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.
Highlights
Type II topoisomerases are essential for faithful cell division in all organisms
We have shown that this residue is a target in vitro for protein kinase C (PKC) and that phosphorylation of topoisomerase II␣ protein purified from HeLa cells by PKC strongly stimulates enzymatic activity in vitro
To provide evidence that PKC is capable of influencing the enzymatic activity of human topoisomerase II␣, HeLa cell nuclear extracts were fractionated by conventional chromatography and fast protein liquid chromatography, and the topoisomerase II␣ protein was purified to near homogeneity
Summary
Type II topoisomerases are essential for faithful cell division in all organisms. In human cells, the ␣ isozyme of topoisomerase II has been implicated in catalyzing mitotic chromosome segregation via its action as a DNA unlinking enzyme. Yeast mutants defective in topoisomerase II activity fail to remove all chromosomal interlinks at mitosis and subsequently incur chromosomal breakage as cell division is attempted in the absence of proper segregation (DiNardo, et al, 1984; Holm, et al, 1985; Uemura and Yanagida, 1986; Uemura, et al, 1987; Holm, et al, 1989; Rose and Holm, 1993; Spell and Holm, 1994). The role of MAP kinase has not been defined in detail, this kinase is implicated in the generation of the mitosis-specific phosphorylated epitope recognized by the MPM-2 antibody (Kuang and Ashorn, 1993; Westendorf et al, 1994) This epitope is found in a number of nuclear proteins, including topoisomerase II␣ and  (Taagepera et al, 1993).
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