scholarly journals Mutation of a PER2 phosphodegron perturbs the circadian phosphoswitch

2019 ◽  
Author(s):  
Shusaku Masuda ◽  
Rajesh Narasimamurthy ◽  
Hikari Yoshitane ◽  
Jae Kyoung Kim ◽  
Yoshitaka Fukada ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Temperature Compensation ◽  
Protein Abundance ◽  
Mrna Levels ◽  
Circadian Period ◽  
Embryonic Fibroblasts ◽  
Casein Kinase 1 ◽  
Ubiquitin E3 Ligase ◽  
Three Phase

AbstractCasein 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 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.

scholarly journals Mutation of a PER2 phosphodegron perturbs the circadian phosphoswitch

2020 ◽  
Vol 117 (20) ◽  
pp. 10888-10896 ◽  
Author(s):  
Shusaku Masuda ◽  
Rajesh Narasimamurthy ◽  
Hikari Yoshitane ◽  
Jae Kyoung Kim ◽  
Yoshitaka Fukada ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Messenger Rna ◽  
Temperature Compensation ◽  
Mrna Levels ◽  
Circadian Period ◽  
Embryonic Fibroblasts ◽  
Casein Kinase 1 ◽  
Ubiquitin E3 Ligase ◽  
Three Phase

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.

scholarly journals TIMELESS mutation alters phase responsiveness and causes advanced sleep phase

2019 ◽  
pp. 201819110 ◽  
Author(s):  
Philip Kurien ◽  
Pei-Ken Hsu ◽  
Jacy Leon ◽  
David Wu ◽  
Thomas McMahon ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Molecular Clock ◽  
Mutant Mice ◽  
Circadian Period ◽  
Negative Regulators ◽  
Mouse Embryonic Fibroblasts ◽  
Sleep Phase ◽  
Embryonic Fibroblasts ◽  
Light Entrainment

Many components of the circadian molecular clock are conserved from flies to mammals; however, the role of mammalian Timeless remains ambiguous. Here, we report a mutation in the human TIMELESS (hTIM) gene that causes familial advanced sleep phase (FASP). Tim CRISPR mutant mice exhibit FASP with altered photic entrainment but normal circadian period. We demonstrate that the mutation prevents TIM accumulation in the nucleus and has altered affinity for CRY2, leading to destabilization of PER/CRY complex and a shortened period in nonmature mouse embryonic fibroblasts (MEFs). We conclude that TIM, when excluded from the nucleus, can destabilize the negative regulators of the circadian clock, alter light entrainment, and cause FASP.

scholarly journals Circadian Clock-Specific Roles for the Light Response Protein WHITE COLLAR-2

2001 ◽  
Vol 21 (8) ◽  
pp. 2619-2628 ◽  
Author(s):  
Michael A. Collett ◽  
Jay C. Dunlap ◽  
Jennifer J. Loros
Keyword(s):  
Circadian Clock ◽  
Temperature Compensation ◽  
Light Response ◽  
White Collar ◽  
Normal Activity ◽  
Relative Increase ◽  
Period Length ◽  
Mrna Levels ◽  
Temperature Dependent ◽  
Protein Levels

ABSTRACT To understand the role of white collar-2 in theNeurospora circadian clock, we examined alleles ofwc-2 thought to encode partially functional proteins. We found that wc-2 allele ER24 contained a conservative mutation in the zinc finger. This mutation results in reduced levels of circadian rhythm-critical clock gene products, frq mRNA and FRQ protein, and in a lengthened period of the circadian clock. In addition, this mutation altered a second canonical property of the clock, temperature compensation: as temperature increased, period length decreased substantially. This temperature compensation defect correlated with a temperature-dependent increase in overall FRQ protein levels, with the relative increase being greater in wc-2(ER24) than in wild type, while overall frq mRNA levels were largely unaltered by temperature. We suggest that this temperature-dependent increase in FRQ levels partially rescues the lowered levels of FRQ resulting from the wc-2 (ER24) defect, yielding a shorter period at higher temperatures. Thus, normal activity of the essential clock component WC-2, a positive regulator offrq, is critical for establishing period length and temperature compensation in this circadian system.

