UC Santa Cruz

Circadian rhythms are generated at the cellular level by a molecular clock oscillator that temporally controls gene expression. These self-sustained biological clocks operate with an intrinsic timing, or period, of ~24-hours but can also respond to external cues, including metabolic signals. Thus, biological rhythms provide organisms with an anticipatory and responsive timekeeping system that aligns physiology to the environment. Circadian rhythm disruption alters the temporal orchestration of clock-controlled genes and can lead to physiologic disorders — demonstrating a need for circadian-related therapeutic development informed by a more holistic understanding of the molecular clock’s mechanistic underpinnings. The mammalian clock is comprised of interlocked transcription/translation-based feedback loops (TTFLs) connected by a core TTFL in which Period 2 (PER2) acts as the stochiometric limiting factor. Because PER2 rhythmic abundance drives clock oscillations, its stability is regulated by multiple forms of post-translational modifications (PTMs). Therefore, resolving the PTM determinants that control PER2 abundance will provide insight to modulate the clock through this regulatory node. Here, we investigate the conformational equilibrium of the Casein Kinase 1 (CK1)-dependent phosphoswitch mechanism in which phosphorylation of a region known as FASP (Familial Advanced Sleep Phase) and a degron region function antagonistically to predominately regulate PER2 stability. We show that CK1 substrate selectivity is controlled through rigid conformational dynamics of its activation loop and requires three residues, including a lysine (K659), to recognize FASP as a substrate. Next, we introduce the acetyl-transferase, CREB Binding Protein (CBP), as a transcriptional coactivator that uses multiple domains to interact with tandem circadian transcription factors for potential multivalent recruitment. Because CBP was shown to interact with PER2 during the repressive phase of the clock, we define their binding interface and map CBP-dependent PER2 acetyl-sites. We found that CBP acetylates K659 which reduces CK1 activity on FASP in vitro. We also reveal that K659 is necessary for proper CK1-dependent regulation of PER2 stability in cells and explore its endogenous acetylation with a validated monospecific antibody. This work aims to deconvolute a mechanistic role for integration of CK1 phosphorylation and CBP acetylation at the stabilizing FASP region of PER2 within the context of the central phosphoswitch.