Functional analysis of Casein Kinase 1 in a minimal circadian system.
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ABSTRACT:
SUBMITTER: van Ooijen G
PROVIDER: S-EPMC3723912 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Zebrafish have become a popular model for studies of the circadian timing mechanism. Taking advantage of its rapid development of a functional circadian clock and the availability of light-entrainable clock-containing cell lines, much knowledge has been gained about the circadian clock system in this species. However, the post-translational modifications of clock proteins, and in particular the phosphorylation of PER proteins by Casein kinase I delta and epsilon (CK1δ and CK1ε), have so far not been examined in the zebrafish. Using pharmacological inhibitors for CK1δ and CK1ε, a pan-CK1δ/ε inhibitor PF-670462, and a CK1ε -selective inhibitor PF-4800567, we show that CK1δ activity is crucial for the functioning of the circadian timing mechanism of zebrafish, while CK1ε plays a minor role. The CK1δ/ε inhibitor disrupted circadian rhythms of promoter activity in the circadian clock-containing zebrafish cell line, PAC-2, while the CK1ε inhibitor had no effect. Zebrafish larvae that were exposed to the CK1δ/ε inhibitor showed no rhythms of locomotor activity while the CK1ε inhibitor had only a minor effect on locomotor activity. Moreover, the addition of the CK1δ/ε inhibitor disrupted rhythms of aanat2 mRNA expression in the pineal gland. The pineal gland is considered to act as a central clock organ in fish, delivering a rhythmic hormonal signal, melatonin, which is regulated by AANAT2 enzymatic activity. Therefore, CK1δ plays a key role in the circadian timing system of the zebrafish. Furthermore, the effect of CK1δ inhibition on rhythmic locomotor activity may reflect its effect on the function of the central clock in the pineal gland as well as its regulation of peripheral clocks.
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Project description:Both casein kinase 1 delta (CK1delta) and epsilon (CK1epsilon) phosphorylate core clock proteins of the mammalian circadian oscillator. To assess the roles of CK1delta and CK1epsilon in the circadian clock mechanism, we generated mice in which the genes encoding these proteins (Csnk1d and Csnk1e, respectively) could be disrupted using the Cre-loxP system. Cre-mediated excision of the floxed exon 2 from Csnk1d led to in-frame splicing and production of a deletion mutant protein (CK1delta(Delta2)). This product is nonfunctional. Mice homozygous for the allele lacking exon 2 die in the perinatal period, so we generated mice with liver-specific disruption of CK1delta. In livers from these mice, daytime levels of nuclear PER proteins, and PER-CRY-CLOCK complexes were elevated. In vitro, the half-life of PER2 was increased by approximately 20%, and the period of PER2::luciferase bioluminescence rhythms was 2 h longer than in controls. Fibroblast cultures from CK1delta-deficient embryos also had long-period rhythms. In contrast, disruption of the gene encoding CK1epsilon did not alter these circadian endpoints. These results reveal important functional differences between CK1delta and CK1epsilon: CK1delta plays an unexpectedly important role in maintaining the 24-h circadian cycle length.
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Project description:Post-translational control of PERIOD stability by Casein Kinase 1δ and ε (CK1) plays a key regulatory role in metazoan circadian rhythms. Despite the deep evolutionary conservation of CK1 in eukaryotes, little is known about its regulation and the factors that influence substrate selectivity on functionally antagonistic sites in PERIOD that directly control circadian period. Here we describe a molecular switch involving a highly conserved anion binding site in CK1. This switch controls conformation of the kinase activation loop and determines which sites on mammalian PER2 are preferentially phosphorylated, thereby directly regulating PER2 stability. Integrated experimental and computational studies shed light on the allosteric linkage between two anion binding sites that dynamically regulate kinase activity. We show that period-altering kinase mutations from humans to Drosophila differentially modulate this activation loop switch to elicit predictable changes in PER2 stability, providing a foundation to understand and further manipulate CK1 regulation of circadian rhythms.
