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Long period phenotype induced by CK1 overexpression. (A) Free-running period was analysed by bioluminescent imaging in 6 independent transgenic lines overexpressing CK1 in the CCA1-LUC background. Lines were compared against the parent line in identical plate positions. In all cases, a significantly (p<0.001) long circadian period was observed (n=8). (B) Example traces of luminescent lines overexpressing CK1 (CK1-OX8, blue) compared to the parent line CCA1-LUC (black) in constant light. doi: 10.1371/journal.pone.0070021.g002
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The Earth's rotation has driven the evolution of cellular circadian clocks to facilitate anticipation of the solar cycle. Some evidence for timekeeping mechanism conserved from early unicellular life through to modern organisms was recently identified, but the components of this oscillator are currently unknown. Although very few clock components a...
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... effect of overexpression on circadian period was analysed by bioluminescent imaging in constant light. A long-period phenotype was associated with CK1 overexpression in all six verified independent transgenic lines ( Figure 2A) as visualised by the CCA1-LUC construct ( Figure 2B), strongly indicating CK1 functions in timekeeping in this organism in spite of the absence of known clock-relevant CK1 targets identified in other taxa. ...
Context 2
... effect of overexpression on circadian period was analysed by bioluminescent imaging in constant light. A long-period phenotype was associated with CK1 overexpression in all six verified independent transgenic lines ( Figure 2A) as visualised by the CCA1-LUC construct ( Figure 2B), strongly indicating CK1 functions in timekeeping in this organism in spite of the absence of known clock-relevant CK1 targets identified in other taxa. ...
Context 3
... observation of period lengthening by both CK1 overexpression (Figure 2) as well as inhibition (Figure 3) appears to be contradictory, as the naive hypothesis would be that these treatments would induce opposite period effects. However, we find that both constitutively heightened CK1 activity by overexpression, as well as constitutively lowered activity by chemical inhibition induces long periodicity, indicating that CK1 activity exhibits complicated effects on timekeeping. ...
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Casein Kinase 1 (CK1) is one of few proteins known to affect cellular timekeeping across metazoans, and the naturally occurring CK1tau mutation shortens circadian period in mammals. Functional conservation of a timekeeping function for CK1 in the green lineage was recently identified in the green marine unicell Ostreococcus tauri, in spite of the a...
Citations
... The structural organization and function of CK1s are conserved across eukaryotes, as they play pivotal roles in a wide spectrum of cellular functions such as cell cycle progression, developmental regulation, and stress responses [3,4,[10][11][12]. In both plants and humans (Homo sapiens), CK1 members share substantial sequence identity (63%-92%) in their catalytic domain ($280 amino acids) with similarly predicted three-dimensional structure and ATPbinding sites [3,13,14]. ...
... (b) Representative photograph of siliques (left; bar, 200 lm) and mean seed abortion rate (right) of Col-0, AEL-OE lines and ael triple mutants from siliques at 7 days after fertilization (DAF). (c) Observations of Col-0, ael123 and c3h17 embryos at the early globular stage(1,6,11,16), late globular stage(2,7,12,17), heart stage(3,8,13,18), torpedo stage(4,9,14,19) and early cotyledon stage(5,10,15,20). Siliques after fertilization (1-7 d) were observed and representative images are shown. ...
Casein kinase 1s (CK1s) are serine/threonine protein kinases highly conserved among eukaryotes that regulate multiple developmental and signaling events through phosphorylation of target proteins. Arabidopsis early flowering 1 (EL1)-like (AELs) are plant-specific CK1s with varied functions, but identification and validation of their substrates is a major bottleneck in elucidating their physiological roles. Here, we conducted a quantitative phosphoproteomic analysis in data-independent acquisition mode to systematically identify CK1 substrates. We extracted proteins from seedlings overexpressing individual AEL genes (AEL1/2/3/4-OE) or lacking AEL function (all ael single mutants and two triple mutants) to identify the high-confidence phosphopeptides with significantly altered abundance compared to wild-type Col-0. Among these, we selected 3985 phosphopeptides with higher abundance in AEL-OE lines or lower abundance in ael mutants compared with Col-0 as AEL-upregulated phosphopeptides, and defined 1032 phosphoproteins. Eight CK1s substrate motifs were enriched among AEL-upregulated phosphopeptides and verified, which allowed us to predict additional candidate substrates and functions of CK1s. We functionally characterized a newly identified substrate C3H17, a CCCH-type zinc finger transcription factor, through biochemical and genetic analyses, revealing a role for AEL-promoted C3H17 protein stability and transactivation activity in regulating embryogenesis. As CK1s are highly conserved across eukaryotes, we searched the rice, mouse, and human protein databases using newly identified CK1 substrate motifs, yielding many more candidate substrates than currently known, largely expanding our understanding of the common and distinct functions exerted by CK1s in Arabidopsis and humans, facilitating future mechanistic studies of CK1-mediated phosphorylation in different species.
