Masato Nakajima

KaiA-stimulated KaiC phosphorylation in circadian timing loops in cyanobacteria

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.

A KaiC-associating SasA–RpaA two-component regulatory system as a major circadian timing mediator in cyanobacteria

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.

CKIε/δ-dependent phosphorylation is a temperature-insensitive, period-determining process in the mammalian circadian clock

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.

Autonomous synchronization of the circadian KaiC phosphorylation rhythm

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.

In vitro regulation of circadian phosphorylation rhythm of cyanobacterial clock protein KaiC by KaiA and KaiB

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.