SIK2调节电离辐射损伤细胞的自吞噬过程及机制研究

Autophagy is a ubiquitous highly conserved pathway in eukaryotic cells that takes place as a response to a variety of conditions, such as nutrient deprivation.It has already been shown that autophagy is one of the cellular responses to ionizing radiation (IR) injury. However, it is still unclear the mechanism of IR-induced autophagy and that how it determines the cell fate. Our recent study have uncovered the interaction of DNA-PKcs, a critical molecule in DNA damage early signaling event, and the salt-inducible kinase 2(SIK2). DNA-PKcs has already well demonstrated to involve in the IR-induced autophagy, while SIK2 was newly shown to plays an important role in the autophagosome maturation processing. What their acting mechanisms are still unknown.In this project, we will start from the interaction of DNA-PKcs and SIK2. Firstly, the role will verified for SIK2 to play on the IR-induced autophagy, and to clear what is the major consequence of SIK2-associated autophagy on cellular radiosensitivity. The SIK2 gene knock-out cells will be constructed by using the TALEN technology. A series of mutants of SIK2 phosphorylation sites and K53 acetalytion site will also been constrcuted and used in this project, to investigate how the IR-induced DNA damage signaling is transduced into the autophagy processing through the DNA-PKcs and SIK2 signal pathway. The regulation mechanism of the translocation and sequestered of SIK2 from whole cytoplasm into the autophagosome, and its effction on autophagy maturation.The results and outcome information from this project will be necessary for the mechanisitc elucidation of IR-induced autophagy. The knowledge from this study will be also useful for the study and development of new radioprotection agents or radiosensitizers targating the autophagy pathway.

自吞噬是真核细胞在营养缺失等压力条件下发生的一种保护性反应，近年研究证实，电离辐射损伤可诱发细胞自吞噬，但其作用机制及对细胞命运的影响还需要更深入研究阐明。我们前期研究发现，DNA损伤信号响应的重要调节分子DNA-PKcs与盐诱导激酶SIK2相互作用，前者早被发现参与放射损伤细胞的自噬调节，后者被最新发现是细胞自吞噬体成熟过程的重要调节物，只是各自机制不清。本项目将以DNA-PKcs与SIK2相互作为切入点，在验证SIK2在电离辐射诱发细胞自吞噬中的作用的基础上，建立和利用SIK2基因敲除细胞、SIK2的磷酸化和乙酰化位点的系列突变体，探讨电离辐射DNA损伤信号是如何由DNA-PKcs或其他激酶并通过SIK2传递到细胞自吞噬途径中，然后再详细阐明SIK2的激活和定位到自吞噬体及参与自吞噬过程的机制。本研究不仅对放射损伤细胞自噬机制有新阐述，还将为针对自吞噬研发辐射增敏或防护剂提供重要信息。

电离辐射通过诱发DNA损伤或其它因素导致细胞自噬，自噬可能起到保护细胞、对抗放射损伤的作用，但也可能是死亡性自噬。盐诱导激酶2（SIK2）是一个对电离辐射响应基因，本研究以SIK2与DNA损伤应答分子DNA-PKcs的相互作用为切入点，开展SIK2在电离辐射诱发细胞自吞噬效应规律、生物学意义和机制等研究。取得如下主要结果：1）明确了细胞电离辐射损伤后SIK2表达的变化规律。发现细胞受照后，SIK2蛋白表达量增加更加明显，而2、4Gy剂量照射对SIK2的诱导表达作用显著大于10Gy大剂量照射；2）明确了SIK2与DNA-PKcs对电离辐射诱发细胞自噬的调节作用。揭示无论敲低SIK2还是DNA-PKcs，均增加细胞放射敏感，但SIK2敲低细胞的增殖活性提高。用3-甲基腺嘌呤抑制细胞自噬，提高了受照射细胞凋亡率，敲低SIK2减弱细胞发生自噬。发现分割照射，增强了辐射诱导的细胞自噬，自噬在此起到保护作用，由此阐述了细胞亚致死性修复的一种新的机制；3）明确了自噬和SIK2在分割照射效应中的作用。为了探讨自噬对受照射细胞是保护还是致死性作用，我们分别设计在照射早期（照射后12h内）和后期（12h以后）抑制自噬，观察对细胞放射敏感性的影响。结果发现，在照射早期抑制细胞自噬，显著增加了细胞凋亡和放射敏感性，而后期抑制自噬对细胞放射增敏作用相对弱，显示照射后早期细胞发生的是保护性自噬。虽然，敲低SIK2后，抑制细胞自噬在一定程度上增加了细胞放射敏感性，其增敏作用比对于SIK2正常表达细胞相对弱；4）SIK2对细胞DNA损伤反应的影响及作用机制。发现SIK2敲低引起细胞G2期到M期的转换时间延长，与转换点调节分子Cdc25C和Cdc2的磷酸化水平升高、无法使下游Cdk1(Cdc2) T15去磷酸化，Cdk1和cyclin B1复合物不能激活有关；5）揭示抑制乙酰转移酶活性，降低了细胞DNA-PKcs 的T2609和S2056位点磷酸化。电离辐射诱导TIP60发生SUMO-2/3修饰，鉴定了TIP60的KBR位点突变及乙酰化负控节其SUMO化修饰，影响DNA-PKcs的S2056自磷酸化，模拟乙酰化的突变体（KBQ）可以抑制DNA-PKcs在电离辐射损伤下的自磷酸化激活及其底物H2AX的磷酸化，DNA-PKcs失活导致SIK2蛋白水平显著降低。本研究为放射损伤防治药物研发提供新的思路。