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Updated: Jun 25, 2026
Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach
Published on: February 21, 2019
Interactions between Casein kinase Iepsilon (CKIepsilon) and two substrates from disparate signaling pathways reveal
Caroline Lund Dahlberg1, Elizabeth Z Nguyen, David Goodlett
1Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
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 CKIepsilon and two substrates from different signaling pathways.
Methodology/Principal Findings:
CKIepsilon, but not CKIalpha, 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 GST-pull-down assays data to show that two key residues in CKIalpha's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIepsilon does not determine Dishevelled's and Period's preference for CKIepsilon nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIepsilon with its substrates. We demonstrate that autophosphorylation of CKIepsilon'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 CKIepsilon and Disheveled, Period, and its own C-terminus lead to models that explain CKIepsilon's specificity and regulation.
Insights
Casein Kinase I epsilon (CKIepsilon) specifically binds and phosphorylates Period and Disheveled proteins. Its C-terminus regulates substrate interaction, revealing CKIepsilon specificity mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Casein Kinase I (CKI) isoforms regulate diverse biological pathways.
- CKI isoforms share conserved kinase domains but have unique N- and C-termini, leading to substrate specificity.
- Understanding how substrates differentiate between CKI isoforms is crucial for deciphering signaling pathways.
Purpose of the Study:
- To investigate the molecular basis of substrate recognition and specificity for Casein Kinase I epsilon (CKIepsilon).
- To elucidate the interaction mechanisms between CKIepsilon and its substrates, Period and Disheveled.
- To determine the role of CKIepsilon's unique C-terminus in substrate binding and regulation.
Main Methods:
- GST-pull-down assays to identify binding interactions.
- Site-directed mutagenesis to probe the role of specific kinase domain residues.
- Mass spectrometry and chemical crosslinking to analyze protein-protein interactions and post-translational modifications.
Main Results:
- CKIepsilon, but not CKIalpha, binds and phosphorylates Period and Disheveled.
- Specific residues in CKIalpha's kinase domain prevent binding of Period and Disheveled.
- CKIepsilon's C-terminus is not essential for substrate binding but stabilizes interactions.
- Autophosphorylation of CKIepsilon's C-terminal tail inhibits substrate binding.
Conclusions:
- Biochemical interactions between CKIepsilon, its substrates (Period, Disheveled), and its C-terminus provide a model for CKIepsilon specificity.
- Phosphorylation-dependent interactions regulate CKIepsilon activity and substrate binding.
- These findings offer insights into the precise regulation of CKIepsilon in various signaling pathways.
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