... Thus, it was postulated that the C. reinhardtii clock may have a contrasting mechanism compared to A. thaliana and O. tauri (Matsuo et al., 2020). This hypothesis is consistent with a different effect on period caused by a knockdown mutant or inhibition of CK1 from O. tauri and A. thaliana compared to C. reinhardtii (Box 1; Schmidt et al., 2006;van Ooijen et al., 2013;Uehara et al., 2019). In general, CK1 and CK2 are involved in posttranslational regulation of the clock in several organisms and are recently even discussed with regard to temperature compensation (Kusakina and Dodd, 2012;Hu et al., 2021;Narasimamurthy and Virshup, 2021). ...
... But we would like to highlight that reversible phosphorylation plays a major role in diverse organisms including not only algae and land plants (Kusakina and Dodd, 2012;Noordally and Millar, 2015;Krahmer et al., 2021), but also fungi (Liu et al., 2000), flies, and mammals (Narasimamurthy and Virshup, 2021). CKs (CK1 and CK2) are well conserved across species and are also found to be of importance in unicellular algae (Mittag et al., 2005;Schmidt et al., 2006;van Ooijen et al., 2013). The role of CKs has been demonstrated in the circadian systems of the chlorophyte algae O. tauri and C. reinhardtii, but also in the A. thaliana angiosperm clock. ...
... The role of CKs has been demonstrated in the circadian systems of the chlorophyte algae O. tauri and C. reinhardtii, but also in the A. thaliana angiosperm clock. A CK1 knockdown shortens the period in C. reinhardtii while inhibition of CK1 in O. tauri and A. thaliana lengthens the period (van Ooijen et al., 2013;Uehara et al., 2019). These effects underline that the clocks of O. tauri and A. thaliana may be closer in their mechanisms than the C. reinhardtii clock (Matsuo et al., 2020). ...
Circadian clocks govern temporal programs in the green lineage (Chloroplastida) as they do in other photosynthetic pro- and eukaryotes, bacteria, fungi, animals, and humans. Their physiological properties, including entrainment, phase responses, and temperature compensation, are well conserved. The involvement of transcriptional/translational feedback loops in the oscillatory machinery and reversible phosphorylation events are also maintained. Circadian clocks control a large variety of output rhythms in green algae and terrestrial plants, adjusting their metabolism and behavior to the day-night cycle. The angiosperm Arabidopsis (Arabidopsis thaliana) represents a well-studied circadian clock model. Several molecular components of its oscillatory machinery are conserved in other Chloroplastida, but their functions may differ. Conserved clock components include at least one member of the CIRCADIAN CLOCK ASSOCIATED1/REVEILLE and one of the PSEUDO RESPONSE REGULATOR family. The Arabidopsis evening complex (EC) members EARLY FLOWERING3, ELF4, and LUX ARRHYTHMO are found in the moss Physcomitrium patens and in the liverwort Marchantia polymorpha. In the flagellate chlorophyte alga Chlamydomonas reinhardtii, only homologs of ELF4 and LUX [(named RHYTHM OF CHLOROPLAST ROC75)] are present. Temporal ROC75 expression in C. reinhardtii is opposite that of the angiosperm LUX, suggesting different clock mechanisms. In the picoalga Ostreococcus tauri, both ELF genes are missing, suggesting that it has a progenitor circadian "green" clock. Clock-relevant photoreceptors and thermosensors vary within the green lineage, except for the CRYPTOCHROMEs, whose variety and functions may differ. More genetically tractable models of Chloroplastida are needed to draw final conclusions about the gradual evolution of circadian clocks within the green lineage.
