Roles of CLOCK Phosphorylation in Suppression of E-Box-Dependent Transcription

Abstract

In mammalian circadian clockwork, the CLOCK-BMAL1 heterodimer activates E-box-dependent transcription, while its activity is suppressed by circadian binding with negative regulators, such as CRYs. Here, we found that the CLOCK protein is kept mostly in the phosphorylated form throughout the day and is partly hyperphosphorylated in the suppression phase of E-box-dependent transcription in the mouse liver and NIH 3T3 cells. Coexpression of CRY2 in NIH 3T3 cells inhibited the phosphorylation of CLOCK, whereas CIPC coexpression markedly stimulated phosphorylation, indicating that CLOCK phosphorylation is regulated by a combination of the negative regulators in the suppression phase. CLOCK-BMAL1 purified from the mouse liver was subjected to tandem mass spectrometry analysis, which identified Ser38, Ser42, and Ser427 as in vivo phosphorylation sites of CLOCK. Ser38Asp and Ser42Asp mutations of CLOCK additively and markedly weakened the transactivation activity of CLOCK-BMAL1, with downregulation of the nuclear amount of CLOCK and the DNA-binding activity. On the other hand, CLOCK⌬19, lacking the CIPC-binding domain, was far less phosphorylated and much more stabilized than wild-type CLOCK in vivo. Calyculin A treatment of cultured NIH 3T3 cells promoted CLOCK phosphorylation and facilitated its proteasomal degradation. Together, these results show that CLOCK phosphorylation contributes to the suppression of CLOCK-BMAL1-mediated transactivation through dual regulation: inhibition of CLOCK activity and promotion of its degradation.Many physiological activities of living organisms show rhythmic changes, with a period of approximately 24 h, even under constant conditions without any external time cues. These daily variations, called circadian rhythms, are generated by the circadian clock, a cell-autonomous time-measuring system that has evolved in a wide variety of organisms, from cyanobacteria to higher plants and humans (9). In mammals, a master clock is located in the hypothalamic suprachiasmatic nucleus, while self-sustaining molecular clocks reside even in the peripheral tissues, such as the liver (18,40,45). In these central and peripheral clock cells, the clock genes form a transcription/ translation-based negative-feedback loop to generate the molecular oscillation with 24-h periodicity. CLOCK and BMAL1 are basic helix-loop-helix (bHLH)-PAS transcription factors, and the CLOCK-BMAL1 heterodimer binds to CACGTG E-box (14) or E-box-like (25, 61) sequences for the transactivation of negative regulatory genes, such as Period (Per1 and Per2) and Cryptochrome (Cry1 and Cry2) genes. Newly translated PERs and CRYs enter the nuclei and bind to the CLOCK-BMAL1 heterodimer, leading to suppression of its transactivation (30).In the mouse liver, the abundances and the phosphorylation states of both PER1 and PER2 show striking temporal changes (32). The total amount of each PER protein shows the lowest level at CT6 (CT is circadian time, with CT0 under the constant-dark [DD] condition corresponding to the lights-on time in the...

Intermolecular recognition revealed by the complex structure of human CLOCK-BMAL1 basic helix-loop-helix domains with E-box DNA

“…It has been reported that three of the four serine residues (CLOCK Ser38, Ser42 and BMAL1 Ser90) can Table S1 for full-length DNA used in ITC measurements. be phosphorylated in vivo [38,39]. However, BMAL1 Ser78 is also very likely to undergo phosphorylation predicted by NetPhos 2.0 Server [40], which is supported by the finding of a mass spectrometry record of phosphorylated BMAL1 Ser78 in PhosphoSite database [41].…”

“…We have shown that the S78E mutant of BMAL1 significantly compromises the ability of DNA binding as well as the transcriptional activity of the complex in cells. Compared with the previously reported inhibitory phosphorylation sites in CLOCK basic regions (Ser38 and Ser42) [38], phosphorylation on BMAL1 Ser78 will be more crucial and efficient in downregulating the transcriptional activity of CLOCK-BMAL1. Considering that many kinases can phosphorylate and regulate the activity of BMAL1 at multiple sites [6,46,47], we propose that BMAL1 Ser78 should be a key residue mediating input signal-regulated transcriptional inhibition for external cues to entrain the circadian clock by kinase cascade.…”

Genetic suppression of the circadian Clock mutation by the melatonin biosynthesis pathway

“…Thus, it is conceivable that melatonin-induced activation of protein kinase C could ultimately affect CLOCK and BMAL1 to suppress the Clock mutation. Recent work has shown that phosphorylation of CLOCKΔ19 mutant protein is reduced relative to wildtype CLOCK, leading to its stabilization (42). Thus, melatonin could differentially affect CLOCKΔ19 phosphorylation to rescue this defect, which could explain at least in part the selective suppression of the Clock mutant phenotype.…”

A Common Origin: Signaling Similarities in the Regulation of the Circadian Clock and DNA Damage Responses

“…The detailed functions of other post-translational modifications of clock proteins have been reviewed elsewhere. 6,33,34) In mammals, the core circadian regulator CLOCK has intrinsic histone acetyltransferase (HAT) activity 35) that it uses to acetylate its heterodimeric partner BMAL1. 36) This CLOCK-mediated acetylation increases the interaction of the CLOCK:BMAL1 complex with CRY1, providing another level of control in the circadian negative feedback loop.…”