Autophosphorylation of the CK1 Kinase Domain Regulates Enzyme Activity and Substrate Specificity

Abstract

CK1 enzymes are conserved, acidophilic serine/threonine kinases with a variety of critical cellular functions; misregulation of CK1 contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Despite this, little is known about how CK1 activity is controlled. CK1 kinases have highly similar catalytic domains, plus a conserved extension to that kinase domain that is important for enzyme stability and activity. In contrast to the catalytic domains, the C-terminal tails of CK1 family members diverge in sequence and length; however, they all appear to serve as substrates of autophosphorylation. The autophosphorylated tails are proposed to inhibit kinase activity by acting as pseudosubstrates. In addition to C-terminal autophosphorylation, autophosphorylation of the CK1Σ kinase domain has been detected, but its effect on CK1 activity and cellular function has never been explored. Here, we addressed the role of kinase domain autophosphorylation in human CK1δ and CK1Σ, as well as Hhp1 and Hhp2, their homologues in Schizosaccharomyces pombe. In each case, we found that autophosphorylation of a conserved threonine residue in the kinase domain inhibited enzyme activity. This site resides in the mobile L-EF loop proximal to the active site, distinct from the well-characterized T-loop autophosphorylation that occurs in other kinase families. We propose that phosphorylation on the L-EF loop inhibits substrate docking with the kinase domain by shielding the positively charged substrate binding pocket. Consistent with this hypothesis, yeast and human enzymes with phosphoablating mutations at this site were hyperactive in vitro. In vivo, mutation of this site protected yeast cells from heat shock, indicating a change in the phosphorylation of substrates in the stress response pathway. To determine whether other pathways may also be rewired by this mutation, we utilized quantitative phosphoproteomics to probe the substrateomes of wildtype and mutant Hhp1 and Hhp2. We found that ablating autophosphorylation in the kinase domain significantly changed the substrate profiles of these enzymes, suggesting that this regulatory mechanism influences substrate specificity in addition to catalytic activity. Due to the strong sequence conservation of this autophosphorylation site and the functional importance of the L-EF loop, which is unique to the CK1 family of kinases, this mechanism is likely to regulate the majority of CK1 enzymes in vivo. We predict that kinase domain autophosphorylation works in conjunction with other mechanisms, such as C-terminal autophosphorylation, to ultimately determine the extent to which different substrates are phosphorylated in different cellular contexts.