Protein kinase CK2 phosphorylates the cell cycle regulatory protein Geminin

Geminin contributes to cell cycle regulation by a timely inhibition of Cdt1p, the loading factor required for the assembly of pre-replication complexes. Geminin is expressed during S and G2 phase of the HeLa cell cycle and phosphorylated soon after its synthesis. We show here that Geminin is an excellent substrate for protein kinase CK2 in vitro; and that the highly specific CK2 inhibitor tetrabromobenzotriazole (TBB) blocks the phosphorylation of Geminin in HeLa protein extracts and HeLa cells in vivo. The sites of CK2 phosphorylation are located in the carboxyterminal region of Geminin, which carries several consensus sequence motifs for CK2. We also show that a minor phosphorylating activity in protein extracts can be attributed to glycogen synthase kinase 3 (GSK3), which most likely targets a central peptide in Geminin. Treatment of HeLa cells with TBB does not interfere with the ability of Geminin to interact with the loading factor Cdt1.