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Project description:The circadian clock provides organisms with the ability to adapt to daily and seasonal cycles. Eukaryotic clocks mostly rely on lineage-specific transcriptional-translational feedback loops (TTFLs). Posttranslational modifications are also crucial for clock functions in fungi and animals, but the posttranslational modifications that affect the plant clock are less understood. Here, using chemical biology strategies, we show that the Arabidopsis CASEIN KINASE 1 LIKE (CKL) family is involved in posttranslational modification in the plant clock. Chemical screening demonstrated that an animal CDC7/CDK9 inhibitor, PHA767491, lengthens the Arabidopsis circadian period. Affinity proteomics using a chemical probe revealed that PHA767491 binds to and inhibits multiple CKL proteins, rather than CDC7/CDK9 homologs. Simultaneous knockdown of Arabidopsis CKL-encoding genes lengthened the circadian period. CKL4 phosphorylated transcriptional repressors PSEUDO-RESPONSE REGULATOR 5 (PRR5) and TIMING OF CAB EXPRESSION 1 (TOC1) in the TTFL. PHA767491 treatment resulted in accumulation of PRR5 and TOC1, accompanied by decreasing expression of PRR5- and TOC1-target genes. A prr5 toc1 double mutant was hyposensitive to PHA767491-induced period lengthening. Together, our results reveal posttranslational modification of transcriptional repressors in plant clock TTFL by CK1 family proteins, which also modulate nonplant circadian clocks.
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Project description:Circadian pacemaking requires the orderly synthesis, posttranslational modification, and degradation of clock proteins. In mammals, mutations in casein kinase 1 (CK1) epsilon or delta can alter the circadian period, but the particular functions of the WT isoforms within the pacemaker remain unclear. We selectively targeted WT CK1epsilon and CK1delta using pharmacological inhibitors (PF-4800567 and PF-670462, respectively) alongside genetic knockout and knockdown to reveal that CK1 activity is essential to molecular pacemaking. Moreover, CK1delta is the principal regulator of the clock period: pharmacological inhibition of CK1delta, but not CK1epsilon, significantly lengthened circadian rhythms in locomotor activity in vivo and molecular oscillations in the suprachiasmatic nucleus (SCN) and peripheral tissue slices in vitro. Period lengthening mediated by CK1delta inhibition was accompanied by nuclear retention of PER2 protein both in vitro and in vivo. Furthermore, phase mapping of the molecular clockwork in vitro showed that PF-670462 treatment lengthened the period in a phase-specific manner, selectively extending the duration of PER2-mediated transcriptional feedback. These findings suggested that CK1delta inhibition might be effective in increasing the amplitude and synchronization of disrupted circadian oscillators. This was tested using arrhythmic SCN slices derived from Vipr2(-/-) mice, in which PF-670462 treatment transiently restored robust circadian rhythms of PER2::Luc bioluminescence. Moreover, in mice rendered behaviorally arrhythmic by the Vipr2(-/-) mutation or by constant light, daily treatment with PF-670462 elicited robust 24-h activity cycles that persisted throughout treatment. Accordingly, selective pharmacological targeting of the endogenous circadian regulator CK1delta offers an avenue for therapeutic modulation of perturbed circadian behavior.
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Project description:Casein kinase 2 (CK2) is a protein kinase that phosphorylates a plethora of cellular target proteins involved in processes including DNA repair, cell cycle control, and circadian timekeeping. CK2 is functionally conserved across eukaryotes, although the substrate proteins identified in a range of complex tissues are often different. The marine alga Ostreococcus tauri is a unicellular eukaryotic model organism ideally suited to efficiently study generic roles of CK2 in the cellular circadian clock. Overexpression of CK2 leads to a slow circadian rhythm, verifying functional conservation of CK2 in timekeeping. The proteome was analysed in wild-type and CK2-overexpressing algae at dawn and dusk, revealing that differential abundance of the global proteome across the day is largely unaffected by overexpression. However, CK2 activity contributed more strongly to timekeeping at dusk than at dawn. The phosphoproteome of a CK2 overexpression line and cells treated with CK2 inhibitor was therefore analysed and compared to control cells at dusk. We report an extensive catalogue of 447 unique CK2-responsive differential phosphopeptide motifs to inform future studies into CK2 activity in the circadian clock of more complex tissues. All MS data have been deposited in the ProteomeXchange with identifier PXD000975 (http://proteomecentral.proteomexchange.org/dataset/PXD000975).