Cre-loxP DNA recombination is possible with only minimal unspecific transcriptional changes and without cardiomyopathy in Tg(αMHC-MerCreMer) mice

2010 ◽  
Vol 299 (5) ◽  
pp. H1671-H1678 ◽  
Author(s):  
Karina Hougen ◽  
Jan Magnus Aronsen ◽  
Mathis K. Stokke ◽  
Ulla Enger ◽  
Ståle Nygård ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Dna Recombination ◽  
Gene Inactivation ◽  
Protein Abundance ◽  
Mrna Levels ◽  
Cardiovascular Research ◽  
Transcriptional Changes ◽  
The One ◽  
Per Oral

Cre-loxP technology for conditional gene inactivation is a powerful tool in cardiovascular research. Induction of gene inactivation can be carried out by per oral or intraperitoneal tamoxifen administration. Unintended transient cardiomyopathy following tamoxifen administration for gene inactivation has recently been reported. We aimed to develop a protocol for tamoxifen-induced gene inactivation with minimal effects on gene transcription and in vivo cardiac function, allowing studies of acute loss of the targeted gene. In mRNA microarrays, 35% of the 34,760 examined genes were significantly regulated in MCM+/0 compared with wild type. In MCM+/0, we found a correlation between tamoxifen dose and degree of gene regulation. Comparing one and four intraperitoneal injections of 40 mg·kg−1·day−1 tamoxifen, regulated genes were reduced to 1/5 in the single injection group. Pronounced alteration in protein abundance and acute cardiomyopathy were observed after the four-injection protocols but not the one-injection protocol. For verification of gene inactivation following one injection of tamoxifen, this protocol was applied to MCM+/0/Serca2fl/fl. Serca2 mRNA levels and protein abundance followed the same pattern of decline with one and four tamoxifen injections. The presence of the MCM transgene induced major alterations of gene expression while administration of tamoxifen induced additional but less gene regulation. Thus nonfloxed MCM+/0 should be considered as controls for mice that carry both a floxed gene of interest and the MCM transgene. One single tamoxifen injection administered to MCM+/0/Serca2fl/fl was sufficient for target gene inactivation, without acute cardiomyopathy, allowing acute studies subsequent to gene inactivation.

scholarly journals Effects of dietary glycerol on the expression of pterin carbinolamine dehydratase in the rat

2003 ◽  
Vol 373 (3) ◽  
pp. 993-997
Author(s):  
Edward CONNOLLY ◽  
John DONLON
Keyword(s):  
Protein Abundance ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Gtp Cyclohydrolase I ◽  
Small Reduction ◽  
Guanosine Triphosphate ◽  
Hepatocyte Nuclear Factor 1Α ◽  
Phenyl Alanine

Earlier studies have shown that the abundance of hepatic phenyl-alanine hydroxylase (PAH) diminishes to 60% of control values in rats fed with a diet composed of 40% (w/w) glycerol [Guerin, Walsh, Donlon and Kaufman (1998) Int. J. Biochem. Cell Biol. 30, 1047–1054]. In this experimental model, there are corresponding decreases in the hepatic concentrations of both the hydroxylase cofactor, tetrahydrobiopterin, and the nucleotide guanosine triphosphate. We now show that the cytoplasmic activities of hepatic pterin-4a-carbinolamine dehydratase (PCD) are also lower in these animals, by approx. 50% compared with control values. Immunoblotting confirmed a diminution of protein abundance in vivo. PCD also functions as a dimerization cofactor (DCoH) for the hepatocyte nuclear factor 1α (HNF1α) and the relative abundance of PCD/DCoH in the nucleus is also decreased. There is a small reduction in the mRNA levels for PAH and for PCD/DCoH in the glycerol-fed animals. In the kidney, there is also a diminution in the abundance of both PAH and PCD proteins. Hepatic GTP cyclohydrolase I activity was not altered and the abundance of hepatic HNF1α remained unchanged. HNF1α is required for the expression of PAH in the liver and our results support a role for PCD/DCoH, through its interaction with HNF1α, in regulating the expression of PAH.

