RESUMO
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.
Assuntos
Caseína Quinase 1 épsilon/metabolismo , Caseína Quinase Idelta/metabolismo , Ritmo Circadiano/fisiologia , Animais , Evolução Biológica , Caseína Quinase 1 épsilon/antagonistas & inibidores , Caseína Quinase 1 épsilon/genética , Caseína Quinase Idelta/antagonistas & inibidores , Caseína Quinase Idelta/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/genética , Cianobactérias/genética , Cianobactérias/fisiologia , Humanos , Cinética , Camundongos , Modelos Biológicos , Células NIH 3T3 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
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.
