脑型肌酸激酶通过定位于纺锤体和中心体参与细胞有丝分裂调控的研究

In the cells, the sites (also called compartments or micro-domains) for ATP generation and consumption are distinct and uncoupled. The spatial separation of these sites and the limited diffusion rate of ATP can not fulfill large energy flux. The phosphocreatine-creatine-kinase (CK) shuttling system has been evolved to buffer the ATP fluctuations at each ATP generation or consumption site. Five decades ago, mitosis has been identified as a process with high-energy demand. However, the source of ATP to complete mitosis is unclear for many years. In the 1980s, CK was shown to have spindle localization and might regulate chromosome movement. This implies that CK may be one of the key enzyme in maintaining ATP homeostasis during mitosis. An unresolved problem is how could the cytosolic CK localize on mitotic spindle and involve in mitosis regulation. We think the key to elucidate the action of CK in mitosis is to identify CK-binding proteins as well as the coupled ATPase. We have screened for CK-binding proteins by yeast-two-hybrid assay and have identified many potential binding proteins. Among them, two proteins were found to have the potency to recruit CK to the mitotic spindle and centrosomes. We are aiming to elucidate how mitosis is regulated by CK via its location at the mitotic spindle and centrosomes by methods of enzymology, protein interactions, cellular functions and identification of the complex/molecular machinaries.

细胞中ATP的产生和消耗部位并非耦联和连续的，产能和耗能部位的ATP稳态依赖于磷酸肌酸-肌酸激酶(CK)系统。五十多年前就发现细胞有丝分裂是一个高耗能且依赖ATP的过程，但对有丝分裂中各个事件所需大量ATP的来源一直存在争议。三十多年前的工作曾提出CK定位于纺锤体且可能参与了有丝分裂中染色体运动的调控，这暗示CK可能在有丝分裂中维持ATP的供给和浓度稳定。一直悬而未决的问题是：CK到底参与了有丝分裂的哪些事件？胞质分布的CK是如何定位于纺锤体并参与有丝分裂的高耗能过程的？鉴定介导CK富集在ATP酶附近的互作蛋白是理解CK工作机制的一个关键点。本项目拟在前期酵母双杂交和细胞定位研究的基础上，利用酶学研究、蛋白质相互作用、细胞功能研究、串联亲和层析纯化以及建立体外工作系统等技术手段，重点研究广泛分布的脑型CK定位于纺锤体和中心体的机制以及如何参与有丝分裂调控。

细胞中产能和耗能部位的ATP稳态依赖于磷酸肌酸-肌酸激酶穿梭系统。五十多年前就发现细胞有丝分裂是一个高耗能且依赖ATP的过程，但对有丝分裂中各个事件所需大量ATP的来源一直存在争议。三十多年前的工作曾提出肌酸激酶定位于纺锤体且可能参与了有丝分裂中染色体运动的调控，但一直悬而未决的问题是：CK到底参与了有丝分裂的哪些事件？胞质分布的CK是如何定位于纺锤体并参与有丝分裂的高耗能过程的？我们用酶学研究、蛋白质相互作用、细胞功能研究、串联亲和层析纯化以及建立体外工作系统等技术手段，发现肌酸激酶的互作蛋白FAM96B介导了肌酸激酶定位于纺锤体，参与了有丝分裂期纺锤体的组装，在细胞中下调FAM96B或肌酸激酶都导致纺锤体的异常组装和细胞死亡。我们还发现，脑型肌酸激酶具有膜定位，膜定位的肌酸激酶与Aki1相互作用参与了Akt跨膜信号传导过程。本项目的研究为长期未解决的科学问题提供了阐释，也为进一步探索重要的高耗能细胞生理活动中的能量代谢机制提供了新的线索。