DYRK1A and Glycogen Synthase Kinase 3β, a Dual-Kinase Mechanism Directing Proteasomal Degradation of CRY2 for Circadian Timekeeping

Abstract

Circadian molecular oscillation is generated by a transcription/translation-based feedback loop in which CRY proteins play critical roles as potent inhibitors for E-box-dependent clock gene expression. Although CRY2 undergoes rhythmic phosphorylation in its C-terminal tail, structurally distinct from the CRY1 tail, little is understood about how protein kinase(s) controls the CRY2-specific phosphorylation and contributes to the molecular clockwork. Here we found that Ser557 in the C-terminal tail of CRY2 is phosphorylated by DYRK1A as a priming kinase for subsequent GSK-3β (glycogen synthase kinase 3β)-mediated phosphorylation of Ser553, which leads to proteasomal degradation of CRY2. In the mouse liver, DYRK1A kinase activity toward Ser557 of CRY2 showed circadian variation, with its peak in the accumulating phase of CRY2 protein. Knockdown of Dyrk1a caused abnormal accumulation of cytosolic CRY2, advancing the timing of a nuclear increase of CRY2, and shortened the period length of the cellular circadian rhythm. Expression of an S557A/S553A mutant of CRY2 phenocopied the effect of Dyrk1a knockdown in terms of the circadian period length of the cellular clock. DYRK1A is a novel clock component cooperating with GSK-3β and governs the Ser557 phosphorylation-triggered degradation of CRY2.

Supplemental material for this article may be found at http://mcb.asm.org/.

We thank Yukiko Gotoh (University of Tokyo) for the Xenopus GSK-3β plasmid, Walter Becker (Medizinische Fakultat der RWTH, Aachen, Germany) for plasmids of GFP-tagged rat DYRK1A, its kinase-negative form, and GST-tagged rat DYRK1A, and Hikari Yoshitane for comments on the manuscript.

This work was supported in part by Grants-in-Aid for Scientific Research and by the 21st century COE and Global COE program (Integrative Life Science Based on the Study of Biosignaling Mechanisms) from MEXT, Japan. N.K. is supported by JSPS Research Fellowships for Young Scientists.