Essential Roles of CKIδ/ε in the Mammalian Circadian Clock

Circadian rhythms in mammals are generated by a negative transcriptional feedback loop in which PERIOD (PER) is rate-limiting for feedback inhibition. Casein kinases Iδ and Iε (CKIδ/ε) can regulate temporal abundance/activity of PER by phosphorylation-mediated degradation and cellular localization. Despite their potentially crucial effects on PER, it has not been demonstrated in a mammalian system that these kinases play essential roles in circadian rhythm generation as does their homolog in Drosophila. To disrupt both CKIδ/ε while avoiding the embryonic lethality of CKIδ disruption in mice, we used CKIδ-deficient Per2Luc mouse embryonic fibroblasts (MEFs) and overexpressed a dominant-negative mutant CKIε (DN-CKIε) in the mutant MEFs. CKIδ-deficient MEFs exhibited a robust circadian rhythm, albeit with a longer period, suggesting that the cells possess a way to compensate for CKIδ loss. When CKIε activity was disrupted by the DN-CKIε in the mutant MEFs, circadian bioluminescence rhythms were eliminated and rhythms in endogenous PER abundance and phosphorylation were severely compromised, demonstrating that CKIδ/ε are indeed essential kinases for the clockwork. This is further supported by abolition of circadian rhythms when physical interaction between PER and CKIδ/ε was disrupted by overexpressing the CKIδ/ε binding domain of PER2 (CKBD-P2). Interestingly, CKBD-P2 overexpression led to dramatically low levels of endogenous PER, while PER-binding, kinase-inactive DN-CKIε did not, suggesting that CKIδ/ε may have a noncatalytic role in stabilizing PER. Our results show that an essential role of CKIδ/ε is conserved between Drosophila and mammals, but CKIδ/ε and DBT may have divergent non-catalytic functions in the clockwork as well. Since reversible phosphorylation events in the circadian clock are thought to be involved in temporal regulation of clock proteins, a dynamic process of clock proteins mediated by protein kinases and phosphatases may be an essential feature in the time-keeping mechanism in mammals. To address these issues more definitively and extend findings that CKIδ/ε are essential for the clockwork, we proposed to explore the dynamics of reversible PER phosphorylation by studying CKIδ/ε conditional mutant mice / cells and by identifying protein phosphatases in targeting PER and characterizing them using genetic and biochemical approaches. We finally validated that CKIδ/ε are essential protein kinases to facilitate driving clockwork based on our findings that CKIδ/ε double KO cells have no circadian rhythms and x they are rescued by transducing CKIε. Moreover, PP1 is highly associated with PER dephosphorylation based on our results in genetic (dominant negative PP1) and chemical approaches (phosphatase inhibitors: OA vs. CA). Therefore, we propose that dynamic and reversible processes mediated by kinases and phosphatases are essential features in the timedriving/ keeping mechanism in mammals.