Abstract
CK1 kinases participate in many signaling pathways; how these enzymes are regulated is therefore of significant biological consequence. CK1s autophosphorylate their C-terminal non-catalytic tails, and eliminating these modifications increases substrate phosphorylation in vitro, suggesting that the autophosphorylated C-termini act as inhibitory pseudosubstrates. To test this prediction, we comprehensively identified the autophosphorylation sites on Schizosaccharomyces pombe Hhp1 and human CK1ε. Peptides corresponding to the C-termini interacted with the kinase domains only when phosphorylated, and phosphoablating mutations increased Hhp1 and CK1ε activity towards substrates. Interestingly, substrates competitively inhibited binding of the autophosphorylated tails to the substrate binding grooves. The presence or absence of tail autophosphorylation influenced the catalytic efficiency with which CK1s targeted different substrates, indicating that tails contribute to substrate specificity. Combining this mechanism with autophosphorylation of the T220 site in the catalytic domain, we propose a displacement specificity model to describe how autophosphorylation regulates substrate specificity for the CK1 family.