Phosphorylation of DNA- binding domains of CLOCK-BMAL1 complex for PER- dependent inhibition in circadian clock of mammalian cells

• 成果类型: Article
• 署名作者: Otobe, Yuta; Jeong, Eui Min; Ito, Shunsuke; Shinohara, Yuta; Kurabayashi, Nobuhiro; Aiba, Atsu; Fukada, Yoshitaka; Kim, Jae Kyoung; Yoshitane, Hikari
• 署名单位: University of Tokyo; Tokyo Metropolitan Institute of Medical Science; Institute for Basic Science - Korea (IBS); Korea Advanced Institute of Science & Technology (KAIST); Hokkaido University; Hokkaido University; University of Tokyo
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-11619
• DOI: 10.1073/pnas.2316858121
• 发表日期: 2024-06-04
• 关键词: molecular-bases; period; cryptochrome; degradation; protein; transcription; Mutation; reveals; oscillation; mechanisms

摘要

In mammals, CLOCK and BMAL1 proteins form a heterodimer that binds to E - box sequences and activates transcription of target genes, including Period ( Per) . Translated PER proteins then bind to the CLOCK-BMAL1 complex to inhibit its transcriptional activity. However, the molecular mechanism and the impact of this PER - dependent inhibition on the circadian clock oscillation remain elusive. We previously identified Ser38 and Ser42 in a DNA - binding domain of CLOCK as phosphorylation sites at the PER - dependent inhibition phase. In this study, knockout rescue experiments showed that nonphosphorylatable (Ala) mutations at these sites shortened circadian period, whereas their constitutive - phospho - mimetic (Asp) mutations completely abolished the circadian rhythms. Similarly, we found that nonphosphorylatable (Ala) and constitutive - phospho - mimetic (Glu) mutations at Ser78 in a DNA - binding domain of BMAL1 also shortened the circadian period and abolished the rhythms, respectively. The mathematical modeling predicted that these constitutive - phospho - mimetic mutations weaken the DNA binding of the CLOCK-BMAL1 complex and that the nonphosphorylatable mutations inhibit the PER - dependent displacement (reduction of DNA - binding ability) of the CLOCK-BMAL1 complex from DNA. Biochemical experiments supported the importance of these phosphorylation sites for displacement of the complex in the PER2 - dependent inhibition. Our results provide direct evidence that phosphorylation of CLOCK-Ser38/Ser42 and BMAL1-Ser78 plays a crucial role in the PER - dependent inhibition and the determination of the circadian period. Significance It has long been established that circadian clock oscillations rely on transcriptional and translational feedback loops but the specific phosphorylation sites underlying the displacement of the CLOCK-BMAL1 complex are still elusive. In this study, we explore the detailed intricate molecular mechanisms governing the circadian clock in mammals, specifically focusing on the roles of phosphorylation events within CLOCK and BMAL1 proteins and their impact on the circadian oscillation. Our research combines mathematical modeling, knockout-rescue experiments, and biochemical experiments to provide direct evidence of the critical role played by their phosphorylation in Period (PER)- dependent displacement mechanisms. These findings shed light on the hitherto elusive details of a PER- dependent displacement mechanism and demonstrate its physiological importance for the circadian clock oscillation.