Abstract 1844 Substrate displacement of CK1 C-termini regulates kinase specificity

CK1 enzymes phosphorylate substrates that participate in a variety of signaling pathways, yet the mechanisms by which these kinases are regulated remain elusive. Human and yeast CK1s autophosphorylate their C-terminal non-catalytic tails, and C-terminal truncation or dephosphorylation of these enzymes has been found to increase substrate phosphorylation in vitro. This has led to a model in which the autophosphorylated C-terminus acts as a pseudosubstrate to inhibit catalytic activity. Here, we have directly evaluated this proposed mechanism in vitro and in vivo using the CK1 homologues Schizosaccharomyces pombe Hhp1 and human CK1 delta and CK1 epsilon. First, we identified the C-terminal autophosphorylation sites, confirming 6 on Hhp1 and 15 on CK1 epsilon. In vivo, Hhp1 autophosphorylated its C-terminus on the sites we identified, and the occupancy of these sites varied under different cellular conditions, indicating that autophosphorylation is dynamic in cells. In vitro, peptides corresponding to the Hhp1 and CK1 epsilon C-termini directly interacted with the kinase domains, but only when these sites were phosphorylated. Mutating these sites to non-phosphorylatable residues increased the activity of both enzymes towards their substrates. Interestingly, we found that substrates have a higher affinity for the kinase domain than the autophosphorylated C-termini, and substrates competitively inhibited tail binding even when the tail was fully autophosphorylated. Mutating the basic residues on the kinase domain that comprise the substrate binding groove prevented both tail binding and substrate phosphorylation, supporting the model that tail and substrate compete for binding to the substrate binding groove. Furthermore, we found that the C-termini contribute to substrate specificity, in combination with the previously described autophosphorylation site at T220 that resides in the catalytic domain. Truncation of the CK1 delta C-terminus relaxed the kinase's preference for an upstream phosphoprimed residue in the linear substrate motif, allowing additional specificity determinants to be observed. Autophosphorylation at T220 affected the likelihood of tail autophosphorylation and binding, demonstrating that these two types of autophosphorylation sites work in concert. Both C-terminal and T220 autophosphorylation affected the specificity constants measured in vitro for different protein substrates of yeast and human CK1s. Based on these data, we propose a displacement specificity model to explain how CK1 autophosphorylation may regulate phosphorylation of specific substrates in different pathways and under different conditions. This work was funded by the National Institutes of Health, R35-GM131799 to KLG and T32-CA119925 to SNC.