scholarly journals Casein kinase 1 family regulates PRR5 and TOC1 in the Arabidopsis circadian clock

2019 ◽  
Vol 116 (23) ◽  
pp. 11528-11536 ◽  
Author(s):  
Takahiro N. Uehara ◽  
Yoshiyuki Mizutani ◽  
Keiko Kuwata ◽  
Tsuyoshi Hirota ◽  
Ayato Sato ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Posttranslational Modifications ◽  
Posttranslational Modification ◽  
Target Genes ◽  
Casein Kinase ◽  
Transcriptional Repressors ◽  
Circadian Period ◽  
Casein Kinase 1 ◽  
Chemical Screening ◽  
Plant Clock

The circadian clock provides organisms with the ability to adapt to daily and seasonal cycles. Eukaryotic clocks mostly rely on lineage-specific transcriptional-translational feedback loops (TTFLs). Posttranslational modifications are also crucial for clock functions in fungi and animals, but the posttranslational modifications that affect the plant clock are less understood. Here, using chemical biology strategies, we show that the Arabidopsis CASEIN KINASE 1 LIKE (CKL) family is involved in posttranslational modification in the plant clock. Chemical screening demonstrated that an animal CDC7/CDK9 inhibitor, PHA767491, lengthens the Arabidopsis circadian period. Affinity proteomics using a chemical probe revealed that PHA767491 binds to and inhibits multiple CKL proteins, rather than CDC7/CDK9 homologs. Simultaneous knockdown of Arabidopsis CKL-encoding genes lengthened the circadian period. CKL4 phosphorylated transcriptional repressors PSEUDO-RESPONSE REGULATOR 5 (PRR5) and TIMING OF CAB EXPRESSION 1 (TOC1) in the TTFL. PHA767491 treatment resulted in accumulation of PRR5 and TOC1, accompanied by decreasing expression of PRR5- and TOC1-target genes. A prr5 toc1 double mutant was hyposensitive to PHA767491-induced period lengthening. Together, our results reveal posttranslational modification of transcriptional repressors in plant clock TTFL by CK1 family proteins, which also modulate nonplant circadian clocks.

scholarly journals Enzymes degraded under high light maintain proteostasis by transcriptional regulation in Arabidopsis

2021 ◽  
Author(s):  
Lei Li ◽  
Owen Duncan ◽  
Diep R Ganguly ◽  
Chun Pong Lee ◽  
Peter A Crisp ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Light Stress ◽  
High Light ◽  
Coupled Analysis ◽  
Protein Abundance ◽  
Mrna Levels ◽  
Transcriptional Responses ◽  
High Light Stress ◽  
Degradation Rates

Photo-inhibitory high light stress in Arabidopsis leads to increases in markers of protein degradation and transcriptional upregulation of proteases and proteolytic machinery, but proteostasis is largely maintained. We find significant increases in the in vivo degradation rate for specific molecular chaperones, nitrate reductase, glyceraldehyde-3 phosphate dehydrogenase, and phosphoglycerate kinase and other plastid, mitochondrial, peroxisomal, and cytosolic enzymes involved in redox shuttles. Coupled analysis of protein degradation rates, mRNA levels, and protein abundance reveal that 57% of the nuclear-encoded enzymes with higher degradation rates also had high light-induced transcriptional responses to maintain proteostasis. In contrast, plastid-encoded proteins with enhanced degradation rates showed decreased transcript abundances and must maintain protein abundance by other processes. This analysis reveals a light-induced transcriptional program for nuclear-encoded genes, beyond the regulation of PSII D1 subunit and the function of PSII, to replace key protein degradation targets in plants and ensure proteostasis under high light stress.  

scholarly journals Tuning the circadian period of cyanobacteria up to 6.6 days by the single amino acid substitutions in KaiC

