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

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.