Department of Entomology and Nematology, College of Agricultural and Environmental Sciences, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA.
Department of Biology, University of Nevada, Reno, NV 89557, USA.
Curr Biol. 2021 Feb 8;31(3):502-514.e7. doi: 10.1016/j.cub.2020.10.061. Epub 2020 Nov 19.
Circadian clocks orchestrate daily rhythms in organismal physiology and behavior to promote optimal performance and fitness. In Drosophila, key pacemaker proteins PERIOD (PER) and TIMELESS (TIM) are progressively phosphorylated to perform phase-specific functions. Whereas PER phosphorylation has been extensively studied, systematic analysis of site-specific TIM phosphorylation is lacking. Here, we identified phosphorylation sites of PER-bound TIM by mass spectrometry, given the importance of TIM as a modulator of PER function in the pacemaker. Among the 12 TIM phosphorylation sites we identified, at least two of them are critical for circadian timekeeping as mutants expressing non-phosphorylatable mutations exhibit altered behavioral rhythms. In particular, we observed that CK2-dependent phosphorylation of TIM(S1404) promotes nuclear accumulation of PER-TIM heterodimers by inhibiting the interaction of TIM and nuclear export component, Exportin 1 (XPO1). We propose that proper level of nuclear PER-TIM accumulation is necessary to facilitate kinase recruitment for the regulation of daily phosphorylation rhythm and phase-specific transcriptional activity of CLOCK (CLK). Our results highlight the contribution of phosphorylation-dependent nuclear export of PER-TIM heterodimers to the maintenance of circadian periodicity and identify a new mechanism by which the negative elements of the circadian clock (PER-TIM) regulate the positive elements (CLK-CYC). Finally, because the molecular phenotype of tim(S1404A) non-phosphorylatable mutant exhibits remarkable similarity to that of a mutation in human timeless that underlies familial advanced sleep phase syndrome (FASPS), our results revealed an unexpected parallel between the functions of Drosophila and human TIM and may provide new insights into the molecular mechanisms underlying human FASPS.
生物钟协调生物体生理和行为的日常节律，以促进最佳表现和适应性。在果蝇中，关键的节律钟蛋白 PERIOD（PER）和 TIMELESS（TIM）逐渐被磷酸化以执行特定相位的功能。虽然 PER 的磷酸化已被广泛研究，但 TIM 特异性磷酸化的系统分析却缺乏。在这里，我们通过质谱法鉴定了 PER 结合 TIM 的磷酸化位点，鉴于 TIM 作为节律钟功能调节剂的重要性。在我们鉴定的 12 个 TIM 磷酸化位点中，至少有两个位点对于生物钟计时至关重要，因为表达不可磷酸化突变的突变体表现出行为节律的改变。特别是，我们观察到 CK2 依赖性 TIM(S1404)磷酸化通过抑制 TIM 与核输出成分 Exportin 1（XPO1）的相互作用，促进 PER-TIM 异二聚体的核积累。我们提出，适当水平的核 PER-TIM 积累对于促进激酶募集以调节每日磷酸化节律和 CLOCK（CLK）的相位特异性转录活性是必要的。我们的结果强调了 PER-TIM 异二聚体的磷酸化依赖性核输出对生物钟周期性维持的贡献，并确定了生物钟负元件（PER-TIM）调节正元件（CLK-CYC）的新机制。最后，由于 tim(S1404A)不可磷酸化突变体的分子表型与导致家族性提前睡眠阶段综合征（FASPS）的人类 timeless 突变非常相似，我们的结果揭示了果蝇和人类 TIM 之间功能的意外相似性，可能为人类 FASPS 的分子机制提供新的见解。
