| Project/Area Number |
18500300
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurochemistry/Neuropharmacology
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| Research Institution | Toho University |
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Principal Investigator |
TAMARU Teruya Toho University, Faculty of Medicine, Teaching assistant (80291706)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,930,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥330,000)
Fiscal Year 2007: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2006: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | circadian / clock / ubiouitin / phosphorylation / transcription factor / acetvlation / protein kinase / BMAL1 / transcription / nuclear entry / pretein kinase / reset |
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| Research Abstract |
Control of the stability of protein is thought to involved in various biological processes, such as cell cycle and circadian clock. MG132, a reversible inhibitor for proteolysis of ubiquitinated (Ub-) proteins; this might be due to inhibition of BMAL1 degradation via Ub-proteasome. I showed that endogenous Ub-BMAL1 accumulated with MG132 during dexamethazone (Dex) -pulse treatment induced cellular clock synchronization/resetting of NIH-3T3 cells. Moreover, BMAL1 ubiquitination was suppressed by U0126, a phosphorylation inhibitor via ERK pathway. Inhibition of Ub-Proteasome during Dex-pulse significantly reduced circadian clock proteins during circadian cycle and damped out-put circadian rhythm of luciferase-reporting Per2 gene expression (Per2-luc rhythm). BMAL1 contains a conserved ERK phosphorylation site (T527) in its PEST-like sequence, which is thought to involved in the controlling protein stability. Ectopic expressed BMAL1 mutants, which lack PEST-like sequence (ΔPEST) or ERK
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phosphorylation site (T527A) , in BMAL1-deficient mouse. Embryonic fibroblasts MEFs) during clock-resetting resulted in dramatically reduced Ub-BMAL1 level in T527A and slightly higher level in ΔPEST. This strongly suggest that ERK phosphorylation in BMAL1-PEST is essential for the ubiquitination-controlled clock-resetting and naive controlled Ub-BMAL1 accumulation of wild type BMAL1 during clock-resetting is Unpaired. In BMAL1-ΔPEST. Transient transfection of GFP-BMAL1/PEST protein, which is expected to perturb ERK phosphorylation mediated ubiquitination during Dex-pulse resulted in reduced level of Ub-BMAL1 accumulation and dramatically damped Per2-luc rhythm. I propose that controlling stability of BMAL1 via ERK phohorylation dependent ubiquitination in its PEST-like sequence have essential roles during resetting/generating circadian clock oscillation.
With collaboration with UCI team (Dept. Pharmacology, USA), I also demonstrated that essential role of circadian BMAL1 acetylation in mammalian clock system (Nature 2007). Less
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