A FRET-based microplate assay for human protein kinase CK2, a target in neoplastic disease

Authors: Gratz, Andreas¹; Götz, Claudia²; Jose, Joachim¹

Source: Journal of Enzyme Inhibition and Medicinal Chemistry, Volume 25, Number 2, April 2010 , pp. 234-239(6)

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Abstract:

Besides cardiovascular diseases, cancer represents the major cause of death in developed countries. In many different human tumors, increased activity of serine/threonine protein kinase CK2 has been detected, and recent in vivo studies support a direct involvement of CK2 in tumor progression. Therefore, potent compounds to decrease CK2 activity to a non-pathogenic level would be a promising effort toward an antineoplastic therapy. In this study, an alternative to the established radiometric phosphorylation assay for quantification of CK2 activity was developed. For this purpose, the substrate peptide RRRDDDSDDD was coupled at the C-terminus to the fluorophore EDANS (5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid) and at the N-terminus to the quencher DABCYL (4-(4-dimethylaminophenylazo)benzoic acid). This resulted in quenched fluorescence of EDANS due to a FRET-based effect. After proteolytic cleavage of the peptide by elastase, the quenching effect was reduced and, as a consequence, fluorescence was increased. Because elastase is supposed to cleave at the S/D site of the peptide, phosphorylation of serine by CK2 hampered substrate binding of elastase and blocked the increase in fluorescence by proteolytic cleavage. This means that the new assay to quantify human CK2 activity is based on the differential accessibility of the proteolytic cleavage site, which is dependent on kinase phosphorylation. It could be used to measure inhibition of the human target in neoplastic diseases by the compounds TBB (4,5,6,7-tetrabromobenzotriazole) and Emodin.

Keywords: Protein kinase; phosphorylation assay; CK2; casein kinase 2; FRET peptide; cancer; porcine pancreatic elastase; protease cleavage

Document Type: Research article

DOI: http://dx.doi.org/10.3109/14756360903170038

Affiliations: 1: 1Bioanalytics, Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany 2: 2Medicinal Biochemistry and Molecular Biology, Saarland University, Homburg-Saarbrücken, Germany

Publication date: 2010-04-01

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A FRET-based microplate assay for human protein kinase CK2, a target in neoplastic disease

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