3.2  The action of IFN-γ on the synthesis and activity of CK2
CK2 exists as a tetramer with two main catalytic (α and α′) and two regulatory β subunits as well as free catalytic subunits [10,22]. In order to investigate whether IFN-γ affects the synthesis of CK2, the levels of the α and α′ polypeptides in J774.2 cells treated with IFN-γ were determined by western blot analysis. Sp1 and Sp3 were also analysed for comparative purposes (Sp1 can also act as a loading control as IFN-γ has no effect on its expression [9]). Fig. 2A shows that IFN-γ does not affect the steady state levels of CK2-α and -α′ polypeptides. In contrast, consistent with our previous studies [9], IFN-γ reduced the polypeptide levels of Sp3 but not Sp1 (Fig. 2A).
Our previous studies on the action of IFN-γ on CK2 activation, which was restricted to the α isoform and a single time point (3 h) [10], showed a dramatic increase in activity following stimulation of the cells with this cytokine. Preliminary time course experiments showed that the activity of both CK2 catalytic subunits was induced within 1 h of incubation of the cells with IFN-γ, peaked at 3 h and was sustained, albeit at reduced levels, for 12–20 h (data not shown). In order to confirm the inhibitory action of apigenin, its effect on the IFN-γ-induced activity of both the catalytic subunits at 3 h was determined. Fig. 2B shows that, consistent with previous studies [e.g. [10,16–19]], apigenin indeed abolishes CK2 activity of both subunits without affecting the expression of the polypeptides.