Site-Specific Labeling of Protein Kinase CK2: Combining Surface Display and Click Chemistry for Drug Discovery Applications.

Christian Nienberg,Thorsten Saenger,Joachim Jose,Anika Retterath,Kira-Sophie Becher,Henning Mootz

doi:10.3390/ph9030036

Christian Nienberg, Thorsten Saenger + Show 4 more

Open Access

https://doi.org/10.3390/ph9030036

Copy DOI

Journal: Pharmaceuticals	Publication Date: Jun 27, 2016
Citations: 14	License type: CC BY 4.0

Affiliation: University of Münster

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.

Highlights

Human protein kinase CK2 was discovered in 1954 by Burnett and Kennedy [1] and is a constitutively active serine/threonine kinase
The kinase activity of CK2 on the substrate peptide RRRDDDSDDD was determined by a capillary electrophoresis assay [23], which is based on a different migration time of the phosphorylated product in contrast to the unphosphorylated substrate through a difference in charge
These results led to the conclusion that unspecific protein modifications as obtained with fluorescein isothiocyanate (FITC) have a negative influence on CK2 activity

Summary

Introduction

Human protein kinase CK2 was discovered in 1954 by Burnett and Kennedy [1] and is a constitutively active serine/threonine kinase. Plenty of inhibitors are known to inhibit the phosphorylation activity of CK2 including dibenzo[b,d]furane- [9] and indeno[1,2-b]indole-derivatives [10]. Most commercially available labeling applications attack lysine and cysteine side chains of proteins. These procedures can lead to modifications at different positions and different protein-to-fluorophore ratios, which can result in heterogeneously labeled products. Unnatural amino acids facilitate bioorthogonal reactions and expand the capabilities of protein chemistry. Among others, these comprise the creation of cyclic peptides by an incorporation of an unnatural amino acid followed by an oxime ligation [17] as well as site-specific chemical-tag labeling of proteins by recombinant split inteins [18]

Methods

Results

Conclusion