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.