It has been suggested that casein kinase II phosphorylates DNA topoisomerase II␣ (topo II␣) in mouse FM3A cells, by comparison of phosphopeptide maps of topo II␣ labeled in intact cells and of topo II␣ phosphorylated by various kinases in vitro. The phosphorylation of purified topo II␣ by casein kinase II, which attached a maximum of two phosphate groups per topo II␣ molecule, had no effect on the activity of topo II␣. Dephosphorylation of purified topo II␣ by potato acid phosphatase, which

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... st completely dephosphorylated the topo II␣, did not reduce the activity of topo II␣. The incubation itself, regardless of phosphorylation or dephosphorylation status, stimulated the enzyme activity in both reactions. Topo II␣ activity was stimulated by incubation in a medium containing low concentrations of glycerol but not in that containing high concentrations of glycerol, such as the 50% in which purified topo II␣ is stored. The stimulation of topo II␣ activity by incubation was dependent on the concentration of topo II␣, requiring a relatively high concentration of topo II␣. DNA topoisomerase II (topo II) 1 is an abundant and essential nuclear enzyme that catalyzes the decatenation and the unknotting of topologically linked DNA circles and the relaxation of supercoiled DNA chains (1, 2). The DNA decatenation activity of the enzyme is essential for the condensation of interphase chromatin into metaphase chromosomes (3-8), and is necessary for the segregation of daughter chromosomes (9 -13). In addition, topo II appears to play important roles in the organization of nuclei and mitotic chromosomes, since it is a component of the nuclear matrix (14) and the mitotic chromosome scaffold (15, 16). Topo II exists as a phosphoprotein in intact cells from a variety of species (16 -24). The phosphorylation of topo II is regulated in a cell cycle dependent manner, reaching the maximal level during the G 2 -M phase (19, 22, 23, 25) . Casein kinase II may phosphorylate topo II in Drosophila cells and yeast (18, 22, 26) . In vitro, topo II is phosphorylated by a number of protein kinases, including casein kinase II (26 -30), protein kinase C (17, 28, 31, 32), and Cdc2 kinase (30) and in all cases, phosphorylation stimulates the enzyme activity. In vertebrate organisms, two isoforms of topo II have been identified, which have been designated topo II␣ and topo II␤, the latter having been discovered later (33). Thus, most reports describing the phosphorylation of topo II of mammalian cells referred to topo II␣, except for a few (16, 24, 34, 35) . While it appears that in yeast and Drosophila melanogaster, there is one enzyme which more closely resembles topo II␣. We reported that an unidentified protein kinase, PKII phosphorylated topo II␣, which stimulated enzyme activity (36). However, the effect of phosphorylation on the activity of topo II varied among preparations. In addition, Shiozaki and Yanagida (37) have reported that yeast topo II without the phosphorylated termini had about 4-fold more catalytic activity than intact topoisomerase II, and that dephosphorylated topo II retained enzymatic activity (38). In this study, to re-evaluate our results and to examine the effect of phosphorylation upon the activity of topo II, we investigated the kinase that phosphorylates topo II␣ in mouse FM3A cells, which dominantly express topo II␣. We found that casein kinase II phosphorylated topo II␣ in FM3A cells and that phosphorylation of topo II␣ by casein kinase II had no effect on the activity of topo II␣ under our experimental conditions. More importantly, we found that the incubation itself stimulated the activity of topo II␣. EXPERIMENTAL PROCEDURES Buffers-Buffer 1 contained 20 mM potassium phosphate buffer, pH 7.5, 0.1 mM Na 3 EDTA, 1 mM 2-mercaptoethanol, 0.25 mM phenylmethylsulfonyl fluoride, and 1% ethanol. Buffer 2 consisted of all components of buffer 1, plus 20% ethylene glycol and 0.01% Triton X-100. Buffer 3 consists of all components of buffer 1, plus 50% glycerol and 0.01% Triton X-100. DNA Topo II Assay-DNA topo II activity was assayed by measuring supercoiled DNA relaxing or DNA unknotting activities. The standard reaction mixture (20 l) for the DNA relaxation assay consisted of 50 mM Tris-HCl, pH 7.9, 100 mM KCl, 10 mM MgCl 2 , 1 mM ATP, 0.5 mM dithiothreitol, 0.5 mM Na 3 EDTA, 100 g/ml bovine serum albumin, and 0.225 mg of supercoiled pUC19 DNA. The incubation proceeded at 30°C, and the reaction was stopped with 5 l of stop solution (5% SDS, 25% Ficoll, and 0.05% bromphenol blue). The sample was incubated for 15 min at 50°C, then loaded on a 0.8% agarose gel in TBE buffer (89 mM Tris borate, pH 8.2, and 2 mM EDTA). After electrophoresis, the gel was stained with ethidium bromide and photographed under UV illumination. DNA unknotting activity was assayed as described above for the relaxing assay except that knotted P4 phage DNA was used as the substrate DNA. Purification of DNA Topo II␣-All operations were performed at 0 -4°C. A total of 8 ϫ 10 9 frozen FM3A cells were thawed, suspended in buffer 1 at a concentration of 1.25 ϫ 10 9 cells/ml, and sonicated 4 times for 10 s at each interval of 20 s with a Branson model 185 sonifier. Buffer 1 (0.1 volume) containing 3.3 M KCl was added dropwise to the sonicate to bring the final concentration of KCl to 0.3 M. After stirring for 30 min, the sonicate was centrifuged for 30 min at 10,000 ϫ g.