... LKP2 has a key role in responses to different photoperiods, acting within the circadian oscillator by interacting with TOC1 and PRR5 proteins 38,49 and contributing to PRR5 degradation 50 . Protein phosphorylation also participates in plant circadian regulation, with roles for CK1-and CK2-family protein kinases in the phosphorylation of components of the Arabidopsis oscillator [51][52][53][54] . There is also extensive circadian regulation of protein phosphorylation. ...
Circadian regulation has a pervasive influence upon plant development, physiology and metabolism, impacting upon components of fitness and traits of agricultural importance. Circadian regulation is inextricably connected to the responses of plants to their abiotic environments, from the cellular to whole plant scales. Here, we review the crosstalk that occurs between circadian regulation and responses to the abiotic environment from the intracellular scale through to naturally fluctuating environments. We examine the spatial crosstalk that forms part of plant circadian regulation, at the subcellular, tissue, organ and whole-plant scales. This includes a focus on chloroplast and mitochondrial signalling, alternative splicing, long-distance circadian signalling and circadian regulation within natural environments. We also consider mathematical models for plant circadian regulation, to suggest future areas for advancing understanding of roles for circadian regulation in plant responses to environmental cues.
... One possibility is that C. reinhardtii has developed a contrasting mechanism of operating the circadian clock to that of A. thaliana and O. tauri. Consistent with this notion, the effect of changes in the casein kinase 1 activity on the circadian clock also shows a sharp contrast; the circadian rhythm is shortened by an RNAi-mediated knockdown in C. reinhardtii [48], whereas it is lengthened by specific inhibitor treatments or misexpression in A. thaliana and O. tauri [49,50]. ...
The circadian clocks in chlorophyte algae have been studied in two model organisms, Chlamydomonas reinhardtii and Ostreococcus tauri. These studies revealed that the chlorophyte clocks include some genes that are homologous to those of the angiosperm circadian clock. However, the genetic network architectures of the chlorophyte clocks are largely unknown, especially in C. reinhardtii. In this study, using C. reinhardtii as a model, we characterized RHYTHM OF CHLOROPLAST (ROC) 75, a clock gene encoding a putative GARP DNA-binding transcription factor similar to the clock proteins LUX ARRHYTHMO (LUX, also called PHYTOCLOCK 1 [PCL1]) and BROTHER OF LUX ARRHYTHMO (BOA, also called NOX) of the angiosperm Arabidopsis thaliana. We observed that ROC75 is a day/subjective day-phase-expressed nuclear-localized protein that associates with some night-phased clock genes and represses their expression. This repression may be essential for the gating of reaccumulation of the other clock-related GARP protein, ROC15, after its light-dependent degradation. The restoration of ROC75 function in an arrhythmic roc75 mutant under constant darkness leads to the resumption of circadian oscillation from the subjective dawn, suggesting that the ROC75 restoration acts as a morning cue for the C. reinhardtii clock. Our study reveals a part of the genetic network of C. reinhardtii clock that could be considerably different from that of A. thaliana.
... Casein kinase I (CK1), a highly conserved serine/threonine (Ser/Thr) protein kinase, has been identified ubiquitously in eukaryotes ranging from yeast to humans since the 1970s [1][2][3][4]. The dual-specificity Ser/Thr kinases evolutionary conserved on both structure and function in eukaryotes have been identified as a family of monomeric kinases [5][6][7][8][9]. In mammals, seven CK1 isoforms (alpha, beta, gamma1-3, delta and epsilon), together with the various splice variants, are involved in a variety of cellular processes, such as chromosome segregation and cellular differentiation, by phosphorylating a wide range of key regulatory proteins [9][10][11][12][13]. ...
