The role of clock protein phosphorylation and degradation in circadian biology

生物钟调节分子振荡，表现为生理和行为节奏。 尽管节律性mRNA表达在昼夜节律振荡器中发挥着重要作用，但一天中的时间特异性 时钟蛋白水平的变化对于昼夜节律起搏器的正常进展也是关键的。 翻译后机制已成为生物钟时间调节的重要因素 蛋白质水平和功能。动物生物钟的一个保守特征是PERIOD（PER）蛋白经历 水平和磷酸化状态的每日波动。在果蝇中，PER蛋白磷酸化状态是 由包括DOUBLETIME（DBT）[酪蛋白激酶le（哺乳动物中的C K I E）]和CK 2的激酶控制，如 以及蛋白磷酸酶1（PP 1）和2A（PP 2A）。过度磷酸化的PER蛋白最终 通过F-box蛋白SLIMB（哺乳动物中p-TrCP的同源物）靶向26 S蛋白酶体。当时- PER蛋白的日特异性磷酸化状态不仅调节PER蛋白的稳定性， 亚细胞定位、转录抑制剂活性和蛋白质-蛋白质相互作用。的总目标 这一建议是为了更好地了解时钟蛋白磷酸化和降解的作用 通过关注dPER（一种中枢时钟蛋白），研究了调节昼夜节律的途径。通过使用果蝇 作为一个模型，我建议（1）检查PER蛋白的磷酸化模式分离 在不同的生理相关条件下通过质谱分析和表征的功能， 使用S2细胞培养和体内转基因动物方法的dPER磷酸化事件。多重dPER 使用这种方法已经成功地鉴定了磷酸化位点簇。其中，我们发现 以dPER（S47）为中心的簇作为触发PER-SLIMB结合的关键磷酸决定簇。我们 目前正在分析其他集群在昼夜节律计时中的作用。此外，我建议（2） 剖析SLIMB介导的dPER降解途径，特别关注SLIMB后的步骤 约束力最后，我计划（3）表征新的dPER相互作用的蛋白质，这些蛋白质是用一步或 串联纯化，并确定它们在dPER磷酸化、代谢和功能中的作用。

Circadian clocks regulate molecular oscillations that manifest into physiological and behavioral rhythms. Despite the prominent role played by rhythmic mRNA expression in circadian oscillators, time-of-day specific changes in clock protein levels are also critical for the normal progression of circadian pacemakers. Posttranslational mechanisms have emerged as significant contributors to the temporal regulation of clock protein levels and function. A conserved feature of animal clocks is that PERIOD (PER) proteins undergo daily oscillations in levels and phosphorylation states. In Drosophiia, PER protein phosphorylation states are controlled by kinases including DOUBLETIME (DBT) [casein kinase le ( C K I E in mammals)] and CK2, as well as protein phosphatase 1 (PP1) and 2A (PP2A). Hyperphosphorylated PER proteins are eventually targeted to the 26S proteasome by the F-box protein SLIMB (a homolog of p-TrCP in mammals). The timeof- day specific phosphorylation states of PER proteins not only regulate PER protein stability, but also affect subcellular localization, transcriptional inhibitor activity, and protein-protein interactions. The overall goal of this proposal is to better understand the contribution of clock protein phosphorylation and degradation pathways in regulating circadian rhythms by focusing on dPER, a central clock protein. By using Drosophiia melanogaster as a model, I propose to (1) examine the phosphorylation profile of PER proteins isolated under different physiologically relevant conditions by mass spectral analysis and characterize the function of dPER phosphorylation events using S2 cell culture and in vivo transgenic animal approaches. Mulitple dPER phosphorylation site clusters have been successfully identified using this approach. Of which, we identified the cluster centered around dPER(S47) as key phosphodeterminants that triggers PER-SLIMB binding. We are currently analyzing other clusters for their role in circadian timekeeping. Furthermore, I propose to (2) dissect the SLIMB-mediated dPER degradation pathway, especially focusing on the steps post-SLIMB binding. Finally, I plan to (3) characterize novel dPER-interacting proteins that was isolated using one-step or tandem purification, and determine their role in dPER phosphorylation, metabolism, and function.