3.3  CK2 associates with, and phosphorylates, Sp1
Our subsequent studies focused on the interaction and action of CK2 on Sp1 because preliminary co-transfection assays showed that Sp1, but not Sp3, was the main activator of the LPL promoter (data not shown). We investigated the interaction of CK2 subunits with Sp1 by co-immunoprecipitation assays. STAT1 was included for comparative purposes. Thus, Sp1 or STAT1 was immunoprecipitated from extracts of J774.2 macrophages that had either been left untreated or stimulated with IFN-γ for 3 h, a period corresponding to dramatic activation of CK2. The immunoprecipitated protein was then subjected to western blot analysis with antibodies against itself or that for CK2-α or α′. Fig. 3A shows that Sp1 interacted with both the catalytic subunits of CK2 and that this association was increased in cells treated with IFN-γ. A similar profile was observed when the analysis was performed with the Sp3 antibody (Supplementary material, Fig. 1). In contrast, no interaction was seen between STAT1 and the CK2 polypeptides (Fig. 3A).
The kinase assays presented in Fig. 2B used the classical β-casein as a substrate. The assay was therefore repeated with recombinant human Sp1 (rhSp1) produced in Sf9 cells using a baculovirus expression system (Promega). The activity of CK2-α or -α′ in terms of their ability to phosphorylate rhSp1 was also induced by IFN-γ (Fig. 3B). Two polypeptide species of 82 kDa and 57 kDa for rhSp1 were observed in this assay, which migrated at an apparently lower molecular weight, due to the lesser extent of glycosylation in Sf9 cells than in mammalian cells (Promega).