Mitotic Phosphorylation of DNA Topoisomerase II α by Protein Kinase CK2 Creates the MPM-2 Phosphoepitope on Ser-1469

Abstract

DNA topoisomerase II␣ is required for chromatin condensation during prophase. This process is temporally linked with the appearance of mitosis-specific phosphorylation sites on topoisomerase II␣ including one recognized by the MPM-2 monoclonal antibody. We now report that the ability of mitotic extracts to create the MPM-2 epitope on human topoisomerase II␣ is abolished by immunodepletion of protein kinase CK2. Furthermore, the MPM-2 phosphoepitope on topoisomerase II␣ can be generated by purified CK2. Phosphorylation of C-truncated topoisomerase II␣ mutant proteins conclusively shows, that the MPM-2 epitope is present in the last 163 amino acids. Use of peptides containing all conserved CK2 consensus sites in this region indicates that only the peptide containing Arg-1466 to Ala-1485 is able to compete with topoisomerase II␣ for binding of the MPM-2 antibody. Replacement of Ser-1469 with Ala abolishes the ability of the phosphorylated peptide to bind to the MPM-2 antibody while a peptide containing phosphorylated Ser-1469 binds tightly. Surprisingly, the MPM-2 phosphoepitope influences neither the catalytic activity of topoisomerase II␣ nor its ability to form molecular complexes with CK2 in vitro. In conclusion, we have identified protein kinase CK2 as a new MPM-2 kinase able to phosphorylate an important mitotic protein, topoisomerase II␣, on Ser-1469.

Interactions between Protein Kinase CK2 and Pin1

“…In these yeast, topoisomerase II is hypo-phosphorylated and inactive, suggesting that it is a bona fide physiological target for CK2. Additionally, it was recently demonstrated in mammalian cells that CK2 phosphorylates residues on topoisomerase II␣, including Thr-1342, which are residues that are maximally phosphorylated in mitotic cells (32)(33)(34). In this study, we demonstrated that Pin1 inhibits the in vitro phosphorylation of Thr-1342 on topoisomerase II␣ by CK2 and that Pin1 interacts with topoisomerase II␣.…”

“…For example, CK2 was recently shown to phosphorylate residues on topoisomerase II␣ that are maximally phosphorylated in mitotic cells (33,34). These observations strongly suggest that topoisomerase II␣ is a mitotic target of CK2 in mammalian cells.…”

“…To explore further the mechanism by which Pin1 inhibits the phosphorylation of topoisomerase II by CK2, we were interested in testing the possibility that topoisomerase II also interacts with Pin1, as has been suggested previously (30,34). To achieve this objective, pulldown assays were performed to determine whether topisomerase II␣ obtained from interphase or mitotic cells can interact with GST-Pin1.…”

Protein kinase CK2 regulates CDC25B phosphatase activity

“…In order to confirm the data obtained in vivo, we used three peptides in kinase assays (Figure 3c). One corresponded to the B domain of CDC25B, the second to a dodecapeptide (RRREDEESDDEE) classically used as CK2 substrate (Escargueil et al, 2000), and the third one to the middle part of the B domain of CDC25B, excluding the putative phosphorylation site (dotted line in Figure 3b). The ability of CK2 to phosphorylate these three peptides was evaluated by autoradiography after incubation with radiolabelled ATP in the presence of recombinant CK2 kinase.…”

“…In addition, CDK1 itself can be phosphorylated by CK2 (Russo et al, 1992) and CK2 has been implicated in the phosphorylation of cyclin B, which regulates its nuclear translocation (Li et al, 1997b). More recently, several proteins including the phosphatase PTP-S2 (Nambirajan et al, 2000), the SIX1 homeodomain (Ford et al, 2000) and the Topoisomerase II (Escargueil et al, 2000), were found to be phosphorylated by CK2 during mitosis. All these data suggest that CK2 plays a role in the control of the cell cycle and, particularly, at the G2/M transition.…”