CK2 Inhibits TIMELESS Nuclear Export and Modulates CLOCK Transcriptional Activity to Regulate Circadian Rhythms

Abstract 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 oscillator. Among the twelve 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 postulate 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 oscillator (PER-TIM) regulate the positive elements (CLK-CYC). Finally, since 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. Significance Statement Phosphorylation is a central mechanism important for the regulation of circadian physiology across organisms. The molecular oscillator is composed of pacemaker proteins that undergo elaborate phosphorylation programs to regulate phase-specific functions. In Drosophila , phosphorylation of TIMELESS (TIM) has been recognized as critical for its function in the oscillator, but a systematic analysis of TIM phosphorylation is lacking. Here, we identified twelve Drosophila TIM phosphorylation sites by mass spectrometry and showed that phosphorylation at TIM(S1404) is necessary for maintaining 24-hour rhythms. Finally, since the molecular phenotype of tim (S1404A) non-phosphorylatable fly mutant exhibits remarkable similarity to that of a mutation in human timeless that underlies FASPS, our results may provide new insights into the molecular underpinnings of human FASPS.