title: Casein kinase II catalyzes a mitotic phosphorylation on threonine 1342 of human DNA topoisomerase IIalpha, which is recognized by the 3F3/2 phosphoepitope antibody.
abstract: The 3F3/2 antibody recognizes a phosphoepitope that is implicated in the mitotic checkpoint regulating the metaphase-to-anaphase transition. Immunoprecipitation and Western blotting revealed that the 3F3/2 antibody binds to human DNA topoisomerase II alpha (HsTIIalpha) from mitotic but not interphase HeLa cells. Extracts from mitotic cells efficiently catalyzed the formation of the 3F3/2 phosphoepitope on fragments of HsTIIalpha expressed in bacteria. Expression and site-directed mutagenesis of various HsTIIalpha protein fragments mapped the 3F3/2 phosphoepitope to the region of HsTIIalpha containing phosphorylated threonine 1342. This threonine lies within a consensus sequence for phosphorylation by casein kinase II (CKII). CKII is present in cellular extracts and is associated with isolated mitotic chromosomes. The 3F3/2 phosphoepitope kinase present in mitotic cell extracts was able to create the epitope using GTP and was inhibited by heparin. A kinase associated with the isolated chromosomes also generated the 3F3/2 phosphoepitope on HsTIIalpha. Recombinant CKII catalyzed the formation of the 3F3/2 phosphoepitope on fragments of HsTIIalpha containing threonine 1342. These results indicate that the mitotic 3F3/2 phosphoepitope kinase activity is attributable to CKII. We suggest that the 3F3/2 phosphoepitope reflects a CKII-catalyzed phosphorylation of threonine 1342 that may regulate mitotic functions of HsTIIalpha.
authors.affiliation: Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Daum
authors.forename: G J
authors.initials: GJ
authors.lastname: Gorbsky
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000378:metabolism; D000951:Antigens, Neoplasm; D047390:Casein Kinase II; D002384:Catalysis; D004250:DNA Topoisomerases, Type II / Q000276:immunology / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D007163:Immunosorbent Techniques; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism*; D008938:Mitosis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D010748:Phosphopeptides / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*
chemical_list: D000906:Antibodies; D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D010446:Peptide Fragments; D010748:Phosphopeptides; D013912:Threonine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D004250:DNA Topoisomerases, Type II