Inhibitory effect of tyrphostin AG114 on recombinant human protein kinase CK2 holoenzyme / 中国药理学与毒理学杂志

ABSTRACT

AIM　To study the direct effect of tyrphostin AG114 on recombinant human protein kinase CK2 holoenzyme and its kinetics. METHODS　Recombinant human protein kinase CK2 α and β subunits were cloned and expressed by genetic engineering, and purified to homogeneity. The two subunits were mixed at equal molar ratio and reconstituted CK2 holoenzyme, which exerted the maximum biological activity. The CK2 activity was assayed by detecting incorporation of 32P of ［γ-32P］ATP or ［γ-32P］GTP into the substrate in various conditions. RESULTS　The recombinant human CK2 was a second messenger (Ca2+, cAMP and cGMP) independent protein kinase, the characterization and function of the reconstituted holoenzyme were consistent with those of native CK2. AG114 strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC50 of 20.8 μmol·L-1, which lay between IC50 of 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole(DRB) and N-(2-aminoethyl)-5-chloronaphthalene-1-sulfonamide(A3), known as CK2 special inhibitors. Kinetic studies of AG114 inhibition on recombinant human CK2 showed that the inhibition was mixed competitive with GTP and noncompetitive with casein. CONCLUSION　AG114 not only is an effective inhibitor of protein tyrosine kinases, but also is a novel potent inhibitor of protein kinase CK2. The recombinant human protein kinase CK2 might be used as a molecular target for simpler screening method and development of more effective inhibitors of CK2.