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ABSTRACT: Picrotoxin is extensively and specifically used to inhibit GABAA receptors and other members of the Cys-loop receptor superfamily. We find that picrotoxin acts independently of known Cys-loop receptors to markedly shorten the period of the circadian clock by specifically advancing the accumulation of PERIOD2 protein. We show that this mechanism is surprisingly tetrodotoxin-insensitive and the effect is larger than any known chemical or genetic manipulation. Notably, our results indicate that picrotoxin's circadian target is common to a variety of human and rodent cell types but not Drosophila, thereby ruling out all conserved Cys-loop receptors and known regulators of mammalian PERIOD protein stability. Given that the circadian clock modulates significant aspects of cell physiology including synaptic plasticity, these results have immediate and broad experimental implications. Furthermore, our data point to the existence of an important and novel target within the mammalian circadian timing system.
Journal of Neurophysiology 04/2013; · 3.32 Impact Factor
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ABSTRACT: Biochemical circadian oscillation of KaiC phosphorylation, by mixing three Kai proteins and ATP, has been proven to be the central oscillator of the cyanobacterial circadian clock. In vivo, the intracellular levels of KaiB and KaiC oscillate in a circadian fashion. By scrutinizing KaiC phosphorylation rhythm in a wide range of Kai protein concentrations, KaiA and KaiB were found to be "parameter-tuning" and "state-switching" regulators of KaiC phosphorylation rhythm, respectively. Our results also suggest a possible entrainment mechanism of the cellular circadian clock with the circadian variation of intracellular levels of Kai proteins.
FEBS letters 03/2010; 584(5):898-902. · 3.54 Impact Factor
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Yasushi Isojima, Masato Nakajima,
Hideki Ukai,
Hiroshi Fujishima,
Rikuhiro G. Yamada,
Koh-hei Masumoto,
Reiko Kiuchi,
Mayumi Ishida,
Maki Ukai-Tadenuma,
Yoichi Minami, [......],
Wataru Kishimoto,
Seung-Hee Yoo,
Kazuhiro Shimomura,
Toshifumi Takao,
Atsuko Takano,
Toshio Kojima,
Katsuya Nagai,
Yoshiyuki Sakaki,
Joseph S. Takahashi,
Hiroki R. Ueda
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ABSTRACT: A striking feature of the circadian clock is its flexible yet robust response to various environmental conditions. To analyze
the biochemical processes underlying this flexible-yet-robust characteristic, we examined the effects of 1,260 pharmacologically
active compounds in mouse and human clock cell lines. Compounds that markedly (>10 s.d.) lengthened the period in both cell
lines, also lengthened it in central clock tissues and peripheral clock cells. Most compounds inhibited casein kinase Iε (CKIε)
or CKIδ phosphorylation of the PER2 protein. Manipulation of CKIε/δ-dependent phosphorylation by these compounds lengthened
the period of the mammalian clock from circadian (24 h) to circabidian (48 h), revealing its high sensitivity to chemical
perturbation. The degradation rate of PER2, which is regulated by CKIε/δ-dependent phosphorylation, was temperature-insensitive
in living clock cells, yet sensitive to chemical perturbations. This temperature-insensitivity was preserved in the CKIε/δ-dependent
phosphorylation of a synthetic peptide in vitro. Thus, CKIε/δ-dependent phosphorylation is likely a temperature-insensitive
period-determining process in the mammalian circadian clock.
