SIK2与DNA-PKcs相互作用调节放射损伤细胞的G2/M阻滞与纺锤体结构稳定性

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 repair 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 and increased radiosensitivity. Therefore, identifying the upstream and downstream regulators of DNA-PKcs and recognizing their functions have great significance in understand the genomic stability mechanism responding to IR-induced DNA damage. In our previous work, the salt-inducible kinase 2 (SIK2) was identified to interact with DNA-PKcs by the yeast two-hybridization. It was reported that SIK2 localizes at centrosomes, plays a key role in regulation of centrosomes separation and the onset of mitosis, via phosphorylating the centrosome linker protein,C-Nap1. Recently we also found DNA-PKcs play roles in regulating mitotic progression in IR-damaged cells, as well as stabilizing spindle and centrosome structures. In this study the cell cycle-dependent interaction between SIK2 and DNA-PKcs will be identified and the interaction domain of SIK2 with DNA-PKcs will be screened by GST pull-down assay. Besides the phosphorylation site(s) of SIK2 by DNA-PKcs will be determined by in vitro phosphorylation assay and in vivo. The effect of DNA-PKcs targeted phosphorylation of SIK2 on centrosome separation，spindle stability and mitotic progression will be further determined. Together with all these, The roles of the interaction between SIK2 and DNA-PKcs in G2/M arrest, spindle structure and mitotic progress will be ascertained, which will extend our knowledge in the molecular mechanism of cell radiosensitivity and genomic stability maintenance。

DNA依赖蛋白激酶催化亚基（DNA-PKcs）是哺乳动物细胞DNA双链断裂非同源末端连接修复中的关键激酶。我们最近发现DNA-PKcs具有调节放射损伤细胞的有丝分裂进程、中心体和纺锤体结构稳定性的功能。进一步通过酵母双杂交技术发现DNA-PKcs与盐诱导激酶2 (SIK2) 有相互作用。而SIK2也参与调控中心体分离和双极纺锤体形成。本课题将首先确定SIK2与DNA-PKcs相互作用的细胞周期时相相关性，并鉴定其相互作用位点；通过体外磷酸化实验确定SIK2为DNA-PKcs的磷酸化底物及其磷酸化位点，验证DNA-PKcs对SIK2体内磷酸化调节作用及与细胞周期进程关系；研究DNA-PKc通过磷酸化SIK2对中心体分离、纺锤体结构及有丝分裂进程的影响。通过此研究将揭示放射损伤细胞G2/M期阻滞和纺锤体结构稳定性新机制，这对全面揭示放射损伤诱发的细胞学反应和基因组稳定性维持机制具有重要意义。

DNA依赖蛋白激酶催化亚基（DNA-PKcs）是DNA双链断裂损伤响应早期阶段的一个重要调节分子，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。 最近报道显示，SIK2也参与调控中心体分离和双极纺锤体形成。.本课题研究首先确定了SIK2与DNA-PKcs的相互作用，并揭示了二者的相互作用持续存在于整个细胞周期，而且电离辐射对二者相互作用没有影响。并通过构建带GST标签的SIK2截短体的GST-pulldown实验，确定了SIK2与DNA-PKcs的相互作用位于400-700氨基酸。此外，还从mRNA、蛋白水平和启动子活性研究了电离辐射对SIK2基因表达的影响、作用规律和相关机制，结果显示电离辐射对SIK2 mRNA表达有诱导作用，但无明显的剂量依赖性。 60Coγ射线诱导SIK2蛋白的表达水平增加，部分作用机制是照射后一定时间范围内SIK2启动子转录活性的增加，由此使mRNA转录增加，进一步加强了SIK2蛋白的表达水平。虽然SIK2参与细胞周期调节，但其 mRNA在照射后期表达增加的原因与辐射诱导的G2/M期阻滞没有关系。.以上研究结果为深入研究DNA-PKcs与SIK2相互作用调节放射损伤细胞M期阻滞和纺锤体结构稳定奠定了基础，这对深入了解辐射所致DNA损伤修复反应机制具有重要意义。