Click chemistry for site specific fluorescence labeling of human protein kinase CK2

Nienberg C, Becher K, Mootz HD, Jose J

Abstract

Human CK2 is a heterotetrameric constitutively active serine / threonine protein kinase and plays an important role in today´s cancer research1. The kinase is composed of two catalytic CK2α subunits andtwo regulatory CK2β subunits. Most protein-protein interaction (PPI) studies or screening assays are based on fluorescence detection and require the labelling of the target enzyme by a fluorophore. Thecatalytic subunit CK2α loses activity after labelling by commercial applications. Furthermore, the labelling ratio of the protein sample differs and is not exactly reproducible.The solution for this problem was the 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 chosen 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 position3. 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 studies4.