AMPK对赤拟谷盗精氨酸激酶的磷酸化修饰和转录调控研究

腺苷酸激活蛋白激酶(AMPK)、叉头框转录因子FOXO和精氨酸激酶（AK）是能量代谢、氧化应激应答等多种细胞过程的重要调节因子。本项目前期研究发现，利用RNA干扰沉默TcAMPKα、TcFOXO和TcAK1与TcAK2后，赤拟谷盗对百草枯诱导氧化应激的耐受性都显著降低，并且AMPK激活剂AICAR处理显著提高了百草枯诱导的TcAK1和TcAK2表达上调倍数，提示三者之间可能存在相互作用。本项目拟在此基础上，应用酵母双杂交、GST pull-down、体外磷酸化、RNA干扰、Western blot、荧光素酶报告基因和染色质免疫共沉淀等分子生物学技术，鉴定TcAMPK对TcAK1与TcAK2的磷酸化位点，分析磷酸化修饰对TcAK1与TcAK2酶学性质的影响，探讨TcAMPK通过TcFOXO对TcAK1与TcAK2的转录调控机制。研究结果对于揭示昆虫响应氧化应激的分子机制具有重要的科学意义。

AMP-activated protein kinase (AMPK), Forkhead box class O (FOXO) and arginine kinase (AK) play key roles in the regulation of multiple cellular processes including energy metabolism and oxidative stress response. Our previous studies have found that RNAi-mediated knockdown of TcAMPKα,TcFOXO, TcAK1 and TcAK2 resulted in decreased tolerance of Tribolium castaneum to paraquat-induced oxidative stress, and paraquat-induced upregulation TcAK1 and TcAK2 was intensified by in vivo treatment with the AMPK-activating compound, AICAR, suggesting the putative interaction among these three proteins. In this project, by using yeast two-hybrid system, GST pull-down, in vitro phosphorylation, RNA interference, Western blot, Luciferase report gene assays, Chromatin immunoprecipitation, and other molecular biology techniques, we will identify the AMPK phosphorylation sites in TcAK1 and TcAK2, analyze the effects of phosphorylation modification on the enzyme activities of TcAK1 and TcAK2, investigate the role of AMPK-FOXO pathway in the transcriptional regulation of TcAK1 and TcAK2. The results of this project will provide novel insights into the molecular mechanisms of insect response to oxidative stress.