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Mutation of a PER2 phosphodegron perturbs the circadian phosphoswitch.

ABSTRACT: 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 ?-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.

SUBMITTER: Masuda S

PROVIDER: S-EPMC7245125 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Mutation of a PER2 phosphodegron perturbs the circadian phosphoswitch.

Masuda Shusaku S Narasimamurthy Rajesh R Yoshitane Hikari H Kim Jae Kyoung JK Fukada Yoshitaka Y Virshup David M DM

Proceedings of the National Academy of Sciences of the United States of America 20200430 20

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 ...[more]

PMID: 32354999

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Project description:Like neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in the brain, single fibroblasts can function as independent oscillators. In the SCN, synaptic and paracrine signaling among cells creates a robust, synchronized circadian oscillation, whereas there is no evidence for such integration in fibroblast cultures. However, interactions among single-cell fibroblast oscillators cannot be completely excluded, because fibroblasts were not isolated in previous work. In this study, we tested the autonomy of fibroblasts as single-cell circadian oscillators in high- and low-density culture, by single-cell imaging of cells from PER2::LUC circadian reporter mice. We found greatly reduced PER2::LUC rhythmicity in low-density cultures, which could result from lack of either constitutive or rhythmic paracrine signals from neighboring fibroblasts. To discriminate between these 2 possibilities, we mixed PER2::LUC wild-type (WT) cells with nonluminescent, nonrhythmic Bmal1-/- cells, so that density of rhythmic cells was low but overall cell density remained high. In this condition, WT cells showed clear rhythmicity similar to high-density cultures. We also mixed PER2::LUC WT cells with nonluminescent, long period Cry2-/- cells. In this condition, WT cells showed a period no different from cells cultured with rhythmic WT cells or nonrhythmic Bmal1-/- cells. In previous work, we found that low K? suppresses fibroblast rhythmicity, and we and others have found that either low K? or low Ca²? suppresses SCN rhythmicity. Therefore, we attempted to rescue rhythmicity of low-density fibroblasts with high K? (21 mM), high Ca²? (3.6 mM), or conditioned medium. Conditioned medium from high-density fibroblast cultures rescued rhythmicity of low-density cultures, whereas high K? or Ca²? medium did not consistently rescue rhythmicity. These data suggest that fibroblasts require paracrine signals from adjacent cells for normal expression of rhythmicity, but that these signals do not have to be rhythmic, and that rhythmic signals from other cells do not affect the intrinsic periods of fibroblasts.

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Mutation of a PER2 phosphodegron perturbs the circadian phosphoswitch.

Publications

Mutation of a PER2 phosphodegron perturbs the circadian phosphoswitch.

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