Essential roles of CKIδ and CKIε in the mammalian circadian clock

摘要

Circadian rhythms in mammals are generated by a negative transcrip-tional feedback loop in which PERIOD (PER) is rate-limiting for feedback inhibition. Casein kinases lδ and lε (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-def icient Per2~(Luc) mouse embryonic f ibroblasts (MEFs) and over-expressed 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 CKIS/e was disrupted by overexpressing the CKIS/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-CKIε did not, suggesting that CKIδ/ε may have a non-catalytic 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.