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 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 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 Tribbles Pseudokinase 3 Contributes to Cancer Stemness of Endometrial Cancer Cells by Regulating β-Catenin Expression

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3785
Author(s):  
Wen-Ling Wang ◽  
Guan-Ci Hong ◽  
Peng-Ju Chien ◽  
Yu-Hao Huang ◽  
Hsueh-Te Lee ◽  
...  
Keyword(s):  
Endometrial Cancer ◽  
Messenger Rna ◽  
Tumor Development ◽  
The Cancer Genome Atlas ◽  
Scaffolding Protein ◽  
Mrna Levels ◽  
Cancer Stemness ◽  
Gynecological Malignancy ◽  
Ec Cells

Endometrial cancer (EC) is the second most common gynecological malignancy worldwide. Tribbles pseudokinase 3 (TRIB3) is a scaffolding protein that regulates intracellular signal transduction, and its role in tumor development is controversial. Here, we investigated the biological function of TRIB3 in EC. We found that the messenger RNA (mRNA) expression level of TRIB3 was significantly and positively correlated with shorter overall survival of EC patients in The Cancer Genome Atlas database. The protein expression of TRIB3 was found to be significantly increased in EC cancer stem cells (CSCs) enriched by tumorsphere cultivation. Knockdown of TRIB3 in EC cells suppressed tumorsphere formation, the expression of cancer stemness genes, and the in vivo tumorigenesis. The expression of β-catenin at both the protein and the mRNA levels was downregulated upon TRIB3 silencing. TRIB3 was found to interact with E74 Like ETS transcription factor 4 (ELF4) in the nucleus and bound to ELF4 consensus sites within the catenin beta 1 (CTNNB1) promoter in EC cell lines. These data indicated that TRIB3 may regulate CTNNB1 transcription by enhancing the recruitment of ELF4 to the CTNNB1 promoter. In conclusion, our results suggest that TRIB3 plays an oncogenic role in EC and positively regulates the self-renewal and tumorigenicity of EC-CSCs. Targeting TRIB3 is considered as a potential therapeutic strategy in future EC therapy.

Asynchronous synthesis of membrane skeletal proteins during terminal maturation of murine erythroblasts

Blood ◽  
1992 ◽  
Vol 80 (2) ◽  
pp. 530-539 ◽  
Author(s):  
M Hanspal ◽  
JS Hanspal ◽  
R Kalraiya ◽  
SC Liu ◽  
KE Sahr ◽  
...  
Keyword(s):  
Blood Cell ◽  
Red Blood Cell ◽  
Messenger Rna ◽  
Membrane Skeleton ◽  
Band 3 ◽  
Metabolic Labeling ◽  
Mrna Levels ◽  
Long Term Stability

To study the changes in the synthesis of the major membrane skeletal proteins, their assembly on the membrane, and their turnover during terminal red blood cell maturation in vivo, we have compared early proerythroblasts and late erythroblasts obtained from the spleens of mice at different times after infection with the anemia-inducing strain of Friend virus (FVA). Metabolic labeling of these cells indicates striking differences between early and late erythroblasts. In early erythroblasts, spectrin and ankyrin are synthesized in large amounts in the cytosol with proportionately high levels of spectrin and ankyrin messenger RNA (mRNA). In contrast, only small amounts of these polypeptides are incorporated into the skeleton, which is markedly unstable. In late erythroblasts, however, the synthesis of spectrin and ankyrin and their mRNA levels are substantially reduced, yet the net amounts of these polypeptides assembled in the membrane skeleton are markedly increased, and the membrane skeleton becomes stable with no detectable protein turnover. The mRNA levels and the synthesis of the band 3 and 4.1 proteins are increased considerably in terminally differentiated normoblasts with a concomitant increase in the net amount and the half-life of the newly assembled spectrin and ankyrin. Thus, the increased accumulation of spectrin and ankyrin at the late erythroblast stage is a consequence of an increased recruitment of these proteins on the membrane and an increase in their stability rather than a transcriptional upregulation. This is in contrast to band 3 and 4.1 proteins, which accumulate in direct proportion to their mRNA levels and rates of synthesis. These results suggest a key role for the band 3 and 4.1 proteins in conferring a long-term stability to the membrane skeleton during terminal red blood cell differentiation.

scholarly journals The Serum- and Glucocorticoid-Induced Kinase Is a Physiological Mediator of Aldosterone Action*

Endocrinology ◽  
2001 ◽  
Vol 142 (4) ◽  
pp. 1587-1594 ◽  
Author(s):  
Aditi Bhargava ◽  
Meryl J. Fullerton ◽  
Kathy Myles ◽  
Timothy M. Purdy ◽  
John W. Funder ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Messenger Rna ◽  
Time Course ◽  
Rat Kidney ◽  
Distal Colon ◽  
Mrna Levels ◽  
Epithelial Tissues ◽  
Sodium And Potassium

