Serine 1524 is a major site of phosphorylation on human topoisomerase II alpha protein in vivo and is a substrate for casein kinase II in vitro.
Topoisomerase II protein is essential for cell proliferation and is known to exist as a phosphoprotein in cells from both lower and higher eukaryotic species. In this paper, we have investigated the phosphorylation 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 tryptic phosphopeptide mapping studies revealed several sites of phosphorylation in vivo, including a major site that was common to topoisomerase II alpha protein from both HeLa and NSCLC-3 cells. To identify sites of phosphorylation, the regulatory C-terminal domain of human topoisomerase II alpha protein was overexpressed in Escherichia coli as a hexahistidine-tagged fusion protein and purified by nickel chelate chromatography. 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-directed mutagenesis studies identified these casein kinase II-specific phosphorylation sites as serine 1524 and serine 1376.
Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, United Kingdom.
N J
NJ
Wells
C M
CM
Addison
A M
AM
Fry
R
R
Ganapathi
I D
ID
Hickson
D001483:Base Sequence; D047390:Casein Kinase II; D017931:DNA Primers; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D011994:Recombinant Proteins; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D004250:DNA Topoisomerases, Type II