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 prophase. This process is temporally linked with the appearance of mitosis-specific phosphorylation sites on topoisomerase IIalpha 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 alpha is abolished by immunodepletion of protein kinase CK2. Furthermore, the MPM-2 phosphoepitope on topoisomerase II alpha can be generated by purified CK2. Phosphorylation of C-truncated topoisomerase II alpha 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 alpha 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 alpha 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 alpha, on Ser-1469.
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Laboratoire de Biologie et Pharmacologie des Tumeurs, CNRS UMR 8532, Institut Gustave-Roussy PR2, Villejuif 94805 Cedex, France.
Plisov
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Filhol
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Cochet
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Larsen
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D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm; D047390:Casein Kinase II; D002384:Catalysis; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D002877:Chromosomes, Human; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006493:Heparin / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D016547:Kinesins; D008938:Mitosis; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism*; D059005:Topoisomerase II Inhibitors
D000951:Antigens, Neoplasm; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D010750:Phosphoproteins; D059005:Topoisomerase II Inhibitors; D012694:Serine; D006160:Guanosine Triphosphate; D006493:Heparin; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; C438388:KIF20B protein, human; D016547:Kinesins; D004250:DNA Topoisomerases, Type II