保幼激素触发Kr-h1磷酸化而激活转录调控功能的分子机制

Krüppel homolog 1 (Kr-h1) serves as an early juvenile hormone (JH) response gene, whose transcription is activated by JH nuclear receptor. Kr-h1 plays critical roles in repressing larval metamorphosis and promoting adult reproduction via transcriptional regulation on target genes. However, it is still unknown that whether Kr-h1 could act on target genes transcription by own phosphorylation. Recently, we have detected phosphorylated Kr-h1 in locust and identified three Kr-h1 phosphorylation sites, whose mutation perturbed the action of Kr-h1 on target gene transcription. Based on these preliminary results, we attempt to: (1) analyze the Kr-h1 phosphorylation under JH deprivation and JHA induction or natural development, and ensure the dependence of Kr-h1 phosphorylation on JH; (2) confirm Kr-h1 phosphorylation sites, and identify candidate protein kinase for the regulation of Kr-h1 phosphorylation; then explore the signal pathway for JH activation on candidate protein kinase to screen crucial cascade factors and understand the molecular mechanisms of JH-triggered Kr-h1 phosphorylation; (3) elucidate the transcriptional regulation of Kr-h1 on target genes by phosphorylation, finally reveal that how Kr-h1 acts through phosphorylation to regulate locust reproduction. The results will give profound insights into our understanding in the molecular mechanisms of JH action.

Krüppel homolog 1（Kr-h1）是保幼激素（JH）的早期响应基因，受JH核受体转录激活后通过调控靶标基因的表达而抑制幼虫变态或促进成虫生殖，但Kr-h1是否通过自身的磷酸化而发挥转录调控作用还未阐述。我们最近发现，飞蝗Kr-h1能被磷酸化，并且鉴定了3个磷酸化位点，同时验证这些磷酸化位点在突变后抑制了Kr-h1的功能发挥。本项目拟在前期研究基础上：（1）分析JH剥夺和诱导及正常发育条件下，Kr-h1磷酸化的变化情况，明确Kr-h1磷酸化修饰对JH的依赖；（2）确认Kr-h1的磷酸化位点，鉴定调控Kr-h1磷酸化的蛋白激酶；探索JH激活候选蛋白激酶的信号通路，识别主要级联因子并揭示JH调控Kr-h1磷酸化的分子基础；（3）解析Kr-h1通过磷酸化而激活靶基因转录的调控机制，阐明Kr-h1磷酸化在飞蝗生殖中的功能。研究结果对剖析JH的分子作用机制具有重要意义。

保幼激素是昆虫变态和生殖调控的关键激素之一。Krüppel homolog-1 (Kr-h1)是JH早期响应基因，也是含8个保守锌指结构（C2H2）的转录因子，能传递JH信号发挥变态阻止和生殖促进的作用。然而，目前关于Kr-h1的调控研究仍然集中在JH-Met/Tai复合体介导的转录水平调控，翻译后修饰水平的研究未曾开展。通过这一项目的研究，申请人结合体内和体外多种磷酸化实验鉴定Ser154是飞蝗Kr-h1的磷酸化位点，并且解析了JH促进Kr-h1磷酸化的级联通路。利用蛋白-DNA互作实验证明了Kr-h1磷酸化对其发挥转录抑制和激活双重作用至关重要。同时证实了Kr-h1发挥双重作用与调节子有关，包括激活子和抑制子，并验证磷酸化修饰对于Kr-h1与调节子结合是必需的。最后，通过在全变态昆虫果蝇、家蚕和赤拟谷盗中实验，确定了Kr-h1磷酸化修饰和位点的保守性。