Site specific labelling of human protein kinase CK2 for drug discovery applications

Nienberg C, Becher KS, Mootz HD, Jose J

Abstract

Human CK2 is a heterotetrameric constitutively active serine / threonine protein kinase, phosphorylating and regulating a variety of cellular processes. In tumor and rapidly proliferating cells the activity ofCK2 is increased. Downregulating the activity of the protein kinase in the affected tumor cells initiates apoptosis [1]. Thus, CK2 represents a promising target in current cancer research. The kinase iscomposed of two catalytic CK2α subunits and two regulatory CK2β subunits. Most protein-protein interaction (PPI) studies or screening assays are based on fluorescence detection and require the labelling ofthe target enzyme by a fluorophore. The catalytic subunit CK2α loses activity after labelling by commercial applications. Furthermore, the labelling ratio of the protein sample differs and is not exactlyreproducible.The solution for this problem was an incorporation of an unnatural amino acid into the CK2α subunit followed by a Strain-Promoted Alkyne-Azide Cycloaddition (SPAAC) [2]. Therefore, a suitable position inthe sequence of CK2α was selected and mutated to the amber nonsense DNA codon, TAG. By suppression of the mutation with an amber suppressor tRNA, the unnatural amino acid para acidophenylalanine(pAzF) could be incorporated at this position [3]. Performing the SPAAC click reaction by the use of dibenzylcyclooctyne fluor 545 (DBCO 545) led to a specifically labelled CK2α and CK2 holoenzyme. Thisspecific kind of labelling does not impair the phosphorylation activity of the CK2α subunit alone nor the holoenzyme, which was evaluated by capillary electrophoresis. The innovatively labelled kinase incombination with the Autodisplay technology could be a significant advancement for inhibitor screening assays by flow cytometry and for CK2α/CK2β interaction studies [4].