The role of clock protein phosphorylation and degradation in circadian biology

生物钟调节表现为生理和行为节律的分子振荡。 尽管节律性的mRNA表达在昼夜节律振荡器中起着显著的作用，但一天中的特定时间 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至关重要的。 翻译后机制已经成为时钟时间调节的重要因素 蛋白质水平和功能。动物时钟的一个保守特征是周期(PER)蛋白质经历 水平和磷酸化状态的每日波动。在Drosophiia中，每种蛋白质的磷酸化状态是 由包括双倍时间(DBT)[酪蛋白激酶LE(哺乳动物中的C K I E)]和CK2，AS 蛋白磷酸酶1(PP1)和蛋白磷酸酶2A(PP2A)。过度磷酸化的PER蛋白最终会 F-box蛋白SLIMB(哺乳动物中p-TrCP的同源蛋白)靶向26S蛋白酶体。时间是- PER蛋白的日特异性磷酸化状态不仅调节PER蛋白的稳定性，而且还影响 亚细胞定位、转录抑制活性和蛋白质-蛋白质相互作用。的总目标是 这项建议是为了更好地了解时钟蛋白的磷酸化和降解的贡献。 通过关注中枢时钟蛋白dPER调节昼夜节律的途径。通过使用Drosophiia 作为一个模型，我建议(1)检测分离的PER蛋白的磷酸化图谱 在不同生理相关条件下进行质谱分析，表征其功能 使用S2细胞培养和体内转基因动物方法的dPER磷酸化事件。多个dPER 使用这种方法已经成功地鉴定了磷酸化位点簇。其中，我们确认了 该簇以dPER(S47)为中心，作为触发Per-SLIMB结合的关键磷酸决定因素。我们 目前正在分析其他星系团在昼夜节律中的作用。此外，我建议(2) 剖析SLIMB介导的dPER降解途径，尤其是SLIMB后的步骤 有约束力的。最后，我计划(3)鉴定新的dPER相互作用蛋白，这些蛋白是用一步或两步法分离的 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.