Isoform-specific C-terminal phosphorylation drives autoinhibition of Casein Kinase 1.

Casein kinase controls essential biological processes including circadian rhythms and Wnt signaling, but how its activity is regulated is not well understood. is inhibited by autophosphorylation of its intrinsically disordered C-terminal tail. Two CK1 splice variants, and , are known to have very different effects on circadian rhythms. These variants differ only in the last 16 residues of the tail, referred to as the extreme C-termini (XCT), but with marked changes in potential phosphorylation sites. Here we test if the XCT of these variants have different effects in autoinhibition of the kinase. Using NMR and HDX-MS, we show that the XCT is preferentially phosphorylated by the kinase and the tail makes more extensive interactions across the kinase domain. Mutation of -specific XCT phosphorylation sites increases kinase activity both and in cells and leads to changes in circadian period, similar to what is reported . Mechanistically, loss of the phosphorylation sites in XCT disrupts tail interaction with the kinase domain. autoinhibition relies on conserved anion binding sites around the CK1 active site, demonstrating a common mode of product inhibition of . These findings demonstrate how a phosphorylation cycle controls the activity of this essential kinase.