2020 ◽  
Vol 117 (34) ◽  
pp. 20926-20931
Author(s):  
Kumiko Ito-Miwa ◽  
Yoshihiko Furuike ◽  
Shuji Akiyama ◽  
Takao Kondo
Keyword(s):  
Amino Acid ◽  
Atpase Activity ◽  
Temperature Compensation ◽  
Period Change ◽  
Single Amino Acid ◽  
Circadian Period ◽  
Clock Proteins ◽  
Wide Range

The circadian clock of cyanobacteria consists of only three clock proteins, KaiA, KaiB, and KaiC, which generate a circadian rhythm of KaiC phosphorylation in vitro. The adenosine triphosphatase (ATPase) activity of KaiC is the source of the 24-h period and temperature compensation. Although numerous circadian mutants of KaiC have been identified, the tuning mechanism of the 24-h period remains unclear. Here, we show that the circadian period of in vitro phosphorylation rhythm of mutants at position 402 of KaiC changed dramatically, from 15 h (0.6 d) to 158 h (6.6 d). The ATPase activities of mutants at position 402 of KaiC, without KaiA and KaiB, correlated with the frequencies (1/period), indicating that KaiC structure was the source of extra period change. Despite the wide-range tunability, temperature compensation of both the circadian period and the KaiC ATPase activity of mutants at position 402 of KaiC were nearly intact. We also found that in vivo and in vitro circadian periods and the KaiC ATPase activity of mutants at position 402 of KaiC showed a correlation with the side-chain volume of the amino acid at position 402 of KaiC. Our results indicate that residue 402 is a key position of determining the circadian period of cyanobacteria, and it is possible to dramatically alter the period of KaiC while maintaining temperature compensation.

scholarly journals Circadian clock regulation of mRNA translation through eukaryotic elongation factor eEF-2

2016 ◽  
Vol 113 (34) ◽  
pp. 9605-9610 ◽  
Author(s):  
Stephen Z. Caster ◽  
Kathrina Castillo ◽  
Matthew S. Sachs ◽  
Deborah Bell-Pedersen
Keyword(s):  
Circadian Clock ◽  
Mapk Pathway ◽  
Elongation Factor ◽  
Mrna Translation ◽  
Radiation Sensitivity ◽  
Mitogen Activated Protein Kinase ◽  
Mrna Levels ◽  
Transcript Accumulation ◽  
Eukaryotic Elongation Factor

The circadian clock has a profound effect on gene regulation, controlling rhythmic transcript accumulation for up to half of expressed genes in eukaryotes. Evidence also exists for clock control of mRNA translation, but the extent and mechanisms for this regulation are not known. In Neurospora crassa, the circadian clock generates daily rhythms in the activation of conserved mitogen-activated protein kinase (MAPK) pathways when cells are grown in constant conditions, including rhythmic activation of the well-characterized p38 osmosensing (OS) MAPK pathway. Rhythmic phosphorylation of the MAPK OS-2 (P-OS-2) leads to temporal control of downstream targets of OS-2. We show that osmotic stress in N. crassa induced the phosphorylation of a eukaryotic elongation factor-2 (eEF-2) kinase, radiation sensitivity complementing kinase-2 (RCK-2), and that RCK-2 is necessary for high-level phosphorylation of eEF-2, a key regulator of translation elongation. The levels of phosphorylated RCK-2 and phosphorylated eEF-2 cycle in abundance in wild-type cells but not in cells deleted for OS-2 or the core clock component FREQUENCY (FRQ). Translation extracts from cells grown in constant conditions show decreased translational activity in the late subjective morning, coincident with the peak in eEF-2 phosphorylation, and rhythmic translation of glutathione S-transferase (GST-3) from constitutive mRNA levels in vivo is dependent on circadian regulation of eEF-2 activity. In contrast, rhythms in phosphorylated eEF-2 levels are not necessary for rhythms in accumulation of the clock protein FRQ, indicating that clock control of eEF-2 activity promotes rhythmic translation of specific mRNAs.

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