Proceedings of the National Academy of Sciences 09/2009; 106(37):15744-15749. · 9.68 Impact Factor
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Yasushi Isojima, Masato Nakajima,
Hideki Ukai,
Hiroshi Fujishima,
Rikuhiro G Yamada,
Koh-hei Masumoto,
Reiko Kiuchi,
Mayumi Ishida,
Maki Ukai-Tadenuma,
Yoichi Minami, [......],
Wataru Kishimoto,
Seung-Hee Yoo,
Kazuhiro Shimomura,
Toshifumi Takao,
Atsuko Takano,
Toshio Kojima,
Katsuya Nagai,
Yoshiyuki Sakaki,
Joseph S Takahashi,
Hiroki R Ueda
[show abstract]
[hide abstract]
ABSTRACT: A striking feature of the circadian clock is its flexible yet robust response to various environmental conditions. To analyze the biochemical processes underlying this flexible-yet-robust characteristic, we examined the effects of 1,260 pharmacologically active compounds in mouse and human clock cell lines. Compounds that markedly (>10 s.d.) lengthened the period in both cell lines, also lengthened it in central clock tissues and peripheral clock cells. Most compounds inhibited casein kinase Iepsilon (CKIepsilon) or CKIdelta phosphorylation of the PER2 protein. Manipulation of CKIepsilon/delta-dependent phosphorylation by these compounds lengthened the period of the mammalian clock from circadian (24 h) to circabidian (48 h), revealing its high sensitivity to chemical perturbation. The degradation rate of PER2, which is regulated by CKIepsilon/delta-dependent phosphorylation, was temperature-insensitive in living clock cells, yet sensitive to chemical perturbations. This temperature-insensitivity was preserved in the CKIepsilon/delta-dependent phosphorylation of a synthetic peptide in vitro. Thus, CKIepsilon/delta-dependent phosphorylation is likely a temperature-insensitive period-determining process in the mammalian circadian clock.
Proceedings of the National Academy of Sciences 09/2009; 106(37):15744-9. · 9.68 Impact Factor
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ABSTRACT: The cyanobacterial circadian oscillator can be reconstituted in vitro by mixing three purified clock proteins, KaiA, KaiB and KaiC, with ATP. The KaiC phosphorylation rhythm persists for at least 10 days without damping. By mixing oscillatory samples that have different phases and analyzing the dynamics of their phase relationships, we found that the robustness of the KaiC phosphorylation rhythm arises from the rapid synchronization of the phosphorylation state and reaction direction (phosphorylation or dephosphorylation) of KaiC proteins. We further demonstrate that synchronization is tightly linked with KaiC dephosphorylation and is mediated by monomer exchange between KaiC hexamers during the early dephosphorylation phase. This autonomous synchronization mechanism is probably the basis for the resilience of the cyanobacterial circadian system against quantitative fluctuations in clock components during cellular events such as cell growth and division.
Nature Structural & Molecular Biology 12/2007; 14(11):1084-8. · 12.71 Impact Factor
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ABSTRACT: KaiA, KaiB, and KaiC clock proteins from cyanobacteria and ATP are sufficient to reconstitute the KaiC phosphorylation rhythm in vitro, whereas almost all gene promoters are under the control of the circadian clock. The mechanism by which the KaiC phosphorylation cycle drives global transcription rhythms is unknown. Here, we report that RpaA, a potential DNA-binding protein that acts as a cognate response regulator of the KaiC-interacting kinase SasA, mediates between KaiC phosphorylation and global transcription rhythms. Circadian transcription was severely attenuated in sasA (Synechococcus adaptive sensor A)- and rpaA (regulator of phycobilisome-associated)-mutant cells, and the phosphotransfer activity from SasA to RpaA changed dramatically depending on the circadian state of a coexisting Kai protein complex in vitro. We propose a model in which the SasA-RpaA two-component system mediates time signals from the enzymatic oscillator to drive genome-wide transcription rhythms in cyanobacteria. Moreover, our results indicate the presence of secondary output pathways from the clock to transcription control, suggesting that multiple pathways ensure a genome-wide circadian system.
Proceedings of the National Academy of Sciences 09/2006; 103(32):12109-14. · 9.68 Impact Factor
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ABSTRACT: KaiA, KaiB, and KaiC are essential proteins of the circadian clock in the cyanobacterium Synechococcus elongatus PCC 7942. The phosphorylation cycle of KaiC that occurs in vitro after mixing the three proteins and ATP is thought to be the master oscillation governing the circadian system. We analyzed the temporal profile of complexes formed between the three Kai proteins. In the phosphorylation phase, KaiA actively and repeatedly associated with KaiC to promote KaiC phosphorylation. High levels of phosphorylation of KaiC induced the association of the KaiC hexamer with KaiB and inactivate KaiA to begin the dephosphorylation phase, which is closely linked to shuffling of the monomeric KaiC subunits among the hexamer. By reducing KaiC phosphorylation, KaiB dissociated from KaiC, reactivating KaiA. We also confirmed that a similar model can be applied in cyanobacterial cells. The molecular model proposed here provides mechanisms for circadian timing systems.
