The Mammalian Circadian Clock Protein Period Counteracts Cryptochrome in Phosphorylation Dynamics of Circadian Locomotor Output Cycles Kaput (CLOCK)

Ritsuko Matsumura,Yoshiki Tsuchiya,Isao Tokuda,Takahiro Matsuo,Miho Sato,Koichi Node,Eisuke Nishida,Makoto Akashi

doi:10.1074/jbc.m114.578278

Abstract

The circadian transcription factor CLOCK exhibits a circadian oscillation in its phosphorylation levels. Although it remains unclear whether this phosphorylation contributes to circadian rhythm generation, it has been suggested to be involved in transcriptional activity, intracellular localization, and degradative turnover of CLOCK. Here, we obtained direct evidence that CLOCK phosphorylation may be essential for autonomous circadian oscillation in clock gene expression. Importantly, we found that the circadian transcriptional repressors Cryptochrome (CRY) and Period (PER) showed an opposite effect on CLOCK phosphorylation; CRY impaired BMAL1-dependent CLOCK phosphorylation, whereas PER protected the phosphorylation against CRY. Interestingly, unlike PER1 and PER2, PER3 did not exert a protective action, which correlates with the phenotypic differences among mice lacking the Per genes. Further studies on the regulatory mechanism of CLOCK phosphorylation would thus lead to elucidation of the mechanism of CRY-mediated transcriptional repression and an understanding of the true role of PER in the negative feedback system.

Highlights

The role of the circadian clock component Period (PER) remains unclear
The mPer2, hPer3, hCry1, hCry2, and hBmal1 regions range from Ϫ2811 to ϩ110, Ϫ779 to ϩ170, Ϫ3195 to ϩ417, Ϫ2925 to ϩ41, and Ϫ3465 to ϩ57, respectively (ϩ1 is the putative transcriptional start site). mPer1-luc was a kind gift from Hajime Tei [10]
Phosphorylation of CLOCK May Be Necessary for Circadian Gene Expression—BMAL1-CLOCK dimerization leads to posttranslational modifications of CLOCK, which correlate with their transcriptional activities [4, 8, 17]

Summary

Introduction

The role of the circadian clock component Period (PER) remains unclear. Results: PER1 and PER2, but not PER3, protected CLOCK phosphorylation against Cryptochrome (CRY). The circadian transcription factor CLOCK exhibits a circadian oscillation in its phosphorylation levels. It remains unclear whether this phosphorylation contributes to circadian rhythm generation, it has been suggested to be involved in transcriptional activity, intracellular localization, and degradative turnover of CLOCK. We obtained direct evidence that CLOCK phosphorylation may be essential for autonomous circadian oscillation in clock gene expression. We found that the circadian transcriptional repressors Cryptochrome (CRY) and Period (PER) showed an opposite effect on CLOCK phosphorylation; CRY impaired BMAL1-dependent CLOCK phosphorylation, whereas PER protected the phosphorylation against CRY. Further studies on the regulatory mechanism of CLOCK phosphorylation would lead to elucidation of the mechanism of CRYmediated transcriptional repression and an understanding of the true role of PER in the negative feedback system

Methods

Results

Conclusion