Wells N J, Addison C M, Fry A M, Ganapathi R, Hickson I D
Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, United Kingdom.
J Biol Chem. 1994 Nov 25;269(47):29746-51.
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.
拓扑异构酶II蛋白对于细胞增殖至关重要，并且已知在低等和高等真核生物的细胞中均以磷蛋白形式存在。在本文中，我们研究了人类拓扑异构酶II的α同工酶的磷酸化情况。在人类肿瘤细胞系HeLa和NSCLC - 3中，拓扑异构酶IIα蛋白主要在丝氨酸残基上发生磷酸化。二维胰蛋白酶磷酸肽图谱研究揭示了体内的几个磷酸化位点，包括一个HeLa细胞和NSCLC - 3细胞的拓扑异构酶IIα蛋白共有的主要位点。为了鉴定磷酸化位点，人类拓扑异构酶IIα蛋白的调节性C末端结构域在大肠杆菌中作为六组氨酸标签融合蛋白过表达，并通过镍螯合色谱法纯化。胰蛋白酶磷酸肽图谱显示，酪蛋白激酶II在体外主要使C末端结构域的2个丝氨酸残基磷酸化，这两个丝氨酸残基在体内被证明是修饰位点。定点诱变研究确定这些酪蛋白激酶II特异性磷酸化位点为丝氨酸1524和丝氨酸1376。
