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Mitogen-activated protein kinase phosphorylates and negatively regulates basic helix-loop-helix-PAS transcription
Kamon Sanada1, Toshiyuki Okano, Yoshitaka Fukada
1Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo and Core Research for Engineering, Science, and Technology, Japan Science and Technology, Tokyo 113-0033, Japan.
The Journal of Biological Chemistry
|November 1, 2001
Summary
Mitogen-activated protein kinase (MAPK) phosphorylates the circadian clock protein BMAL1, inhibiting its activity. This discovery reveals a molecular link between MAPK signaling and the core circadian oscillator.
Area of Science:
- Chronobiology
- Molecular Biology
- Biochemistry
Background:
- Mitogen-activated protein kinase (MAPK) shows circadian activation in vertebrate clock structures.
- MAPK is implicated in the timekeeping mechanisms of the circadian clock.
- BMAL1 is a key positive regulator in the circadian oscillator's feedback loop.
Purpose of the Study:
- To investigate the interaction between MAPK and BMAL1.
- To determine if MAPK affects BMAL1's function in circadian rhythm regulation.
- To elucidate the molecular mechanism linking MAPK to the circadian clock.
Main Methods:
- In vitro phosphorylation assays to identify MAPK targets on BMAL1.
- Transactivation assays in 293 cells using BMAL1:CLOCK and MAPK signaling components.
- Site-directed mutagenesis of BMAL1 phosphorylation sites.
Main Results:
- MAPK directly phosphorylates BMAL1 at multiple sites, including Ser-527, Thr-534, and Ser-599.
- Constitutively active MAPK kinase inhibits BMAL1:CLOCK-mediated transcription.
- This inhibition is reversed by a kinase-dead MAPK mutant or by mutating BMAL1 at Thr-534.
Conclusions:
- MAPK negatively regulates BMAL1:CLOCK-induced transcription via phosphorylation at Thr-534.
- This study establishes a molecular link between circadian-activated MAPK and the core circadian oscillator.
- Phosphorylation of BMAL1 by MAPK is a key regulatory step in circadian rhythm maintenance.