| S-EPMC4716292 | biostudies-literature
Project description:Casein Kinase 2 (CK2) is an evolutionarily conserved kinase protein that phosphorylates a plethora of cellular target protein involved in processes including DNA repair, cell cycle control, and circadian rhythms. CK2 is functionally conserved across all eukaryotes, although the substrate proteins identified in targeted experiments on a diverse range of complex tissues are often different. A growing interest exists to identify conserved, core signalling principles essential to eukaryotic life. To efficiently study generic roles of CK2 in the cellular circadian clock, we used a minimal eukaryotic model organism (Ostreococcus tauri). Overexpression of CK2 leads to a slow circadian rhythm, verifying functional conservation of CK2 in cellular timekeeping. CK2 activity contributes more strongly to the clock around dusk than around dawn, and analysis of the phospho-proteome at this time revealed a strong overrepresentation of potential CK2 sites. Large changes to the phospho-proteome are reported here upon overexpression of CK2, identifying a set of CK2-responsive phospho-sites. To identify more CK2-specific phosphorylation events, the phospho-proteome of cells treated with CK2 inhibitor were analysed and compared to the overexpression lines. We thus report a full inventory of the CK2-responsive phospho-proteome to inform studies into CK2 activity in the circadian clock of more complex tissues, as science moves to understand and modulate cellular circadian rhythms to help us negate the negative effects of shiftwork or cross-continental travel.
2015-08-11 | PXD000975 | Pride
Project description:The circadian clock is a timekeeping system for regulation of numerous biological daily rhythms. One characteristic of the circadian clock is that period length remains relatively constant in spite of environmental fluctuations, such as temperature change. Here, using the curated collection of in-house small molecule chemical library (ITbM chemical library), we show that small molecule 3,4-dibromo-7-azaindole (B-AZ) lengthened the circadian period of Arabidopsis thaliana (Arabidopsis). B-AZ has not previously been reported to have any biological and biochemical activities. Target identification can elucidate the mode of action of small molecules, but we were unable to make a molecular probe of B-AZ for target identification. Instead, we performed other analysis, gene expression profiling that potentially reveals mode of action of molecules. Short-term treatment of B-AZ decreased the expression of four dawn- and morning-phased clock-associated genes, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), PSEUDO-RESPONSE REGULATOR 9 (PRR9) and PRR7. Consistently, amounts of PRR5 and TIMING OF CAB EXPRESSION 1 (TOC1) proteins, transcriptional repressors of CCA1, LHY, PRR9 and PRR7 were increased upon B-AZ treatment. B-AZ inhibited Casein Kinase 1 family (CK1) that phosphorylates PRR5 and TOC1 for targeted degradation. A docking study and molecular dynamics simulation suggested that B-AZ interacts with the ATP-binding pocket of human CK1 delta, whose amino acid sequences are highly similar to those of Arabidopsis CK1. B-AZ-induced period-lengthening effect was attenuated in prr5 toc1 mutants. Collectively, this study provides a novel and simple structure CK1 inhibitor that modulates circadian clock via accumulation of PRR5 and TOC1.
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Project description:Temperature compensation and period determination by casein kinase 1 (CK1) are conserved features of eukaryotic circadian rhythms, whereas the clock gene transcription factors that facilitate daily gene expression rhythms differ between phylogenetic kingdoms. Human red blood cells (RBCs) exhibit temperature-compensated circadian rhythms, which, because RBCs lack nuclei, must occur in the absence of a circadian transcription-translation feedback loop. We tested whether period determination and temperature compensation are dependent on CKs in RBCs. As with nucleated cell types, broad-spectrum kinase inhibition with staurosporine lengthened the period of the RBC clock at 37°C, with more specific inhibition of CK1 and CK2 also eliciting robust changes in circadian period. Strikingly, inhibition of CK1 abolished temperature compensation and increased the Q10 for the period of oscillation in RBCs, similar to observations in nucleated cells. This indicates that CK1 activity is essential for circadian rhythms irrespective of the presence or absence of clock gene expression cycles.
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Project description:The molecular oscillator that keeps circadian time is generated by a negative feedback loop. Nuclear entry of circadian regulatory proteins that inhibit transcription from E-box-containing promoters appears to be a critical component of this loop in both Drosophila and mammals. The Drosophila double-time gene product, a casein kinase I epsilon (CKIepsilon) homolog, has been reported to interact with dPER and regulate circadian cycle length. We find that mammalian CKIepsilon binds to and phosphorylates the murine circadian regulator mPER1. Unlike both dPER and mPER2, mPER1 expressed alone in HEK 293 cells is predominantly a nuclear protein. Two distinct mechanisms appear to retard mPER1 nuclear entry. First, coexpression of mPER2 leads to mPER1-mPER2 heterodimer formation and cytoplasmic colocalization. Second, coexpression of CKIepsilon leads to masking of the mPER1 nuclear localization signal and phosphorylation-dependent cytoplasmic retention of both proteins. CKIepsilon may regulate mammalian circadian rhythm by controlling the rate at which mPER1 enters the nucleus.
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