CELL-CYCLE PHASE-SPECIFIC PHOSPHORYLATION OF HUMAN TOPOISOMERASE II-ALPHA - EVIDENCE OF A ROLE FOR PROTEIN-KINASE-C

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 a ction as a DNA unlinking enzyme. Here, we have shown that the enzymati c activity of topoisomerase II alpha protein purified from HeLa cell n uclei was strongly enhanced following phosphorylation by protein kinas e C. We have investigated the possibility that this kinase is involved in cell cycle phase-specific phosphorylation of topoisomerase II alph a in HeLa cells. Two-dimensional tryptic phosphopeptide tide mapping r evealed that topoisomerase II alpha protein immunoprecipitated from me tabolically labeled HeLa cells was differentially phosphorylated durin g the G(2)/M phases of the cell cycle. To identify sites of phosphoryl ation and the kinase(s) responsible for this modification, oligohistid ine-tagged recombinant domains of topoisomerase II alpha protein were overexpressed in Escherichia coil and purified by affinity chromatogra phy. Phosphorylation of a short fragment of the N-terminal ATPase doma in of topoisomerase II alpha by protein kinase C in vitro generated tw o phosphopeptides that co-migrated with prominent G(2)/M phase-specifi c phosphopeptides from the HeLa cell-derived topoisomerase II alpha pr otein. Site-directed mutagenesis studies indicated that phosphorylatio n of serine 29 generated both of these phosphopeptides. Our results im plicate protein kinase C in the cell cycle phase-dependent modulation of topoisomerase II alpha enzymatic activity in human cells.