Abstract Aldosterone plays a major role in regulating sodium and potassium flux in epithelial tissues such as kidney and colon. Recent evidence suggests that serum- and glucocorticoid-regulated kinase (SGK) is induced by aldosterone and acts as a key mediator of aldosterone action in epithelial tissues. Induction of SGK messenger RNA (mRNA) has previously been shown within 30 min of addition of supraphysiological doses of aldosterone to Xenopus A6 cells and within 4 h in rat kidney in vivo. In this study we determined the time course of SGK induction, at doses of aldosterone in the physiological range, in rat kidney and colon, using Northern and Western blot analyses and in situ hybridization and determined concurrent changes in urinary sodium and potassium excretion by Kagawa bioassay. On Northern blot analysis, SGK mRNA levels were significantly elevated in both kidney and colon 60 min after the injection of aldosterone. SGK protein in late distal colon was significantly elevated 2 and 4 h after aldosterone treatment. In situ hybridization showed SGK mRNA to be induced in renal collecting ducts and distal tubular elements in both cortex and medulla by doses of aldosterone of 0.1 μg/100 g BW or more within 30 min of steroid treatment. Significant changes in urinary composition were similarly seen with an aldosterone dose of 0.1 μg/100 g BW from 90 min after aldosterone injection. The early onset of SGK induction in kidney and colon and the correlation with urinary changes in terms of both time course and dose response suggest that SGK plays an important role in mediating the effects of aldosterone on sodium homeostasis in vivo.

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.

Effect of serum thyrotropin levels on the concentration of messenger RNA for thyroid peroxidase in the rat

1992 ◽  
Vol 126 (5) ◽  
pp. 460-466 ◽  
Author(s):  
Ronald P Magnusson ◽  
Bo Yu ◽  
Veronica Brennan
Keyword(s):  
Messenger Rna ◽  
Thyroid Peroxidase ◽  
Mrna Levels ◽  
Hypophysectomized Rats ◽  
Steady State Levels ◽  
Low Levels ◽  
Rat Thyroid ◽  
Serum Tsh ◽  
Bovine Tsh

The effect of serum TSH on rat thyroid peroxidase mRNA levels was studied in order to investigate the regulation of thyroid peroxidase gene expression in vivo. A nearly full-length rat thyroid peroxidase cDNA clone was isolated from a bacteriophage cDNA library synthesized using poly A+ RNA isolated from the thyroids of propylthiouracil-treated rats. cDNA probes derived from this clone were used to study rat thyroid peroxidase mRNA levels in response to the level of serum TSH. Two major rat thyroid peroxidase mRNA bands were detected on Northern blots of total cellular RNA (at 3.2 kb and at 3.7kb). Injection of thyroxine, which lowered the levels of serum TSH, also lowered the steady-state levels of both rat thyroid peroxidase mRNAs, whereas treatment with methimazole, which increased serum TSH, increased both rat thyroid peroxidase mRNA levels. In hypophysectomized rats 10 days postoperative, very low levels of thyroid peroxidase mRNA were observed. Injection of bovine TSH (1 IU/day) increased rat thyroid peroxidase mRNA expression, preferentially in the 3.2 kb band. These results clearly demonstrate that TSH regulates rat thyroid peroxidase mRNA levels in vivo.

The in vivo kinetics of tissue factor messenger RNA expression during human endotoxemia: relationship with activation of coagulation

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 554-559 ◽  
Author(s):  
Rendrik F. Franco ◽  
Evert de Jonge ◽  
Pascale E. P. Dekkers ◽  
Janneke J. Timmerman ◽  
C. Arnold Spek ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Tissue Factor ◽  
Messenger Rna ◽  
Thrombin Generation ◽  
Mrna Levels ◽  
Total Blood ◽  
Human Endotoxemia ◽  
Lps Challenge ◽  
Kinetics Of

Abstract Triggering of the tissue factor (TF)-dependent coagulation pathway is considered to underlie the generation of a procoagulant state during endotoxemia. To determine the in vivo pattern of monocytic TF messenger RNA (mRNA) expression during endotoxemia, 10 healthy volunteers were injected with lipopolysaccharide (LPS, 4 ng/kg) and blood was collected before and 0.5, 1, 2, 3, 4, 6, 8, and 24 hours after LPS administration. Total blood RNA was isolated and amplified by NASBA (nucleic acid sequence-based amplification), followed by quantitation of TF mRNA by an electrochemiluminescence (ECL) assay. To compare the pattern of coagulation activation with the kinetics of monocytic TF mRNA expression, we measured plasma levels of markers of thrombin generation, thrombin-antithrombin (TAT) complexes, and prothrombin fragment 1 + 2 (F1 + 2). Baseline value (mean ± SEM) of the number of TF mRNA molecules per monocytic cell was 0.08 ± 0.02. A progressive and significant (P < .0001) increase in TF expression was observed after LPS injection (+0.5 hour: 0.3 ± 0.1, +1 hour: 1.3 ± 0.9, +2 hours: 4.1 ± 0.9), peaking at +3 hours (10 ± 1.9 TF mRNA molecules per monocyte). As TF mRNA levels increased, thrombin generation was augmented. Peak levels of TAT and F1 + 2 were reached later (at t +4 hours) than those of TF mRNA. TF mRNA, TAT, and F1 + 2 levels returned to baseline after 24 hours. In conclusion, we used a NASBA/ECL-based technique to quantify TF mRNA in whole blood during human endotoxemia and observed a 125-fold increase in TF mRNA levels. Our data demonstrate a pivotal role for enhanced TF gene activity in the activation of coagulation after LPS challenge.

