Abstract
Circadian rhythms in mammals are generated by a negative transcriptional feedback loop in which PERIOD (PER) is rate-limiting for feedback inhibition. Casein kinases Id and Ie(CKId/e) 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 CKId/e while avoiding the embryonic lethality of CKId disruption in mice, we used CKId-deficient Per2Luc mouse embryonic fibroblasts (MEFs) and overexpressed a dominant-negative mutant CKIe (DN-CKIe) in the mutant MEFs. CKId-deficient MEFs exhibited a robust circadian rhythm, albeit with a longer period, suggesting that the cells possess a way to compensate for CKId loss. When CKIe activity was disrupted by the DN-CKIe in the mutant MEFs, circadian bioluminescence rhythms were eliminated and rhythms in endogenous PER abundance and phosphorylation were severely compromised, demonstrating that CKId/e are indeed essential kinases for the clockwork. This is further supported by abolition of circadian rhythms when physical interaction between PER and CKId/e was disrupted by overexpressing the CKId/e binding domain of PER2 (CKBD-P2). Interestingly, CKBD-P2 overexpression led to dramatically low levels of endogenous PER, while PER-binding, kinase-inactive DN-CKIe did not, suggesting that CKId/e may have a noncatalytic role in stabilizing PER. Our results show that an essential role of CKId/e is conserved between Drosophila and mammals, but CKId/e 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 CKId/e are essential for the clockwork, we proposed to explore the dynamics of reversible PER phosphorylation by studying CKId/e conditional mutant mice / cells and by identifying protein phosphatases in targeting PER and characterizing them using genetic and biochemical approaches. We finally validated that CKId/e are essential protein kinases to facilitate driving clockwork based on our findings that CKId/e double KO cells have no circadian rhythms and they are rescued by transducing CKIe. 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 time driving/keeping mechanism in mammals.
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