RACK1与MCM7相互作用在肺癌细胞DNA复制中的作用及其机制

DNA replication licensing system and Pre-RC formation and activation are regulated to allow only a single round of replication during each cell cycle. MCM7 is a critical component of the DNA replication licensing complex, and control cell cycle progression from G1 to S phase. RACK1 is one of the MCM7 interaction proteins screening from the lung epithelial cDNA library by yeast two hybrid system. RACK1 is identified as an anchoring protein of protein kinase C; it is a scaffolding protein playing an essential role in the spatial and temporal regulation of individual enzymes and provides the link between extracellular events and intracellular compartments. In this study we provide the first evidence that RACK1, through its interaction with MCM7 with the N-terminal regions, is critical for the cell cycle progression. It enters the nuclear, and plays a role in MCM7 protein phosphorylation and binding with chromatin without detaching. Then what we are interested are in lung cancer cell, what is the significance of RACK1 associated with MCM7 in DNA replication licensing system? And will it cause DNA replicate licensing system regulation disorder? Here, this project aims at demonstration of interaction of RACK1 and MCM7, finding out their binding motif, and exploring the function of their binding to lung cancer cell replication and cell cycle progression. We want to know the mechanisms of RACK1 entering nuclear. Then, we would demonstrate the RACK1 and MCM7 association would affect lung cancer cell proliferation and prognosis by clinical lung cancer samples and animal models. Overall our project would find a new function and a signal pathway of RACK1; and would explore the mechanism of DNA replication re-licensing in lung cancer.

DNA复制执照系统确保每个细胞周期的DNA只复制一次，MCM7为该系统的重要成员。RACK1是我们筛选出的新的MCM7结合蛋白。我们发现：脚手架蛋白RACK1入核后与MCM7结合并使其磷酸化、持续结合在染色质上，促进细胞周期、DNA多倍体形成。但RACK1是如何入核的，其与MCM7结合对造成DNA复制执照系统功能失调的分子机制等尚不清楚。我们拟使用GST pull down、CoIP、酵母双杂交等，明确和验证二者作用域；磷酸激酶实验以明确RACK1的入核机制；双向调控RACK1表达、构建结合域缺失突变体及MCM7磷酸化位点点突变，利用染色质结合实验、激酶实验和流式细胞术等，阐明二者结合对肺癌细胞复制执照系统的影响及机制；采用克隆形成及裸鼠移植等方法，验证RACK1与MCM7相互作用对肺癌细胞增殖的影响。研究结果将为发现RACK1、MCM7的新功能、寻找防治肺癌的新靶点打下理论和实验基础。

项目的背景.RACK1是我们发现的MCM7新的相互作用蛋白。RACK1是活化的蛋白激酶C受体，也是细胞内的支架蛋白--细胞内纷繁复杂的信号途径进行时空调控的中心，但其在肿瘤中作用尚不清楚。前期工作发现，RACK1在肺癌组织和细胞系中高表达，并且与T1期肺癌的预后相关，促进肺癌细胞增殖和细胞周期进展。MCM7是DNA复制执照系统的重要成员，肿瘤中染色质DNA异常是由复制执照系统调节失常所致。.主要研究内容.1、.RACK1与MCM7蛋白相互作用，应用酵母双杂交实验、免疫共沉淀、激光共聚焦扫描显微镜证实RACK1与MCM7在细胞内相互作用.2、.RACK1在肺癌细胞中生物学作用及机制探讨，以及RACK1与MCM7结合对MCM7与染色质结合、复制执照活性影响.3、.临床肺癌组织样本中RACK1和MCM7蛋白的表达及与临床病理因素间关系.重要结果：提出并证实：肺癌细胞中RACK1通过新的MCM7/RACK1/AKT活化途径，进入细胞核内，参与调解磷酸化MCM7蛋白，使MCM7以及复制执照复合体持续与染色质结合，无法解离，阻碍复制执照蛋白的正常功能，致DNA复制再执照、异常复制。.关键数据.1、.RCAK1促进NSCLC细胞增殖.2、.RACK1调节MCM7与染色质的结合及MCM复合体的形成.3、.RACK1通过RACK1/MCM7/AKT复合体介导MCM7的磷酸化.4、.RACK1和MCM7在NSCLC的表达.科学意义.理论意义 ①我们首次提出肺癌细胞中RACK1-MCM7信号传导途径，及其与DNA复制执照间关系和作用机制；②揭示RACK1移位到细胞核内的机制，可望发现一个新的调控RACK1的信号途径；③为进一步揭示复制执照蛋白在肿瘤中失调节再执照的机制提供理论基础。.现实意义 针对肿瘤细胞中染色质DNA异常复制这一普遍现象，利用重要的复制执照蛋白仅在肿瘤细胞中失控再执照，导致细胞无限增殖的机理，阐明MCM7的作用蛋白及生物学作用意义重大，以其为靶点有望成为肿瘤治疗的“利器”。.综上，本研究揭示在肺癌中DNA复制执照蛋白失调的新机制--支架蛋白RACK1募集AKT，磷酸化MCM7，通过RACK1/MCM7/AKT途径致复制执照系统失调节，DNA复制失控，促进肺癌发生发展，同时也可为据此寻找肺癌的治疗靶点提供新的理论依据。

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