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      The CK2 Kinase Stabilizes CLOCK and Represses Its Activity in the Drosophila Circadian Oscillator

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          The CK2 kinase plays diverse roles in the Drosophila circadian clock by controlling stability of the CLOCK transcription factor itself, besides its known role in degrading CLOCK repressors.


          Phosphorylation is a pivotal regulatory mechanism for protein stability and activity in circadian clocks regardless of their evolutionary origin. It determines the speed and strength of molecular oscillations by acting on transcriptional activators and their repressors, which form negative feedback loops. In Drosophila, the CK2 kinase phosphorylates and destabilizes the PERIOD (PER) and TIMELESS (TIM) proteins, which inhibit CLOCK (CLK) transcriptional activity. Here we show that CK2 also targets the CLK activator directly. Downregulating the activity of the catalytic α subunit of CK2 induces CLK degradation, even in the absence of PER and TIM. Unexpectedly, the regulatory β subunit of the CK2 holoenzyme is not required for the regulation of CLK stability. In addition, downregulation of CK2α activity decreases CLK phosphorylation and increases per and tim transcription. These results indicate that CK2 inhibits CLK degradation while reducing its activity. Since the CK1 kinase promotes CLK degradation, we suggest that CLK stability and transcriptional activity result from counteracting effects of CK1 and CK2.

          Author Summary

          The CK2 kinase is an ancient enzyme known to be at the heart of self-sustaining circadian clocks in animals, plants, and fungi. Circadian clocks are responsible for daily circadian rhythms in molecular, physiological, and behavioral processes. Their mechanism relies on transcriptional activators and repressors that constitute a feedback loop. The CLOCK (CLK) activator is required to initiate the transcription of the period ( per) and timeless ( tim) genes in the late day. The PER and TIM repressors accumulate in a delayed manner and translocate to the nucleus to repress their own gene. Degradation of these repressors allows the activator to start a new cycle. In the fruit fly Drosophila melanogaster, CK2-mediated phosphorylation of the PER and TIM repressors targets them for degradation. Here we find that the CLK activator is also regulated by CK2. In contrast to PER and TIM, CLK is stabilized by CK2α phosphorylation; we also show that PER and TIM are dispensable for this stabilization of CLK. The identification of CK2–CLK protein complexes and the in vitro phosphorylation of CLK by CK2 hint at a direct action of the kinase on the activator. Although transcription factor stabilization is generally expected to be associated with increased activity, genetic analysis indicates that CK2 represses CLK. Hence CK2 phosphorylation seems to be a key signal for the transcriptional activator complex to adopt a proper activation state at a given point of the circadian cycle.

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          Most cited references 64

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          double-time is a novel Drosophila clock gene that regulates PERIOD protein accumulation.

          We have isolated three alleles of a novel Drosophila clock gene, double-time (dbt). Short- (dbtS) and long-period (dbtL) mutants alter both behavioral rhythmicity and molecular oscillations from previously identified clock genes, period and timeless. A third allele, dbtP, causes pupal lethality and eliminates circadian cycling of per and tim gene products in larvae. In dbtP mutants, PER proteins constitutively accumulate, remain hypophosphorylated, and no longer depend on TIM proteins for their accumulation. We propose that the normal function of DOUBLETIME protein is to reduce the stability and thus the level of accumulation of monomeric PER proteins. This would promote a delay between per/tim transcription and PER/TIM complex function, which is essential for molecular rhythmicity.
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            Clock mutants of Drosophila melanogaster.

            Three mutants have been isolated in which the normal 24-hour rhythm is drastically changed. One mutant is arrhythmic; another has a period of 19 hr; a third has a period of 28 hr. Both the eclosion rhythm of a population and the locomotor activity of individual flies are affected. All these mutations appear to involve the same functional gene on the X chromosome.
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              A mutant Drosophila homolog of mammalian Clock disrupts circadian rhythms and transcription of period and timeless.

              We report the identification, characterization, and cloning of a novel Drosophila circadian rhythm gene, dClock. The mutant, initially called Jrk, manifests dominant effects: heterozygous flies have a period alteration and half are arrhythmic, while homozygous flies are uniformly arrhythmic. Furthermore, these flies express low levels of the two clock proteins, PERIOD (PER) and TIMELESS (TIM), due to low per and tim transcription. Mapping and cloning of the Jrk gene indicates that it encodes the Drosophila homolog of mouse Clock. The mutant phenotype results from a premature stop codon that eliminates much of the putative activation domain of this bHLH-PAS transcription factor, thus explaining the dominant features of Jrk. The remarkable sequence conservation strongly supports common clock components present in the common ancestor of Drosophila and mammals.

                Author and article information

                Role: Academic Editor
                PLoS Biol
                PLoS Biol
                PLoS Biology
                Public Library of Science (San Francisco, USA )
                August 2013
                August 2013
                27 August 2013
                : 11
                : 8
                [1 ]Institut de Neurobiologie Alfred Fessard, Centre National de la Recherche Scientifique Unité Propre de Recherche 3294, Gif-sur-Yvette, France
                [2 ]Département de Biologie, Université Paris Sud, Orsay, France
                [3 ]Heidelberg University, Biochemistry Center (BZH), Im Neuenheimer Feld 328, Heidelberg, Germany
                [4 ]Institut de Chimie des Substances Naturelles, CNRS UPR2301, Gif-sur-Yvette, France
                [5 ]IMAGIF, Centre de Recherche de Gif, Gif-sur-Yvette, France
                [6 ]University of Wuerzburg, Institute of Medical Radiation and Cell Research, Wuerzburg, Germany
                University of Geneva, Switzerland
                Author notes

                The authors have declared that no competing interests exist.

                The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: AS FW PP FR. Performed the experiments: AS CP PP DZ. Analyzed the data: AS CP PP DZ FW FR. Contributed reagents/materials/analysis tools: TR. Wrote the paper: AS FR.


                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 16
                This work was funded by Agence Nationale de la Recherche “DrosoClock”, “ClockGene” and “FunGenDroso”, “Equipe FRM” program of Fondation pour la Recherche Médicale, and European Union 6th Framework Programme “EUCLOCK”. AS was partly supported by Association pour la Recherche sur le Cancer and FR is supported by Institut National de la Santé et des Etudes et Recherches Médicales. TR was funded by the DFG SFB1047 grant from Deutsche Forschungsgemeinschaft. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article
                Model Organisms
                Animal Models
                Drosophila Melanogaster
                Molecular Neuroscience

                Life sciences


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