Control of Mammalian Circadian Rhythm by CKIε-Regulated Proteasome-Mediated PER2 Degradation

Abstract

The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKI (casein kinase I) has been postulated to prime PER2 for degradation. Supporting this idea, CKI inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKI inhibition also slows the degradation of PER2 in cells. CKI-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein ␤-TrCP to a specific site, and dominant negative ␤-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.Diverse organisms from prokaryotes to mammals coordinate behavioral and physiological rhythms with the daily dark-light cycle by means of a circadian clock. In mammals, the master circadian clock is located in the suprachiasmatic nucleus of the brain, and it entrains peripheral cell-autonomous clocks throughout the body. In mice, a positively acting heterodimeric transcription factor composed of the PAS-bHLH proteins CLOCK (CLK) and BMAL1 drives transcription of tissuespecific circadian output genes, as well as its own negative regulators, the Period (denoted mPer1, mPer2, and mPer3), and Cryptochrome (mCry1 and mCry2) genes. The mammalian PER and CRY proteins form multimeric complexes that enter the nucleus and repress the transcriptional activity of CLK/ BMAL1, modulating circadian output (reviewed in references 29 and 41). Additional stabilizing feedback loops, including inhibition of Bmal1 transcription by REV-ERB␣ (37), further contribute to the timing and robustness of the cycle. The daily rhythmic degradation of PERIOD proteins leading to derepression of CLK/BMAL1 is postulated to be critical to the proper functioning of the clock. Therefore, the mechanism and control of this process are of great interest.Genetic studies have identified CKIε (casein kinase Iε) as a key regulator of metazoan circadian rhythm and both genetic and biochemical studies suggest that the PER proteins are important substrates (reviewed in reference 10). CKIε was first implicated as a circadian regulator in Drosop...

Molecular characterization of the transition to mid-life in Caenorhabditis elegans

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Dependence of the period on the rate of protein degradation in minimal models for circadian oscillations

“…Among the multiple molecular mechanisms that influence the period of circadian rhythms, a major role is played by those that control clock protein degradation (Liu et al 2000;Eide et al 2005;Gallego et al 2006;Busino et al 2007;Reischl et al 2007). Here we compared three minimal models proposed for circadian clocks, showed that they predict opposite profiles of period as a function of the clock protein degradation rate, and indicated how to reconcile these contradictory predictions.…”

Circadian regulation of lipid metabolism

“…Hence, PER and CRY levels are higher during the day and lower at night, similar to the SCN neural activity rhythm. The circadian time course of gene expression is regulated by post-translational mechanisms, including phosphorylation-dependent ubiquitination and proteosomal degradation of PER and CRY proteins in the cytoplasm (36)(37)(38)(39)(40) , allowing for a new cycle of transcription by relieving inhibition of the BMAL1:CLOCK/NPAS2 transcriptional complex.…”