scholarly journals Asynchronous synthesis of membrane skeletal proteins during terminal maturation of murine erythroblasts

Blood ◽  
1992 ◽  
Vol 80 (2) ◽  
pp. 530-539 ◽  
Author(s):  
M Hanspal ◽  
JS Hanspal ◽  
R Kalraiya ◽  
SC Liu ◽  
KE Sahr ◽  
...  
Keyword(s):  
Blood Cell ◽  
Red Blood Cell ◽  
Messenger Rna ◽  
Membrane Skeleton ◽  
Band 3 ◽  
Metabolic Labeling ◽  
Mrna Levels ◽  
Long Term Stability

Abstract To study the changes in the synthesis of the major membrane skeletal proteins, their assembly on the membrane, and their turnover during terminal red blood cell maturation in vivo, we have compared early proerythroblasts and late erythroblasts obtained from the spleens of mice at different times after infection with the anemia-inducing strain of Friend virus (FVA). Metabolic labeling of these cells indicates striking differences between early and late erythroblasts. In early erythroblasts, spectrin and ankyrin are synthesized in large amounts in the cytosol with proportionately high levels of spectrin and ankyrin messenger RNA (mRNA). In contrast, only small amounts of these polypeptides are incorporated into the skeleton, which is markedly unstable. In late erythroblasts, however, the synthesis of spectrin and ankyrin and their mRNA levels are substantially reduced, yet the net amounts of these polypeptides assembled in the membrane skeleton are markedly increased, and the membrane skeleton becomes stable with no detectable protein turnover. The mRNA levels and the synthesis of the band 3 and 4.1 proteins are increased considerably in terminally differentiated normoblasts with a concomitant increase in the net amount and the half-life of the newly assembled spectrin and ankyrin. Thus, the increased accumulation of spectrin and ankyrin at the late erythroblast stage is a consequence of an increased recruitment of these proteins on the membrane and an increase in their stability rather than a transcriptional upregulation. This is in contrast to band 3 and 4.1 proteins, which accumulate in direct proportion to their mRNA levels and rates of synthesis. These results suggest a key role for the band 3 and 4.1 proteins in conferring a long-term stability to the membrane skeleton during terminal red blood cell differentiation.

scholarly journals Regulation of Corticotropin-Releasing Factor Receptor Type 2β Messenger Ribonucleic Acid in the Rat Cardiovascular System by Urocortin, Glucocorticoids, and Cytokines*

Endocrinology ◽  
2000 ◽  
Vol 141 (7) ◽  
pp. 2285-2293 ◽  
Author(s):  
Kazunori Kageyama ◽  
Georges E. Gaudriault ◽  
Margaret J. Bradbury ◽  
Wylie W. Vale
Keyword(s):  
Cardiovascular System ◽  
Mrna Expression ◽  
Messenger Rna ◽  
Physical Restraint ◽  
Receptor Type ◽  
Mrna Levels ◽  
Immune Challenge ◽  
Messenger Ribonucleic Acid

CRF receptor type 2 (CRF R2) messenger RNA (mRNA) expression in the rodent heart is modulated by exposure to both the bacterial endotoxin lipopolysaccharide (LPS) and glucocorticoids. In this study we examined the roles of glucocorticoids, cytokines, and CRF R2β ligands in the regulation of CRF R2β expression in the cardiovascular system both in vivo and in vitro. Using ribonuclease protection assays, we found that, in addition to the injection of LPS or corticosterone, physical restraint caused a decrease in CRF R2β mRNA levels in the rat heart and aorta. Adrenalectomy with corticosterone replacement at constant levels partially blocked LPS-induced decreases in CRF R2β mRNA expression in the heart. Thus, elevations of endogenous circulating corticosterone could contribute to the down-regulation of CRF R2β mRNA expression in heart. To identify other putative modulating factors, we examined CRF R2β expression in the aorta- derived A7R5 cell line. Incubation with CRF R2 ligands or dexamethasone reduced CRF R2β mRNA levels. In addition, incubation with a variety of cytokines, proteins released during immune challenge, also reduced CRF R2β mRNA expression. The multifactorial regulation of CRF R2β mRNA expression in the cardiovascular system may serve to limit the inotropic and chronotropic effects of CRF R2 agonists such as urocortin during prolonged physical or immune challenge.

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