKL募集到Kinetochore是必需的，并且PLKL和Aurora-B对Incenp的复杂形成可能在调节染色体动力学的调节中起关键作用。