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

Casein kinase 1 δ (CK1δ) controls essential biological processes, including circadian rhythms and Wnt signaling, but how its activity is regulated is not well understood. CK1δ is inhibited by autophosphorylation of its intrinsically disordered C-terminal tail. Two CK1 splice variants, δ1 and δ2, differ only in the last 16 residues of the tail and have different activity in vivo, suggesting that the extreme C-terminus (XCT) of CK1 plays a role in autoinhibition. Using NMR and HDX-MS, we show that the δ1 XCT is preferentially phosphorylated by the kinase and the δ1 tail makes more extensive interactions across the kinase domain. Mutation of δ1-specific XCT phosphorylation sites disrupts its interaction with the kinase domain and increases kinase activity in vitro and in cells. δ1 autoinhibition relies on conserved anion binding sites around the CK1 active site, demonstrating a common mode of product inhibition of CK1δ. These findings demonstrate how a phosphorylation cycle controls the activity of this essential kinase. ### Competing Interest Statement The authors have declared no competing interest.