CKIε/δ-dependent phosphorylation is a temperature-insensitive, period-determining process in the mammalian circadian clock

Abstract

T he circadian clock is a molecular mechanism underlying endogenous, self-sustained oscillations with a period of Ϸ24 h, manifested in diverse physiological and metabolic processes (1-3). The most striking feature of circadian clock is its flexible yet robust response to various environmental conditions. For example, circadian periodicity varies with light intensity (4-6) while remaining robust over a wide range of temperatures (''temperature compensation'') (1, 3, 7-9). This flexible-yetrobust characteristic is evolutionarily conserved in organisms ranging from photosynthetic bacteria to warm-blooded mammals (3, 10-12), and has interested researchers from a broad range of disciplines. However, despite many genetic and molecular studies (13-22), the detailed biochemical mechanism underlying this characteristic remains poorly elucidated (3).The simplest explanation for this flexible-yet-robust property is that the key period-determining reactions are insensitive to temperature but responsive to other environmental conditions. Indeed, Pittendrigh proposed the existence of a temperatureinsensitive component in the clock system in 1954 (7), and in 1968, he and his colleagues demonstrated that both the wave form and the period of circadian oscillations are invariant with temperature (23). However, the idea of a temperatureinsensitive biochemical reaction is counterintuitive, as elementary chemical processes are highly temperature-sensitive. One exception is the cyanobacterial clock, in which temperatureinsensitive enzymatic reactions are observed (24,25). However, the cyanobacterial clock is quite distinct from other clock systems, and this biochemical mechanism has not been demonstrated in other clocks.Recently, a chemical-biological approach was proposed to help elucidate the basic processes underlying circadian clocks (26), and high-throughput screening of a large chemical compound library was performed (27). In this report, to analyze systematically the fundamental processes involved in determining the period length of mammalian clocks, we tested 1,260 pharmacologically active compounds for their effect on period length in mouse and human clock cell lines, and found 10 compounds that most markedly lengthened the period of both clock cell lines affected both the central and peripheral circadian clocks. Most compounds inhibited CKI or CKI␦ activity, suggesting that CKI /␦-dependent phosphorylation is an important period-determining process in the mammalian circadian clock. Surprisingly, the degradation rate of endogenous PER2, which is regulated by CKI -dependent phosphorylation (28) and probably by CKI␦-dependent phosphorylation, was temperatureinsensitive in the living clock cells, and the temperatureinsensitivity was preserved even for the in vitro CKI /␦-dependent phosphorylation of a synthetic peptide derived from PER2. These results suggest that this period-determining process is flexible in response to chemical perturbation yet robust in the face of temperature perturbations. Based on these findings, we prop...

Identification of diverse modulators of central and peripheral circadian clocks by high-throughput chemical screening

“…S4A). Given the clock-lengthening effects of CKI inhibitors (27,29,35), we performed in vitro casein kinase Iε inhibition assays. Three compounds (1-3, Fig.…”

“…Several recent studies have employed cell-based chemical screening strategy to identify small molecules with clock-altering activities. For example, screening of off-patent drug collections has revealed multiple casein kinase I inhibitors that markedly lengthened the circadian period (27) and a period-shortening compound that acts on glycogen synthase kinase-3β (GSK-3β) (28). More recently, screening of a large collection (170,000) of uncharacterized compounds identified another period-lengthening compound that interacts with multiple kinases, including CKIα previously unknown to be involved in clock regulation (29).…”

Entrainment of disrupted circadian behavior through inhibition of casein kinase 1 (CK1) enzymes

“…Nonetheless, rhythmicity persists in these cells and targeting of both CK1ε and CK1δ isoforms is necessary to render MEFs arrhythmic (19). RNA interference targeting of either isoform results in significant period lengthening in a U2OS cell line, but significantly greater effects are observed with CK1δ-directed RNAi (17). These studies suggest that, although both isoforms are likely regulators of the circadian clock, accurate cellular circadian timing can persist in the absence of CK1ε.…”

“…In humans, familial advanced sleep phase syndrome (FASPS) is associated with a T44A missense mutation in CK1δ, leading to hypophosphorylation of PER2 in vitro (12), although in Syrian hamsters and mice, the CK1ε tau mutation results in hyperphosphorylation and subsequent destabilization of its PER targets with significant shortening of behavioral activity cycles to 20 h (13-16). Finally, extensive in vitro screening of pharmacologic compounds shows that the majority of compounds that lengthen the period do so by inhibiting CK1ε/CK1δ-mediated phosphorylation of PER (17).…”

A simple method to measure CLOCK-BMAL1 DNA binding activity in tissue and cell extracts

“…Pharmacological inhibition of CKIϵ/δ has previously been shown to severely disrupt circadian rhythms ( Isojima et al , 2009; Meng et al , 2010). In this study, mouse hepatoma cells in culture were incubated with a specific kinase inhibitor of CKIϵ/δ (PF670462, IC50 for CKIϵ and CKIδ are 7.7 nM and 14 nM, respectively) or DMSO (vehicle) for 48 hours.…”