Roles of CLOCK Phosphorylation in Suppression of E-Box-Dependent Transcription

Abstract

In mammalian circadian clockwork, the CLOCK-BMAL1 heterodimer activates E-box-dependent transcription, while its activity is suppressed by circadian binding with negative regulators, such as CRYs. Here, we found that the CLOCK protein is kept mostly in the phosphorylated form throughout the day and is partly hyperphosphorylated in the suppression phase of E-box-dependent transcription in the mouse liver and NIH 3T3 cells. Coexpression of CRY2 in NIH 3T3 cells inhibited the phosphorylation of CLOCK, whereas CIPC coexpression markedly stimulated phosphorylation, indicating that CLOCK phosphorylation is regulated by a combination of the negative regulators in the suppression phase. CLOCK-BMAL1 purified from the mouse liver was subjected to tandem mass spectrometry analysis, which identified Ser38, Ser42, and Ser427 as in vivo phosphorylation sites of CLOCK. Ser38Asp and Ser42Asp mutations of CLOCK additively and markedly weakened the transactivation activity of CLOCK-BMAL1, with downregulation of the nuclear amount of CLOCK and the DNA-binding activity. On the other hand, CLOCKΔ19, lacking the CIPC-binding domain, was far less phosphorylated and much more stabilized than wild-type CLOCK in vivo. Calyculin A treatment of cultured NIH 3T3 cells promoted CLOCK phosphorylation and facilitated its proteasomal degradation. Together, these results show that CLOCK phosphorylation contributes to the suppression of CLOCK-BMAL1-mediated transactivation through dual regulation: inhibition of CLOCK activity and promotion of its degradation.

ACKNOWLEDGMENTS

We thank Joseph S. Takahashi (Northwestern University, Evanston, IL) for the generous gifts of Clock mutant mice and Takeshi Todo (Osaka University) for the anti-CRY2 antibody. We also thank Munenori Komori, Shumpei Yamauchi, and Hiroshi Kiyota for technical assistance and Naohiro Kon for critical reading of the manuscript.

This work was supported in part by grants-in-aid and by the Global COE program (Integrative Life Science Based on the Study of Biosignaling Mechanisms) from MEXT, Japan. This work was performed partly under the Cooperative Research Program of the Institute for Protein Research, Osaka University. H.Y. is supported by JSPS Research Fellowships for Young Scientists.