Mitotic phosphorylation of DNA topoisomerase II alpha by protein kinase CK2 creates the MPM-2 phosphoepitope on Ser-1469

DNA. topoisomerase II alpha is required for chromatin condensation during p rophase, This process is temporally linked with the appearance of mitosis-s pecific phosphorylation sites on topoisomerase II alpha including one recog nized by the MPM-2 monoclonal antibody. We now report that the ability of m itotic extracts to create the MPM-2 epitope on human topoisomerase II alpha is abolished by immunodepletion of protein kinase CK2. Furthermore, the MP M-2 phosphoepitope on topoisomerase II alpha can be generated by purified C R2. Phosphorylation of C-truncated topoisomerase II alpha mutant proteins c onclusively 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 alpha for binding of the MPM-2 antibo dy. Replacement of Ser-1469 with Ala abolishes the ability of the phosphory lated peptide to bind to the MPM-2 antibody while a peptide containing phos phorylated Ser-1469 binds tightly. Surprisingly, the MPM-2 phosphoepitope i nfluences neither the catalytic activity of topoisomerase II alpha nor its, ability to form molecular complexes with CR2 in vitro. In conclusion, we h ave identified protein kinase CR2 as a new MPM-2 kinase able to phosphoryla te an important mitotic protein, topoisomerase II alpha; on Ser-1469.