A PER2‐Derived Mechanism‐Based Bisubstrate Analog for Casein Kinase 1ε

Abstract

Casein kinase 1ε (CK1ε) plays an important regulatory role in various cellular processes including circadian rhythms. Mutations in CK1ε or the recognition site on its substrate PER2 result in modulation of the circadian period length. In particular, the tau mutation (R178C) in the catalytic domain of CK1ε was identified as the molecular basis for a dose-dependent heritable shortened circadian period in hamsters. However, the biochemical basis for the physiological effects of the tau mutant remains unclear. It has been reported that the tau mutation has reduced in vitro activity against some substrates but increased in vitro activity against other substrates. To better understand the effects of the CK1ε tau mutation, an ATP-phosphopeptide conjugate was synthesized to yield a transition-state bisubstrate analog. Kinase activity assays determined that the tau mutant has 80% reduced activity and a fourfold decrease in sensitivity to the bisubstrate analog compared to wild type. This confirms that Arg178 is important in the recognition of the preferred phosphosubstrates of CK1ε.

PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period

“…Combining the total intensities for phosphoserines 4 and 5 from states C and D ( Figure 2g ) confirms that the reaction kinetics proceed as predicted by the model outlined in Figure 2b . To probe the ordered distributive mechanism, we utilized a mutant form of the kinase, K224D (Shinohara et al, 2017), that disrupts the anion binding pocket Site 1 near the active site thought to anchor primed substrates and facilitate kinase activity on downstream consensus sites (Longenecker et al, 1996; Venkatesan et al, 2019; Zeringo and Bellizzi, 2014). The K224D mutant retains its ability to prime the FASP region with kinetics similar to the WT kinase but has decreased activity on subsequent consensus-based sites (Philpott et al, 2020).…”

Casein Kinase 1 dynamics underlie the PER2 circadian phosphoswitch

“…The mutation eliminates a positively charged residue in the first of three CK1 family-specific pockets (Sites 1, 2, and 3) that coordinate anions in the crystallographic structure ( Figure 1C) (Longenecker et al, 1996). The Site 1 pocket where R178 sits is located adjacent to the active site and has been postulated to bind the phosphorylated 170 priming site to position serines of the consensus motif at the active site (Longenecker et al, 1996;Zeringo and Bellizzi, 2014). Based on this model, it is predicted that tau should preferentially disrupt phosphorylation of the downstream consensus sites in the FASP region due to its inability to recruit the primed substrate.…”