Interactions between Casein Kinase Iε (CKIε) and Two Substrates from Disparate Signaling Pathways Reveal Mechanisms for Substrate-Kinase Specificity

Background: Members of the Casein Kinase I (CKI) family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKI epsilon and two substrates from different signaling pathways. Methodology/Principal Findings: CKI epsilon, but not CKI alpha, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GSTpull-down assays data to show that two key residues in CKI alpha's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKI epsilon does not determine Dishevelled's and Period's preference for CKI epsilon nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKI epsilon with its substrates. We demonstrate that autophosphorylation of CKI epsilon's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding. Conclusions/Significance: The biochemical interactions between CKI epsilon and Disheveled, Period, and its own C-terminus lead to models that explain CKI epsilon's specificity and regulation.