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. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2010.. / October 15, 2010. / Mouse embryonic fibroblast, Dominant negative, Casein kinase, Circadian clock / Includes bibliographical references. / Choogon Lee, Professor Directing Dissertation; James Fadool, University Representative; Mohamed Kabbaj, Committee Member; James Olcese, Committee Member; Yoichi Kato, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_181497 |
| Contributors | Lee, Hyeong-Min (authoraut), Lee, Choogon (professor directing dissertation), Fadool, James (university representative), Kabbaj, Mohamed (committee member), Olcese, James (committee member), Kato, Yoichi (committee member), Department of Biomedical Sciences (degree granting department), Florida State University (degree granting institution) |
| Publisher | Florida State University, Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text |
| Format | 1 online resource, computer, application/pdf |
| Rights | This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). The copyright in theses and dissertations completed at Florida State University is held by the students who author them. |
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