DNA-PKcs与SIK2相互作用在放射损伤细胞有丝分裂灾变中的作用及机制研究

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a significant role in the process of non-homologous end-joining (NHEJ) repair, which is one of the major pathways for the IR-induced DSBs in mammalian cells. Cells that lack or have inactive DNA-PKcs have defects in repairing DSBs, disordered regulation of mitotic progress，which could even lead to mitotic catastrophe. Therefore, identifying the upstream and downstream regulators of DNA-PKcs and recognizing their functions have great significance in understand the repairing mechanism of IR-induced DNA damage. In our previous experiments, the salt-inducible kinase 2 (SIK2) was identified to have interaction with DNA-PKcs by the yeast two-hybridization assay. It was reported that SIK2 was located at centrosome, played a key role in the initiation of mitosis, could phosphorylate the centrosome linker protein, C-Nap1. Overexpression of SIK2 induced centrosome splitting and depletion of SIK2 delayed mitotic progression. Recently we also found DNA-PKcs played roles in regulating mitotic progression of IR-induced cell, stabilizing spindle and centrosome. In our previous study, the interaction of SIK2 and DNA-PKcs has been identified by GST-pulldown assay and Co-IP. In this study we plan to elucidate how DNA-PKcs interacts with SIK2 and whether their interaction would functions in centrosome separation，spindle stability and mitotic progression post irradiation. Moreover whether the disruption of their interaction would lead to mitotic catastrophe post irradiation will be determined. Together with all these, The roles of the interaction of SIK2 and DNA-PKcs in G2/M arrest, spindle structure, mitotic progress and mitotic catastrophe post irradiationwill be ascertained, which will extend our understanding in molecular mechanism of cell radiosensitivity，and also provide us theoretical basis of SIK2 as the potential molecalar target for therapy in cancer.

DNA-PKcs不仅在电离辐射诱导的DNA双链断裂损伤修复中有关键作用，还具有调节电离辐射损伤细胞的有丝分裂进程和中心体与纺锤体稳定的功能，SIK2是最近报导的另一调控中心体分离和双极纺锤体形成的分子。我们前期研究发现DNA-PKcs与SIK2有相互作用，二者是否协同调节电离辐射损伤细胞的有丝分裂进程反应及机制值得关注。本课题就DNA-PKcs与SIK2相互作用方式，及其相互作用在放射损伤细胞的纺锤体结构稳定和G2/M检查点功能的作用，其缺失是否引起放射损伤细胞有丝分裂灾变进行深入探讨。通过本课题的研究不仅可以阐明DNA-PKcs与SIK2参与有丝分裂灾变的分子机制，而且为传统化疗药物有耐药性的肿瘤的治疗开辟了新的途径，有望成为开发新抗癌药物的分子靶向目标。

DNA-PKcs不仅在电离辐射诱导的DNA双链断裂损伤修复中有关键作用，还具有调节电离辐射损伤细胞的有丝分裂进程和中心体与纺锤体稳定的功能，SIK2是最近报导的另一调控中心体分离和双极纺锤体形成的分子。我们前期研究发现DNA-PKcs与SIK2有相互作用，二者是否协同调节电离辐射损伤细胞的有丝分裂进程反应及机制值得关注。本课题拟就DNA-PKcs与SIK2相互作用方式，及其相互作用在放射损伤细胞的纺锤体结构稳定和G2/M检查点功能的作用，其缺失是否引起放射损伤细胞有丝分裂灾变进行深入探讨。. 本课题首先明确SIK2敲低, 正如DNA-PKcs敲低一样，均能诱发辐射诱导的细胞有丝分裂灾变，且DNA-PKcs通过与SIK2互作用调节放射损伤细胞有丝分裂灾变。然后证明了γ射线不能影响SIK2与DNA-PKcs的相互作用，DNA-PKcs通过APC/Cdc20途径调控SIK2的降解来调节放射损伤细胞有丝分裂灾变。此外课题还明确了SIK2敲低引起放射损伤细胞G2/M期阻滞，SIK2能参与辐射诱发的DNA损伤修复。SIK2的DNA损伤修复能力在Cre-LoxP重组酶系统构建的SIK2基因敲除（SIK+/-）小鼠中得到证实，SIK2基因敲除后小鼠及其MEFs细胞的辐射敏感性增强。此外，体外生化实验还证实：DNA-PKcs可以磷酸化SIK2，磷酸化作用位点为SIK2的534位丝氨酸，S534；SIK2可以结合DNA，并且DNA-PKcs能够增强SIK2与DNA的结合。SIK2作为新的参与辐射诱导有丝分裂灾变的蛋白，有望成为开发新抗癌药物的分子靶向目标。