... Application of CKI-7 has effectively eliminated the catalytic activity of CK1 in a wide spectrum of plant species, such as rice, broccoli and Chlamydomonas [14,30,31]. PF670462, a highly CK1-selective inhibitor, and small molecule IC261 have been applied in investigation of CKL-mediated circadian rhythm [7,32]. Recent chemical screening has demonstrated that PHA767491, an animal CDC7 (cell division control protein 7) inhibitor, and analogs, such as AMI-23, -212 and -331, inhibited CK1 activity [20,33]. ...
... CK1 has been known to affect timekeeping across metazoans and fungi [9,62]. Early evidence of the implication of plant CK1 in circadian clock was found in a unicellular marine algal species Ostreococcus tauri [7]. The basal alga of the green lineage contained only one CK1 with diurnal fluctuation. ...
: Casein kinase I (CK1), a ubiquitous serine/threonine (Ser/Thr) protein kinase in eukaryotes, plays pivotal roles in a wide spectrum of cellular functions including metabolism, cell cycle progression, developmental control and stress responses. Plant CK1 evolves a lineage expansion, resulting in a unique branch of members exclusive to the kingdom. Among them, Arabidopsis Mut9p-LIKE KINASEs (MLKs) target diverse substrates including histones and the key regulatory proteins involving in physiological processes of light signaling, circadian rhythms, phytohormone and plant defense. Deregulation of the kinase activity by mutating the enzyme or the phosphorylation sites of substrates causes developmental disorders and susceptibility to adverse environmental conditions. MLKs have evolved as a general kinase that modifies transcription factors or primary regulatory proteins in a dynamic way. Here, we summarize the current knowledge of the roles of MLKs and MLK orthologs in several commercially important crops.
... Despite the importance of CK1 for circadian timing in eukaryotic organisms from humans to Drosophila, Neurospora, and green algae (Gö rl et al., 2001;Kloss et al., 1998;van Ooijen et al., 2013;Xu et al., 2005), little is known about how its activity is regulated on clock protein substrates. CK1 is thought of as an anion-or phosphate-directed kinase, relying on negative charge on the substrate to template activity in the pSxxS consensus motif (Flotow et al., 1990). ...
... Despite its powerful control over the timing of circadian rhythms in eukaryotes from humans to green algae (van Ooijen et al., 2013;Xu et al., 2005), very little is known about the molecular determinants of CK1d substrate selectivity and activity. We discovered a conformational switch in the activation loop of CK1 that regulates its activity on two regulatory regions in PER2 that control its stability and circadian timing in mammals (Zhou et al., 2015). ...
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.
... Las proteínas circadianas están sujetas a regulación por varias cascadas de señalización alternativas, como DBP, ROR y REV-ERB 15 . Asimismo, las proteínas circadianas son susceptibles a modificación enzimática luego del proceso de traducción génica, especialmente a través de la actividad de la protein-fosfatasa 1 (PP1) y la caseinkinasa 1 δ/ε (CK1δ/ε) 16 . ...
Chronic non-communicable diseases
dominate the current world epidemiologic landscape, with cardiovascular disease and type 2 diabetes mellitus being responsible
for high morbidity and mortality. The metabolic syndrome
(MS) is notorious for grouping a constellation of risk factors for these entities. Recent research has underlined the
importance of behavioral aspects as a new dimension of the
psychobiological habits which may promote or prevent the
development of MS. Among these, the impact of circadian
rhythms in the pathophysiology of MS has been a key point
of scientific interest. The mechanisms underlying this circadian adaptation are centered on the modulation of genetic
expression throughout the day, in response to distinct synchronizing cues. When these stimuli are natural, adaptation
to the medium is preserved, whereas the influence of deleterious cues may result in maladaptation to the environment.
Circadian dysregulation impacts each component of the MS
in a particular and specific manner, including carbohydrate
and lipid metabolism, blood pressure regulation, and numerous other aspects. Evidence describing the impact of circadian rhythms on the pathophysiology of MS is abundant,
justifying their consideration in the preventive and therapeutic approach to this entity. This may yield a significant
improvement in patients’ quality of life. The future appears
to bring many other opportunities for intervention upon the
chronobiologic molecular mechanisms, which would represent a major revolution in medical therapeutics. This review
summarizes current evidence on the pathogeny of the MS
from a chronobiologic perspective, as well as their therapeutic implications.
