Mutation of a PER2 phosphodegron perturbs the circadian phosphoswitch

Casein kinase 1 (CK1) plays a central role in regulating the period of the circadian clock. In mammals, PER2 protein abundance is regulated by CK1-mediated phosphorylation and proteasomal degradation. On the other hand, recent studies have questioned whether the degradation of the core circadian mac...

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Published in:Proceedings of the National Academy of Sciences - PNAS 2020-05, Vol.117 (20), p.10888-10896
Main Authors: Masuda, Shusaku, Narasimamurthy, Rajesh, Yoshitane, Hikari, Kim, Jae Kyoung, Fukada, Yoshitaka, Virshup, David M.
Format: Article
Language:English
Subjects:
Biological clocks
Biological Sciences
Casein
Casein kinase I
Circadian rhythm
Circadian rhythms
Compensation
Cryptochromes
Cytoplasm
Degradation
Embryo fibroblasts
Fibroblasts
Kinases
mRNA
Mutation
Period 1 protein
Period 2 protein
Phosphorylation
Proteasomes
Proteins
Temperature compensation
Ubiquitin
Ubiquitin-protein ligase
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Summary:Casein kinase 1 (CK1) plays a central role in regulating the period of the circadian clock. In mammals, PER2 protein abundance is regulated by CK1-mediated phosphorylation and proteasomal degradation. On the other hand, recent studies have questioned whether the degradation of the core circadian machinery is a critical step in clock regulation. Prior cell-based studies found that CK1 phosphorylation of PER2 at Ser478 recruits the ubiquitin E3 ligase β-TrCP, leading to PER2 degradation. Creation of this phosphodegron is regulated by a phosphoswitch that is also implicated in temperature compensation. However, in vivo evidence that this phosphodegron influences circadian period is lacking. Here, we generated and analyzed PER2-Ser478Ala knock-in mice. The mice showed longer circadian period in behavioral analysis. Molecularly, mutant PER2 protein accumulated in both the nucleus and cytoplasm of the mouse liver, while Per2 messenger RNA (mRNA) levels were minimally affected. Nuclear PER1, CRY1, and CRY2 proteins also increased, probably due to stabilization of PER2-containing complexes. In mouse embryonic fibroblasts derived from PER2-Ser478Ala::LUC mice, three-phase decay and temperature compensation of the circadian period was perturbed. These data provide direct in vivo evidence for the importance of phosphorylation-regulated PER2 stability in the circadian clock and validate the phosphoswitch in a mouse model.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.2000266117