Casein kinase II catalyzes a mitotic phosphorylation on threonine 1342 of human DNA topoisomerase IIalpha, which is recognized by the 3F3/2 phosphoepitope antibody

J Biol Chem. 1998 Nov 13;273(46):30622-9. doi: 10.1074/jbc.273.46.30622.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Antibodies / metabolism
  • Antigens, Neoplasm
  • Casein Kinase II
  • Catalysis
  • DNA Topoisomerases, Type II* / immunology
  • DNA Topoisomerases, Type II* / metabolism*
  • DNA-Binding Proteins
  • HeLa Cells
  • Humans
  • Immunosorbent Techniques
  • Isoenzymes / immunology
  • Isoenzymes / metabolism*
  • Mitosis*
  • Molecular Sequence Data
  • Molecular Weight
  • Peptide Fragments / immunology
  • Peptide Fragments / metabolism
  • Phosphopeptides / immunology
  • Phosphopeptides / metabolism
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism*
  • Threonine / metabolism*

Substances

  • Antibodies
  • Antigens, Neoplasm
  • DNA-Binding Proteins
  • Isoenzymes
  • Peptide Fragments
  • Phosphopeptides
  • Threonine
  • Casein Kinase II
  • Protein-Serine-Threonine Kinases
  • DNA Topoisomerases, Type II