Circadian clocks are molecular timers that measure 24-hour periods through a series of slow but precise biochemical processes. While circadian clocks of cyanobacteria are driven by interactions and ordered phosphorylation of highly structured protein assemblies, timekeeping by circadian clocks of eukaryotes involves slow progressive hyperphosphorylation of a large number of redundant sites in intrinsically disordered regions of clock proteins.

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.