Search

In the intracellular environment, the circadian oscillator is exposed to molecular noise. Nevertheless, cellular rhythms are robust and show almost constant period length for several weeks. To find which molecular processes modulate the stability, we examined the effects of a sublethal dose of inhibitors for processes in the molecular clock. Inhibition of PER1/2 phosphorylation by CKIε/δ led to reduced amplitude and enhancement of damping, suggesting that inhibition of this process destabilized oscillation. In contrast, moderate inhibition of translation led to stabilization of the circadian oscillation. Moreover, inhibition of translation also reduced magnitude of phase shift. These results suggest that some specific molecular processes are crucial for stabilizing the circadian rhythm, and that the molecular clock may be stabilized by optimizing parameters of some crucial processes in the primary negative feedback loop. Moreover, our findings also suggested that rhythm stability is closely associated with phase stability against stimuli., (Copyright © 2015 Elsevier Inc. All rights reserved.) more...

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., (Copyright (c) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.) more...

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. more...

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. more...

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. more...

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. more...

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. more...

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. more...

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. more...