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Phosphorylation Regulating the Ratio of Intracellular CRY1 Protein Determines the Circadian Period

The core circadian oscillator in mammals is composed of transcription/translation feedback loop, in which cryptochrome (CRY) proteins play critical roles as repressors of their own gene expression. Although post-translational modifications, such as phosphorylation of CRY1, are crucial for circadian...

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Autores principales: Liu, Na, Zhang, Eric Erquan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033960/
https://www.ncbi.nlm.nih.gov/pubmed/27721804
http://dx.doi.org/10.3389/fneur.2016.00159
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author Liu, Na
Zhang, Eric Erquan
author_facet Liu, Na
Zhang, Eric Erquan
author_sort Liu, Na
collection PubMed
description The core circadian oscillator in mammals is composed of transcription/translation feedback loop, in which cryptochrome (CRY) proteins play critical roles as repressors of their own gene expression. Although post-translational modifications, such as phosphorylation of CRY1, are crucial for circadian rhythm, little is known about how phosphorylated CRY1 contributes to the molecular clockwork. To address this, we created a series of CRY1 mutants with single amino acid substitutions at potential phosphorylation sites and performed a cell-based, phenotype-rescuing screen to identify mutants with aberrant rhythmicity in CRY-deficient cells. We report 10 mutants with an abnormal circadian period length, including long period (S280D and S588D), short period (S158D, S247D, T249D, Y266D, Y273D, and Y432D), and arrhythmicity (S71D and S404D). When expressing mutated CRY1 in HEK293 cells, we show that most of the mutants (S71D, S247D, T249D, Y266D, Y273D, and Y432D) exhibited reduction in repression activity compared with wild-type (WT) CRY1, whereas other mutants had no obvious change. Correspondingly, these mutants also showed differences in protein stability and cellular localization. We show that most of mutants are more stable than WT, except S158D, T249D, and S280D. Although the characteristics of the 10 mutants are various, they all impair the ratio balance of intracellular CRY1 protein. Thus, we conclude that the mutations caused distinct phenotypes most likely through the ratio of functional CRY1 protein in cells.
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spelling pubmed-50339602016-10-07 Phosphorylation Regulating the Ratio of Intracellular CRY1 Protein Determines the Circadian Period Liu, Na Zhang, Eric Erquan Front Neurol Neuroscience The core circadian oscillator in mammals is composed of transcription/translation feedback loop, in which cryptochrome (CRY) proteins play critical roles as repressors of their own gene expression. Although post-translational modifications, such as phosphorylation of CRY1, are crucial for circadian rhythm, little is known about how phosphorylated CRY1 contributes to the molecular clockwork. To address this, we created a series of CRY1 mutants with single amino acid substitutions at potential phosphorylation sites and performed a cell-based, phenotype-rescuing screen to identify mutants with aberrant rhythmicity in CRY-deficient cells. We report 10 mutants with an abnormal circadian period length, including long period (S280D and S588D), short period (S158D, S247D, T249D, Y266D, Y273D, and Y432D), and arrhythmicity (S71D and S404D). When expressing mutated CRY1 in HEK293 cells, we show that most of the mutants (S71D, S247D, T249D, Y266D, Y273D, and Y432D) exhibited reduction in repression activity compared with wild-type (WT) CRY1, whereas other mutants had no obvious change. Correspondingly, these mutants also showed differences in protein stability and cellular localization. We show that most of mutants are more stable than WT, except S158D, T249D, and S280D. Although the characteristics of the 10 mutants are various, they all impair the ratio balance of intracellular CRY1 protein. Thus, we conclude that the mutations caused distinct phenotypes most likely through the ratio of functional CRY1 protein in cells. Frontiers Media S.A. 2016-09-23 /pmc/articles/PMC5033960/ /pubmed/27721804 http://dx.doi.org/10.3389/fneur.2016.00159 Text en Copyright © 2016 Liu and Zhang. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Neuroscience
Liu, Na
Zhang, Eric Erquan
Phosphorylation Regulating the Ratio of Intracellular CRY1 Protein Determines the Circadian Period
title Phosphorylation Regulating the Ratio of Intracellular CRY1 Protein Determines the Circadian Period
title_full Phosphorylation Regulating the Ratio of Intracellular CRY1 Protein Determines the Circadian Period
title_fullStr Phosphorylation Regulating the Ratio of Intracellular CRY1 Protein Determines the Circadian Period
title_full_unstemmed Phosphorylation Regulating the Ratio of Intracellular CRY1 Protein Determines the Circadian Period
title_short Phosphorylation Regulating the Ratio of Intracellular CRY1 Protein Determines the Circadian Period
title_sort phosphorylation regulating the ratio of intracellular cry1 protein determines the circadian period
topic Neuroscience
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033960/
https://www.ncbi.nlm.nih.gov/pubmed/27721804
http://dx.doi.org/10.3389/fneur.2016.00159
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