Molecular Cell 08/2006; 23(2):161-71. · 14.18 Impact Factor
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ABSTRACT: Kai proteins globally regulate circadian gene expression of cyanobacteria. The KaiC phosphorylation cycle, which persists even without transcription or translation, is assumed to be a basic timing process of the circadian clock. We have reconstituted the self-sustainable oscillation of KaiC phosphorylation in vitro by incubating KaiC with KaiA, KaiB, and adenosine triphosphate. The period of the in vitro oscillation was stable despite temperature change (temperature compensation), and the circadian periods observed in vivo in KaiC mutant strains were consistent with those measured in vitro. The enigma of the circadian clock can now be studied in vitro by examining the interactions between three Kai proteins.
Science 05/2005; 308(5720):414-5. · 31.20 Impact Factor
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ABSTRACT: An autoregulatory transcription-translation feedback loop is thought to be essential in generating circadian rhythms in any model organism. In the cyanobacterium Synechococcus elongatus, the essential clock protein KaiC is proposed to form this type of transcriptional negative feedback. Nevertheless, we demonstrate here temperature-compensated, robust circadian cycling of KaiC phosphorylation even without kaiBC messenger RNA accumulation under continuous dark conditions. This rhythm persisted in the presence of a transcription or translation inhibitor. Moreover, kinetic profiles in the ratio of KaiC autophosphorylation-dephosphorylation were also temperature compensated in vitro. Thus, the cyanobacterial clock can keep time independent of de novo transcription and translation processes.
Science 02/2005; 307(5707):251-4. · 31.20 Impact Factor
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Taeko Nishiwaki,
Yoshinori Satomi, Masato Nakajima,
Cheolju Lee,
Reiko Kiyohara,
Hakuto Kageyama,
Yohko Kitayama,
Mioko Temamoto,
Akihiro Yamaguchi,
Atsushi Hijikata,
Mitiko Go,
Hideo Iwasaki,
Toshifumi Takao,
Takao Kondo
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ABSTRACT: In the cyanobacterium Synechococcus elongatus PCC 7942, KaiA, KaiB, and KaiC are essential proteins for the generation of a circadian rhythm. KaiC is proposed as a negative regulator of the circadian expression of all genes in the genome, and its phosphorylation is regulated positively by KaiA and negatively by KaiB and shows a circadian rhythm in vivo. To study the functions of KaiC phosphorylation in the circadian clock system, we identified two autophosphorylation sites, Ser-431 and Thr-432, by using mass spectrometry (MS). We generated Synechococcus mutants in which these residues were substituted for alanine by using site-directed mutagenesis. Phosphorylation of KaiC was reduced in the single mutants and was completely abolished in the double mutant, indicating that KaiC is also phosphorylated at these sites in vivo. These mutants lost circadian rhythm, indicating that phosphorylation at each of the two sites is essential for the control of the circadian oscillation. Although the nonphosphorylatable mutant KaiC was able to form a hexamer in vitro, it failed to form a clock protein complex with KaiA, KaiB, and SasA in the Synechococcus cells. When nonphosphorylatable KaiC was overexpressed, the kaiBC promoter activity was only transiently repressed. These results suggest that KaiC phosphorylation regulates its transcriptional repression activity by controlling its binding affinity for other clock proteins.
Proceedings of the National Academy of Sciences 10/2004; 101(38):13927-32. · 9.68 Impact Factor
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ABSTRACT: Cyanobacterial clock proteins KaiA and KaiC are proposed as positive and negative regulators in the autoregulatory circadian kaiBC expression, respectively. Here, we show that activation of kaiBC expression by kaiA requires KaiC, suggesting a positive feedback control in the cyanobacterial clockwork. We found that robust circadian phosphorylation of KaiC. KaiA was essential for in vivo KaiC phosphorylation and activated in vitro KaiC autophosphorylation. These effects of KaiA were attenuated by the kaiA2 long period mutation. Both the long period phenotype and the abnormal KaiC phosphorylation in this mutant were suppressed by a previously undocumented kaiC mutation. We propose that KaiA-stimulated circadian KaiC phosphorylation is important for circadian timing.
Proceedings of the National Academy of Sciences 12/2002; 99(24):15788-93. · 9.68 Impact Factor
