UC Santa Cruz

Casein Kinase 1δ influences the timing of the mammalian clock by regulating PER2 stability through post-translational modifications. CK1δ regulates PER2 through a phosphoswitch mechanism, in which phosphorylation of the FASP (Familial Advanced Sleep Phase) region of PER2 stabilizes the protein to lengthen clock period, while phosphorylation of the degron promotes its degradation to shorten clock period. This phosphoswitch is held in delicate balance to control proper timing of the clock; mutations in CK1δ and PER2 influence the selectivity of the kinase on this switch and result in sleep phase disorders. Two CK1δ splice variants, δ1 and δ2, that differ only in the last 15 residues, show different kinase activity towards the FASP region, which suggests that the extreme C-terminus of CK1δ is a regulator of this switch. Although autophosphorylation of the disordered C-terminus of CK1δ is known to inhibit its kinase activity, it is not clear how this might differ between the CK1δ splice variants. Using Hydrogen/Deuterium Exchange-Mass Spectrometry (HDX-MS), we have begun to map the differences in tail interaction with the catalytic kinase domain of CK1δ. Nuclear Magnetic Resonance (NMR) studies have also been used to measure autophosphorylation rates of the δ1 tail in trans and in cis, and to shed light on potential differences in intramolecular interactions of the tail isoforms. Coupled with biochemical assays using full length kinase mutants, we demonstrate that anion binding sites on the kinase domain may influence the difference in activity between the splice isoforms. Inhibition assays with tail phosphopeptides and full-length tail mutants have also shown a difference in phosphorylation dependence between the splice variants. A deeper comparison of intermolecular interactions in CK1δ and the effects of phosphorylation in splice variants will shed light on how they exhibit differential activity on the PER2 phosphoswitch to control circadian timing.