... Despite the importance of CK1 for circadian timing in eukaryotic organisms from humans to Drosophila, Neurospora, and green algae (Gö rl et al., 2001;Kloss et al., 1998;van Ooijen et al., 2013;Xu et al., 2005), little is known about how its activity is regulated on clock protein substrates. CK1 is thought of as an anion-or phosphate-directed kinase, relying on negative charge on the substrate to template activity in the pSxxS consensus motif (Flotow et al., 1990). ...
... Despite its powerful control over the timing of circadian rhythms in eukaryotes from humans to green algae (van Ooijen et al., 2013;Xu et al., 2005), very little is known about the molecular determinants of CK1d substrate selectivity and activity. We discovered a conformational switch in the activation loop of CK1 that regulates its activity on two regulatory regions in PER2 that control its stability and circadian timing in mammals (Zhou et al., 2015). ...
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 activation loop to define substrate selectivity on mammalian PER2, thereby directly regulating its 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.
... thus often possess only one copy of genes that are multigene families in other photosynthetic organisms, so alteration of individual genes by directed mutagenesis immediately gives rise to new phenotypes [14,15]. Various kinds of biological analyses involving these algae are being undertaken, for example, the functional analysis of casein kinase 1 in cell cycle [16], nitrogen status sensing by calcium-dependent protein kinases (CDPKs) [17], ...
Ostreococcustauri is an easily cultured representative of unicellular algae (class Mamiellophyceae) that abound in oceans worldwide. Eight complete 13–22 Mb genomes of phylogenetically divergent species within this class are available, and their DNA sequences are nearly always present in metagenomic data produced from marine samples. Here we describe a simplified and robust transformation protocol for the smallest of these algae (O. tauri). Polyethylene glycol (PEG) treatment was much more efficient than the previously described electroporation protocol. Short (2 min or less) incubation times in PEG gave >104 transformants per microgram DNA. The time of cell recovery after transformation could be reduced to a few hours, permitting the experiment to be done in a day rather than overnight as used in previous protocols. DNA was randomly inserted in the O. tauri genome. In our hands PEG was 20–40-fold more efficient than electroporation for the transformation of O. tauri, and this improvement will facilitate mutagenesis of all of the dispensable genes present in the tiny O. tauri genome.
... CASEIN KINASE (CK) 1 is an evolutionarily conserved kinase that regulates circadian periodicity in fungi, animals, and algae, but the substrates of CK1 differ greatly across lineages. CK1 phosphorylates FREQUENCY (FRQ) in fungi (5) and PERIOD in mice (animals) (6), but the substrates of CK1 in algae are as yet unknown (7,8). ...
... Two independent experiments showed that protein kinases, including the CK1 family (CKL), the shaggy-related protein kinase 3 (GSK3) family, AT2G32850, 5-METHYLTHIORIBOSE KINASE1 (MTK1), and other proteins were bound by PHA beads (SI Appendix, Table S1). CK1 is involved in clocks other than in land plants (e.g., fungi, flies, animals, and algae) (5)(6)(7)(8)19), but CKL clock-related functioning in Arabidopsis was unknown, likely due to the limits of traditional genetic analysis with functional redundancy among CKL family that contains 13 members in Arabidopsis (SI Appendix, Fig. S2). Arabidopsis homologs of the mammalian CDC7/CDK9 [CDKC1 (AT5G10270) and CDKC2 (AT5G64960) (47% identity, and Expected {E} value lower than 1e−100 to human CDK9) (20), AT4G16970 (27% identity, and E value 4e−34 to human CDC7)] were not enriched by PHA-bead binding, despite the fact that PHA767491 is an inhibitor of CDC7/CDK9 in mammals. ...
... Genes. Although CK1 is involved in the control of circadian clocks in green algae, the mechanism of CK1 activity in clock control in plants is unknown (7,8). To reveal possible action mechanisms of CK1 for clock control, we examined short-term (3 h) effects of PHA767491 on clock gene expression. ...
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.
