SERINE-1524 IS A MAJOR SITE OF PHOSPHORYLATION ON HUMAN TOPOISOMERASEII-ALPHA PROTEIN IN-VIVO AND IS A SUBSTRATE FOR CASEIN KINASE-II IN-VITRO

Topoisomerase II protein is essential for cell proliferation and is kn own to exist as a phosphoprotein in cells from both lower and higher e ukaryotic species. In this paper, we have investigated the phosphoryla tion of the alpha isozyme of human topoisomerase II. The topoisomerase II alpha protein was phosphorylated predominantly on serine residues in the human tumor cell lines HeLa and NSCLC-3. Two-dimensional trypti c phosphopeptide mapping studies revealed several sites of phosphoryla tion in vivo, including a major site that was common to topoisomerase II alpha protein from both HeLa and NSCLC-3 cells. To identify sites o f phosphorylation, the regulatory C-terminal domain of human topoisome rase II alpha protein was overexpressed in Escherichia coli as a hexah istidine-tagged fusion protein and purified by nickel chelate chromato graphy. Tryptic phosphopeptide mapping revealed that casein kinase II phosphorylated the C-terminal domain primarily on 2 serine residues in vitro, which were shown to be sites of modification in vivo. Site-dir ected mutagenesis studies identified these casein kinase II-specific p hosphorylation sites as serine 1524 and serine 1376.