Cell cycle phase-specific phosphorylation of human topoisomerase II alpha. Evidence of a role for protein kinase C

J Biol Chem. 1995 Nov 24;270(47):28357-63. doi: 10.1074/jbc.270.47.28357.

Abstract

Type II topoisomerases are essential for faithful cell division in all organisms. In human cells, the alpha isozyme of topoisomerase II has been implicated in catalyzing mitotic chromosome segregation via its action as a DNA unlinking enzyme. Here, we have shown that the enzymatic activity of topoisomerase II alpha protein purified from HeLa cell nuclei was strongly enhanced following phosphorylation by protein kinase C. We have investigated the possibility that this kinase is involved in cell cycle phase-specific phosphorylation of topoisomerase II alpha in HeLa cells. Two-dimensional tryptic phosphopeptide mapping revealed that topoisomerase II alpha protein immunoprecipitated from metabolically labeled HeLa cells was differentially phosphorylated during the G2/M phases of the cell cycle. To identify sites of phosphorylation, and the kinase(s) responsible for this modification, oligohistidine-tagged recombinant domains of topoisomerase II alpha protein were overexpressed in Escherichia coli and purified by affinity chromatography. Phosphorylation of a short fragment of the N-terminal ATPase domain of topoisomerase II alpha by protein kinase C in vitro generated two phosphopeptides that co-migrated with prominent G2/M phase-specific phosphopeptides from the HeLa cell-derived topoisomerase II alpha protein. Site-directed mutagenesis studies indicated that phosphorylation of serine 29 generated both of these phosphopeptides. Our results implicate protein kinase C in the cell cycle phase-dependent modulation of topoisomerase II alpha enzymatic activity in human cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Cycle*
  • Cell Division
  • DNA Primers
  • DNA Topoisomerases, Type II / chemistry
  • DNA Topoisomerases, Type II / isolation & purification
  • DNA Topoisomerases, Type II / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • G2 Phase
  • HeLa Cells
  • Humans
  • Isoenzymes / chemistry
  • Isoenzymes / isolation & purification
  • Isoenzymes / metabolism*
  • Mitosis
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide Fragments / chemical synthesis
  • Peptide Fragments / chemistry
  • Peptide Mapping
  • Phosphopeptides / chemistry
  • Phosphopeptides / isolation & purification
  • Phosphorylation
  • Polymerase Chain Reaction
  • Protein Kinase C / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism

Substances

  • DNA Primers
  • Isoenzymes
  • Peptide Fragments
  • Phosphopeptides
  • Recombinant Proteins
  • Protein Kinase C
  • DNA Topoisomerases, Type II