中心体结合蛋白Cep88在中心体复制及斑马鱼早期胚胎发育中的功能研究

Centrosome is the main microtubule organizer of mammalian cells, and is crucial for many biological processes such as formation of the mitotic spindle and cilia. Centrosome dysfunction or abnormal centrosome number is intimately implicated in tumorogenesis as well as certain types of develomental defects. Currently, over 200 new centrosomal proteins have been identified, however, most of their functions in centrosomal regulation are unknown. We have identified Cep88 （Centrosomal Protein 88 kDa） as an interacting protein of Nek2A （NIMA-related kinase 2A）, which is an important serine/threonine kinase responsible for the centrosome segregation in G2/M phase. In order to investigate this novel protein with unkown function, we have raised rabbit anti-Cep88 antibody,and have established two stable U2OS cell lines expressing GFP-Cep88 and CETN1-GFP。 We have also determined the centrosomal localization of Cep88. We have established the interaction between the endogenous Cep88 and Nek2, and have displayed their colocalization in the cells. Overexpressed Cep88 significantly reduces pericentriole materials and leads to the prematured centrosome separation, suggesting that Cep88 may be involved in the regulation of centrosome structure and funtion. We also found that Cep88 can directly bind to Nek2A and inhibit Nek2A kinase activity. We have found that zebrafish homologue zCep88 is expressed in the early -staged embryos. Elevation of zCep88 protein levels or reduction of zNek2 protein levels will cause similar developmental defects in the gastulation and segmentation processes in the zebrafish embryos, suggesting that these two protein are intimately cooperate to regulate the early development of zebrafish. We will further invesigate the regulatory roles of Cep88 in the structure and function of centrosomes in cell cycle, as well as in the early development of zebrafish. We believe that our studies will unveil the functions of this novel gene in centrosomal regulation as and embryogeneis.

Nek2是调控中心体复制的关键激酶，如何调控其激酶活性是当今的研究热点。我们在寻找与Nek2结合的蛋白中分离并鉴定了Cep88。在此基础上，我们制备了抗Cep88的抗体，建立了GFP-Cep88及CETN1-GFP两种细胞系；确定了內源的Cep88与Nek2的相互作用以及在细胞内的共定位；发现了Cep88可直接结合并抑制Nek2的激酶活性；确定了Cep88在细胞内的分布及中心体上的定位；初步研究了Cep88在调控中心体结构和功能上的作用。我们在斑马鱼中确立了Cep88在胚胎发育各个时期的表达谱，发现过量表达Cep88和敲低Nek2对斑马鱼胚胎发育的影响作用非常相似。在本项目中，我们将进一步研究Cep88与Nek2 协同调控中心体结构和功能的分子机制以及它们在斑马鱼早期胚胎发育中的作用。本项目的开展将揭示Cep88这个功能未知蛋白的生物学功能，必将有助于更好地阐明中心体复制及调控的分子机制。

Nek2是调控中心体复制与分离的关键激酶，Nek2的活性过高将导致细胞中心体提早分离的现象。因此，Nek2激酶活性的调控机制是当今的研究热点。我们在寻找与Nek2结合的蛋白中分离并鉴定了Cep85蛋白。Cep85 蛋白和Nek2 的一个亚型Nek2A 共定位，两个蛋白分布于中心粒近端85蛋白，形成颗粒状的网络结构。和Nek2A 的作用相反的是， 过表达Cep85 蛋白， 抑制了马达蛋白Eg5（KIF11）介导的中心体的分离。相反地，损耗了活性的Cep85导致中心体的提早分离。我们还鉴定了Cep85 蛋白和Nek2A相互作用， 以及Cep85在中心体上的定位的结构域。 总之， 我们的研究证实了Cep85 是一个新的Nek2 作用蛋白，他们都定位在中心粒的远端，和PP1γ 协同作用拮抗Nek2A 的活性，以维持细胞间期中心体的完整性。