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Updated: Mar 18, 2026
Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach
Published on: February 21, 2019
Site-Specific Labeling of Protein Kinase CK2: Combining Surface Display and Click Chemistry for Drug Discovery
Christian Nienberg1, Anika Retterath2, Kira-Sophie Becher3
1Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, D-48149 Münster, Germany. christian.nienberg@uni-muenster.de.
Abstract:
Human CK2 is a heterotetrameric constitutively active serine/threonine protein kinase and is an emerging target in current anti-cancer drug discovery. The kinase is composed of two catalytic CK2α subunits and two regulatory CK2β subunits. In order to establish an assay to identify protein-protein-interaction inhibitors (PPI) of the CK2α/CK2β interface, a bioorthogonal click reaction was used to modify the protein kinase α-subunit with a fluorophore. By expanding the genetic code, the unnatural amino acid para azidophenylalanine (pAzF) could be incorporated into CK2α. Performing the SPAAC click reaction (Strain-Promoted Azide-Alkyne Cycloaddition) by the use of a dibenzylcyclooctyne-fluorophore (DBCO-fluorophore) led to a specifically labeled human protein kinase CK2α. This site-specific labeling does not impair the phosphorylation activity of CK2, which was evaluated by capillary electrophoresis. Furthermore a dissociation constant (KD) of 631 ± 86.2 nM was determined for the substrate αS1-casein towards CK2α. This labeling strategy was also applied to CK2β subunit on Escherichia coli, indicating the site-specific modifications of proteins on the bacterial cell surface when displayed by Autodisplay.
Insights
Researchers developed a bioorthogonal click chemistry method to label human protein kinase CK2 alpha subunits with a fluorophore. This site-specific labeling enables the development of assays for anti-cancer drug discovery targeting CK2 protein-protein interactions.
Area of Science:
- Biochemistry
- Molecular Biology
- Chemical Biology
Background:
- Human CK2 is a constitutively active serine/threonine protein kinase and a key target in anti-cancer drug discovery.
- CK2 comprises two catalytic CK2α and two regulatory CK2β subunits, with the CK2α/CK2β interface being a target for protein-protein interaction inhibitors (PPI).
Purpose of the Study:
- To establish an assay for identifying PPI inhibitors of the CK2α/CK2β interface.
- To develop a site-specific protein labeling strategy for CK2 subunits using bioorthogonal click chemistry.
Main Methods:
- Incorporation of the unnatural amino acid para-azidophenylalanine (pAzF) into the CK2α subunit via genetic code expansion.
- Site-specific labeling of CK2α with a dibenzylcyclooctyne-fluorophore (DBCO-fluorophore) using Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) click reaction.
- Evaluation of CK2 phosphorylation activity using capillary electrophoresis and determination of dissociation constant (KD) for αS1-casein.
Main Results:
- Successfully achieved site-specific fluorophore labeling of human CK2α without impairing its kinase activity.
- Determined a dissociation constant (KD) of 631 ± 86.2 nM for the substrate αS1-casein towards CK2α.
- Demonstrated the applicability of the labeling strategy to the CK2β subunit in Escherichia coli using Autodisplay for bacterial cell surface display.
Conclusions:
- The developed bioorthogonal click chemistry approach enables site-specific labeling of CK2 subunits.
- This labeling strategy is valuable for developing assays to identify CK2 protein-protein interaction inhibitors for anti-cancer drug discovery.
- The method shows potential for modifying proteins on bacterial cell surfaces for various applications.
