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J Biol Chem. 2018 Jun 8;293(23):9126-9136. doi: 10.1074/jbc.RA117.000773. Epub 2018 Mar 27.

CLOCK phosphorylation by AKT regulates its nuclear accumulation and circadian gene expression in peripheral tissues.

Author information

1
From the Departments of Pharmacology and.
2
the Vascular Biology and Therapeutics Program (VBT), Yale University School of Medicine, New Haven, Connecticut 06520 and.
3
Cell Biology and.
4
the Department of Internal Medicine, Veterans Affairs Connecticut Healthcare System, West Haven, Connecticut 06516.
5
From the Departments of Pharmacology and william.sessa@yale.edu.

Abstract

Circadian locomotor output cycles kaput (CLOCK) is a transcription factor that activates transcription of clock-controlled genes by heterodimerizing with BMAL1 and binding to E-box elements on DNA. Although several phosphorylation sites on CLOCK have already been identified, this study characterizes a novel phosphorylation site at serine 845 (Ser-836 in humans). Here, we show that CLOCK is a novel AKT substrate in vitro and in cells, and this phosphorylation site is a negative regulator of CLOCK nuclear localization by acting as a binding site for 14-3-3 proteins. To examine the role of CLOCK phosphorylation in vivo, ClockS845A knockin mice were generated using CRISPR/Cas9 technology. ClockS845A mice are essentially normal with normal central circadian rhythms and hemodynamics. However, examination of core circadian gene expression from peripheral tissues demonstrated that ClockS845A mice have diminished expression of Per2, Reverba, Dbp, and Npas2 in skeletal muscle and Per2, Reverba, Dbp, Per1, Rora, and Npas2 in the liver during the circadian cycle. The reduction in Dbp levels is associated with reduced H3K9ac at E-boxes where CLOCK binds despite no change in total CLOCK levels. Thus, CLOCK phosphorylation by AKT on Ser-845 regulates its nuclear translocation and the expression levels of certain core circadian genes in insulin-sensitive tissues.

KEYWORDS:

Akt PKB; circadian rhythm; mouse genetics; phosphorylation; substrate specificity

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