정의민 (KAIST, 기초과학연구원) | 한빛사논문 > 한빛사

한빛사논문

조회389

KAIST, 기초과학연구원

Proc. Natl. Acad. Sci. USA, Jun 04 2024, 121 (23) | https://doi.org/10.1073/pnas.2316858121 Phosphorylation of DNA-binding domains of CLOCK-BMAL1 complex for PER-dependent inhibition in circadian clock of mammalian cells

Yuta Otobe^a,b,1, Eui Min Jeong^c,d,1, Shunsuke Ito^a,b,1, Yuta Shinohara^e, Nobuhiro Kurabayashi^b, Atsu Aiba^a,f, Yoshitaka Fukada^a,b,2, Jae Kyoung Kim^c,d,2, and Hikari Yoshitane^a,b,2

^aDepartment of Biological Sciences, School of Science, The University of Tokyo, Bunkyo ku, Tokyo 113-0033, Japan;

^bCircadian Clock Project, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan;

^cBiomedical Mathematics Group, Pioneer Research Center for Mathematical and Computational Sciences, Institute for Basic Science, Daejeon 34141, Republic of Korea;

^dDepartment of Mathematical Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea;

^eDivision of Molecular Psychoimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Kita-Ku, Sapporo 060-0815, Japan; and

^fLaboratory of Animal Resources, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan

¹Y.O., E.M.J., and S.I. contributed equally to this work.

²To whom correspondence may be addressed. : Yoshitaka Fukada, Jae Kyoung Kim, Hikari Yoshitane

Abstract

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.

논문정보

형식Research article
게재일2024년 06월 (BRIC 등록일 2024-05-29)
연구진국내(교신)+국외 연구진
분야 생명과학 > 세포생물학

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