Casein kinase 1 delta regulates the pace of the mammalian circadian clock.

Circadian rhythms are rhythms in gene expression, metabolism, physiology, and behavior that persist in constant environmental conditions with a cycle length near 24 h. In mammals, the circadian timing system is hierarchical. The primary pacemaker regulating circadian behavioral rhythms is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Most cell types express circadian clock genes and will express rhythmicity in vitro. In vivo, the SCN entrains peripheral oscillators through a complex set of physiological and hormonal rhythms (31, 32, 36). At the molecular level, circadian oscillations are governed by a cell-autonomous negative-feedback loop in which transcription factors drive the expression of their own negative regulators, leading to oscillation between periods of transcriptional activation and repression (reviewed in references 32 and 36). The bHLH-PAS containing transcription factors CLOCK or NPAS2 form heterodimers with BMAL1. These heterodimers binds to E-box elements within regulatory regions of Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) genes to stimulate their transcription. Approximately 12 h after transcriptional activation, PER and CRY proteins reach concentrations sufficient to form repressor complexes that inhibit the activity of the CLOCK/NPAS2:BMAL1 heterodimer, reducing the transcription of Per and Cry genes and subsequently relieving PER/CRY-mediated negative feedback. E-box-mediated expression of other transcription factors, including members of the DBP/HLF/TEF and nuclear orphan receptor families (e.g., Rev-Erbα and ROR-A), provides a mechanism for clock control of genes with diverse promoters and with gene expression peaks occurring at a variety of phases. Posttranslational modifications of circadian clock proteins play a well-established role in the regulation of circadian cycle length. In both flies and mammals, phosphorylation of PER proteins by casein kinase 1 (CK1) proteins is thought to play a key step in determining the speed of the circadian clock (reviewed in reference 5). Mammalian cell culture studies indicate that the phosphorylation of PER proteins by CK1 epsilon (CK1ɛ) regulates their subcellular localization, likely affects their transcription repression capability, and promotes their degradation through a proteasomal pathway dependent upon the F-box proteins β-TrCP1 and β-TrCP2 (12, 29, 34, 35). Interference with β-TrCP1 activity lengthens circadian period in oscillating fibroblasts (27). The CK1 inhibitor IC261 and the proteasome inhibitors MG132 and lactacystin also lengthen period in fibroblasts (12). CRY proteins are also subjected to phosphorylation and degradation cycles that regulate circadian period. The F-box protein FBXL3 plays a key role in regulating CRY1 stability; mutations inactivating this gene increase circadian cycle length (6, 16, 30). Collectively, these studies indicate that the duration of activity of the PER/CRY repressor complex, regulated primarily by the stability of PER and CRY proteins, dictates the cycle length of the molecular oscillator (15). Genetic studies also support an important role for casein kinase action on PER proteins in regulating circadian period. A mutation in the Syrian hamster CK1ɛ gene, tau, shortens the circadian period of behavioral rhythms. Biochemically, the tau mutation (CK1ɛtau, a T178C substitution) differentially affects the activity of the kinase protein, reducing general kinase activity while increasing activity at specific residues of the PER proteins (14, 23). The tau mutation is a gain-of-function mutation with respect to circadian substrates, resulting in decreased PER stability and a reduction in circadian period length in tau mutant hamsters and mice (14, 24). In humans, familial advanced sleep phase syndrome (FASPS) is a circadian-based sleep disorder, in which affected individuals have a short circadian period and an advanced phase of the sleep-wake cycle. One study identified a FASPS pedigree with a mutation in human PER2 (hPER2; S662G mutation); this mutation prevents a priming phosphorylation, thus preventing CK1-mediated phosphorylation (33). A second study identified a dominant mutation within the kinase domain of CK1δ in a family with FASPS (38). Modeling this mutation in mice and flies revealed alterations in period length (38). In the circadian field, much of the attention on mammalian casein kinases has focused on CK1ɛ. The few studies examining CK1δ suggest that it plays a role similar to CK1ɛ. For example, CK1δ, like CK1ɛ, phosphorylates PER proteins, reducing their stability in vitro (7, 38), and both CK1δ and CK1ɛ are present in PER/CRY repressor complexes in vivo (21). Despite these similarities, the role of CK1δ in the molecular clockwork is not well understood. We report here results demonstrating important differences between CK1δ and CK1ɛ in the regulation of circadian cycle length, based on studies utilizing mice in which these genes have been inactivated.