Circadian clocks are timing systems that rhythmically adjust physiology and metabolism to the 24-h day–night cycle. Eukaryotic circadian clocks are based on transcriptional–translational feedback loops (TTFLs). Yet TTFL-core components such as Frequency (FRQ) in Neurospora and Periods (PERs) in animals are not conserved, leaving unclear how a 24-h period is measured on the molecular level. Here, we show that CK1 is sufficient to promote FRQ and mouse PER2 (mPER2) hyperphosphorylation on a circadian timescale by targeting a large number of low-affinity phosphorylation sites. Slow phosphorylation kinetics rely on site-specific recruitment of Casein Kinase 1 (CK1) and access of intrinsically disordered segments of FRQ or mPER2 to bound CK1 and on CK1 autoinhibition. Compromising CK1 activity and substrate binding affects the circadian clock in Neurospora and mammalian cells, respectively. We propose that CK1 and the clock proteins FRQ and PERs form functionally equivalent, phospho-based timing modules in the core of the circadian clocks of fungi and animals.
Theory predicts that self-sustained oscillations require robust delays and nonlinearities (ultrasensitivity). Delayed negative feedback loops with switch-like inhibition of transcription constitute the core of eukaryotic circadian clocks. The kinetics of core clock proteins such as PER2 in mammals and FRQ in Neurospora crassa is governed by multiple phosphorylations. We investigate how multiple, slow and random phosphorylations control delay and molecular switches. We model phosphorylations of intrinsically disordered clock proteins (IDPs) using conceptual models of sequential and distributive phosphorylations. Our models help to understand the underlying mechanisms leading to delays and ultrasensitivity. The model shows temporal and steady state switches for the free kinase and the phosphoprotein. We show that random phosphorylations and sequestration mechanisms allow high Hill coefficients required for self-sustained oscillations.
Timing by the circadian clock of Neurospora is associated with hyperphosphorylation of frequency (FRQ), which depends on anchoring casein kinase 1a (CK1a) to FRQ. It is not known how CK1a is anchored so that approximately 100 sites in FRQ can be targeted. Here, we identified two regions in CK1a, p1 and p2, that are required for anchoring to FRQ. Mutation of p1 or p2 impairs progressive hyperphosphorylation of FRQ. A p1‐mutated strain is viable but its circadian clock is non‐functional, whereas a p2‐mutated strain is non‐viable. Our data suggest that p1 and potentially also p2 in CK1a provide an interface for interaction with FRQ. Anchoring via p1‐p2 leaves the active site of CK1a accessible for phosphorylation of FRQ at multiple sites.
Casein kinase 1δ (CK1δ) is a simple monomeric enzyme involved in the regulation of a variety of functions, including signal transduction, the circadian clock, and the cell cycle, and is a known target of the ubiquitin ligase APC/CCdh1. How CK1δ is regulated to exert its multiple functions is not understood. Here, we have characterized the posttranslational regulation of CK1δ. We show that newly synthesized CK1δ is highly susceptible to proteasomal degradation in the nucleus and shuttles between the cytosol and nucleus in search of assembly partners that stabilize CK1δ. Kinase activity supports two competing processes: export from the nucleus to ensure distribution of CK1δ between its clients, and degradation in the nucleus to keep the amount of active, potentially deleterious orphan kinase low. During mitosis, CK1δ is inhibited by (auto)phosphorylation, stabilizing the CK1δ released from centrosomes to preserve the kinase for the subsequent G1 phase. Our data show that all active CK1δ is associated with stabilizing partners.
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