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Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus
Published on: February 16, 2011
Periodically fluctuating protein kinases phosphorylate CLOCK, the putative target in the suprachiasmatic nucleus
1Department of Physiology, Toho University School of Medicine, Tokyo, Japan.
Abstract:
We studied nuclear protein phosphorylation in the rat suprachiasmatic nucleus (SCN) and found that a nuclear fraction of the SCN contained histone H1 kinase activity that periodically fluctuated with a diurnal rhythm, reaching a maximum at the midpoint of the light phase and a minimum at the midpoint of the dark phase. A p13suc1-bound fraction from the SCN nuclear fraction also exhibited diurnally fluctuating histone H1 kinase activity. Using in situ kinase assay, three histone H1 kinases, p45PFK, p100PFK, and p200PFK (termed periodically fluctuating protein kinases, or PFKs) were found in the p13suc1-bound fractions. p45PFK exhibited the highest level of light/dark cycle phosphorylation activity fluctuation. p45PFK highly phosphorylated the Ser-Pro-rich region of CLOCK, the putative physiological target. These results suggest that PFKs, especially p45PFK, are involved in circadian clock-related signal transduction and gene expression, through the phosphorylation of target proteins such as CLOCK.
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