Plk1激酶调控中心体分离的新途径研究

Timely separation of centrosome is critical for bipolar spindle formation to ensure chromosome stability. Nek2A is the key kinase directly responsible for the phosphorylation and thereby destruction of proteinaceous linker, mainly consisting of C-Nap1, Cep68, Rootletin and LRRC45. Recently, we have demonstrated that Cep85, a previously uncharacterized centrosomal protein, binds to and inhibits Nek2A to prevent centrosome precocious disjunction in interphases. As the protein levels of Cep85 is elevated but not reduced in the late G2, it is unknown how Nek2A could escape from Cep85 inhibition to enhance its kinase activity to promote centrosome disjunction. We have found that Cep85 greatly reduces its binding affinity to Nek2A in the mid–G2 stage when centrosomes start to separate. The mechanism underlying this observation is unknown. To address it, we have also found that Plk1 interacts with Cep85 in vitro and in vivo. They can form a complex in cells and co-localized at the proximal end of centrosome. Overexpression of Plk1 can cause the phosphorylation of Cep85 and phosphorylated Cep85 reduces binding affinity to Nek2A. Meanwhile, incubation of Cep85-Nek2A complex with Plk1 in an in vitro kinase reaction leads to the dissociation of Nek2A from the complex. Thus, these preliminary data suggest that Plk1 is a potential candidate that is involved in regulating the destruction of Cep85-Nek2A complex in G2/M phase by phosphorylating Cep85. We will carry out further investigation to confirm this possibility. First, we will identify the phosphorylation sites in Cep85 and raise antibody against those phosphoamino acids within Nek2A binding domain in Cep85. Then, we will investigate when and how Cep85 is phosphorylated in cell cycle as well as the effects of Cep85 phosphorylation on centrosome disjunction. Finally, we will purify Plk1, Cep85 and Nek2A to study the procedure of Cep85 phosphorylation and Nek2A activation in details. We believe that our study will further shed light on a new molecular mechanism regarding Nek2A regulation and centrosome disjunction.

中心体适时分离是维持染色体组稳定性的重要基础，Nek2A是直接调控中心体分离的关键激酶。但是如何在G2/M期解除Cep85对Nek2A的抑制促进中心体分离是目前尚未解决的关键问题。我们前期研究发现：在G2中期Cep85-Nek2A开始解离；Plk1结合Cep85，共定位于中心体上；Plk1直接结合并且磷酸化Cep85，导致Cep85-Nek2A解离。因此，我们推测在G2后期Plk1通过磷酸化Cep85，促进Cep85-Nek2A解离，在中心体分离过程中发挥主导作用。本项目将首先鉴定Cep85磷酸化位点，制备磷酸化抗体；接着研究Cep85在细胞周期中的磷酸化状态及不同磷酸化状态的Cep85对Nek2A活性和中心体分离的影响；最后体外重组Plk1磷酸化Cep85促进Cep85-Nek2A解离的生化过程, 彻底阐明Plk1在G2/M期修饰Cep85、提高Nek2A活性、促进中心体分离这一新途径。

由Nek2A主导的中心体在G2/M期的适时分离是分裂期双极纺锤体的形成及随后的染色体及时、准确分离的内在前提。先前的研究发现Nek2A在G2后期被高度磷酸化，而磷酸化的Nek2A才能起始中心体的分离。但是，对于为何Nek2A激酶只是在G2后期表现出高活性以及如何控制Nek2A的激酶活性还有待于进一步的研究。 在本项目的研究中，我们发现Nek2A上游的激酶Plk1发挥了关键的调节作用。Plk1能够结合并且磷酸化Cep85，但是Cep85的磷酸只有在Cep85预先结合Nek2A形成Cep85-Nek2A复合体后才会发生。另外，这个过程不依赖于Nek2A的激酶活性,也与其是否磷酸化无关。 Plk1介导的Cep85磷酸化反过来导致磷酸化的Cep85与磷酸化的Nek2A解离。由于Plk1的活性和Nek2A磷酸化的程度在G2后期都才显著升高，因此，Cep85的磷酸化以及与磷酸化的Nek2A从Cep85-Nek2A复合体分离只能发生在G2后期。大量细胞内源的实验也证实了这个结论。游离的磷酸化的Nek2A因被再被Cep85抑制而起始了G2后期中心体间蛋白连丝的磷酸化和解聚从，而促成了中心体在G2/M期的分离。我们之前发表的文章已经揭示在细胞间期的大部分时间里如何通过Cep85来抑制Nek2A活性，确保了中心体不分离而成为细胞内主要的微管组织中心。本项目则是在此基础上进一步揭示了当中心体需要分开的时刻（G2/M期）Nek2A如何摆脱Cep85的抑制，提高激酶活性，启动中心体的分离的分子机制。通过本项目的研究我们揭示了Plk1-Cep85-Nek2A这一调节中心体分离的新途径。这大大地提升了我们对于Cep85这个我们最早鉴定的蛋白能够的了解，也极大地提升了我们对细胞周期中中心体分离这一重要事件的发生过程以及分子机制的了解，丰富和完善了细胞周期调控的基础知识。

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