Essential roles of CKIδ and CKIε 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 I delta and I epsilon (CKI delta/epsilon) 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 CKI delta/epsilon while avoiding the embryonic lethality of CKI delta disruption in mice, we used CKI delta-deficient Per2(Luc) mouse embryonic fibroblasts (MEFs) and over-expressed a dominant-negative mutant CKI epsilon (DN-CKI epsilon) in the mutant MEFs. CKI delta-deficient MEFs exhibited a robust circadian rhythm, albeit with a longer period, suggesting that the cells possess a way to compensate for CKI delta loss. When CKI epsilon activity was disrupted by the DN-CKI epsilon in the mutant MEFs, circadian bioluminescence rhythms were eliminated and rhythms in endogenous PER abundance and phosphorylation were severely compromised, demonstrating that CKI delta/epsilon are indeed essential kinases for the clockwork. This is further supported by abolition of circadian rhythms when physical interaction between PER and CKI delta/epsilon was disrupted by overexpressing the CKI delta/epsilon binding domain of PER2 (CKBD-P2). Interestingly, CKBD-P2 overexpression led to dramatically low levels of endogenous PER, while PER-binding, kinase-inactive DN-CKI epsilon did not, suggesting that CKI delta/epsilon may have a non-catalytic role in stabilizing PER. Our results show that an essential role of CKI delta/epsilon is conserved between Drosophila and mammals, but CKI delta/epsilon and DBT may have divergent noncatalytic functions in the clockwork as well.