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

Abstract

ircadian ocomotorutput yclesaput (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 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, knockin mice were generated using CRISPR/Cas9 technology. mice are essentially normal with normal central circadian rhythms and hemodynamics. However, examination of core circadian gene expression from peripheral tissues demonstrated that mice have diminished expression of and in skeletal muscle and, and in the liver during the circadian cycle. The reduction in 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.

Supply of methionine and arginine alters phosphorylation of mechanistic target of rapamycin (mTOR), circadian clock proteins, and α-s1-casein abundance in bovine mammary epithelial cells

“…This would occur via phosphorylating CLOCK (Ser 845 ) and ARNTL (Ser 42 ), which subsequently influences their nuclear/cytoplasmic shuttling. 75,76 In the present study, the lack of statistical changes in ARNTL protein abundance due to AA treatments was inconsistent with previous work where AKT deficiency decreased ARNTL abundance. 77 Lower p-AKT protein with increased supply of Arg could alleviate inhibition on clock function, but the opposite response of CLOCK protein abundance with increased Met or Arg alone or in combination might have occurred because AKT might not be the sole regulator of CLOCK and ARNTL in mammary cells.…”

RETRACTED: Melatonin Attenuates Dysregulation of the Circadian Clock Pathway in Mice With CCl4-Induced Fibrosis and Human Hepatic Stellate Cells

“…In fact, information on the crosstalk between melatonin and clock genes in pathological situations is very scarce; the most detailed data have been obtained in breast cancer, having been found that melatonin, via activation of MT1, represses the transcriptional activity of RORα to suppress BMAL1 promoter activity ( Xiang et al, 2012 ). Regulation of clock through changes in CLOCK phosphorylation which could modify the transactivation activity of CLOCK-BMAL1 ( Luciano et al, 2018 ) is another interesting possibility worthy to be explored in the future.…”

The Cancer Clock Is (Not) Ticking: Links between Circadian Rhythms and Cancer

“…Activation of AKT has also been found to act on the molecular clock, lengthening the circadian period [148]. AKT-driven phosphorylation of CLOCK directs it for nuclear translocation, affecting the expression of downstream metabolic genes [149]. A number of metabolites display circadian oscillation [150][151][152] and important regulators of metabolism, such as Ras, c-Myc, nuclear hormone receptors and p53 are also associated with circadian rhythms (reviewed in [142,153,154]).…”