Casein Kinase 1 Alpha (CK1a) Promotes Nuclear Entry of PERIOD and Represses CLOCK transcriptional activity in the Drosophila melanogaster Circadian Clock

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Circadian clocks endow organisms with the ability to temporally coordinate and synchronize their physiology with the surrounding environment, thereby conferring an adaptive advantage. Although the specific clock genes are not highly conserved, the basic design and regulation of the circadian oscillator is remarkably similar across species. In Drosophila melanogaster, the central oscillator exists as interconnected transcriptional translational feedback loops that drive and sustain rhythm. Essential to this regulatory network are the transcriptional activator and repressor: CLOCK (CLK) and PERIOD (PER), respectively, as mutations in these proteins result in arrhythmicity. Functional PER and CLK, however, undergo daily cycle of phosphorylation that regulates both the abundance and the timing for which they act as regulators of the circadian transcriptome. To comprehensively identify proteins responsible for these phosphorylation events, we performed proteomics studies using affinity purification (AP) and label‐free quantitative mass spectrometry (MS) using Drosophila S2 cells. We annotate our result by incorporating published PPIs, knowledge about common AP‐MS contaminants, as well as protein coevolution data. We identified Casein Kinase 1 alpha (CK1α) as a clock kinase that phosphorylates CLK to promote CLK clearance at the per promoter and thereby terminating CLK transcriptional activity. Furthermore, CK1α regulates the timing of PER nuclear entry in the cytoplasm, whereas in the nucleus, CK1α phosphorylates and destabilizes PER.