Phosphorylation-independent Stimulation of DNA Topoisomerase II Activity

It has been suggested that casein kinase II phosphorylates DNA topoisomerase IIα (topo IIα) in mouse FM3A cells, by comparison of phosphopeptide maps of topo IIα labeled in intact cells and of topo IIα phosphorylated by various kinases in vitro. The phosphorylation of purified topo IIα by casein kinase II, which attached a maximum of two phosphate groups per topo IIα molecule, had no effect on the activity of topo IIα. Dephosphorylation of purified topo IIα by potato acid phosphatase, which almost completely dephosphorylated the topo IIα, did not reduce the activity of topo IIα. The incubation itself, regardless of phosphorylation or dephosphorylation status, stimulated the enzyme activity in both reactions. Topo IIα 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 IIα is stored. The stimulation of topo IIα activity by incubation was dependent on the concentration of topo IIα, requiring a relatively high concentration of topo IIα.

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 almost 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)(4)(5)(6)(7)(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␣.
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. Labeling Topo II␣ in Intact Cells-Exponentially growing FM3A cells were washed once with phosphate-free RPMI 1640 and inoculated into 60-mm plastic dishes at a density of 1.5 ϫ 10 6 cells/ml with 5 ml of above medium supplemented with 10% dialyzed fetal bovine serum. The cells were incubated for 1 h at 37°C, then labeled with [ 32 P]orthophosphate (60 Ci/ml, 8500 -9100 Ci/mmol) for 2 h at 37°C. 32 P-Labeled topo II␣ was immunoprecipitated with anti-topo II antibody by the procedure of Saijo et al. (23).
For phosphopeptide mapping, purified topo II␣ (1 g) was phosphorylated by the indicated kinases as follows. Phosphorylation by casein kinase II proceeded in a reaction mixture containing 20 mM Hepes, pH 7.4, 150 mM NaCl, 10 mM MgCl 2 , 1 mM dithiothreitol, and 10 M Phosphopeptide Mapping-32 P-Labeled topo II␣ was resolved by SDS-PAGE and stained with Coomassie Brilliant Blue R-250. The stained band of topo II␣ was cut and washed with 25% isopropyl alcohol 5 times, with 10% methanol twice, and with double distilled water twice at 30-min intervals. The gel slice was crushed and incubated in 50 l of medium containing 0.1 mg/ml Achrombacter protease 1 and 50 mM NH 4 HCO 4 , pH 9.0, or 0.1 mg/ml V8 protease and 50 mM NH 4 HCO 4 , pH 7.8, or 0.1 mg/ml endoproteinase Asp-N and 50 mM Tris-HCl, pH 7.5, at 37°C for 12 h. The crashed gels were removed from the medium by centrifugation for 10 min at 10,000 ϫ g, then the supernatant was recovered and dried. A solution (40 l) containing 62.5 mM Tris-HCl, pH 6.8, 1% SDS, 1% 2-mercaptoethanol, 10% glycerol, and 0.01% bromphenol blue was added to dissolve the digested peptides and boiled for 30 s. The sample was resolved by Tris-Tricine SDS-PAGE according to Schagger and Jagow (39), and phosphopeptides were detected with an image analyzer (BAS 2000 Fuji Photofilm, Tokyo, Japan).
Phosphatase Treatment of Topo II␣-Topo II␣ (60 ng) was incubated with 0.2 units of purified potato acid phosphatase (PAP) as described by Shiozaki and Yanagida (38) or incubated with 25 units of calf intestine alkaline phosphatase (CIAP) at 30°C for 30 min.

RESULTS
The Kinase Responsible for Phosphorylating Topo II␣ in FM3A Cells-We purified topo II␣ and casein kinase II from mouse FM3A cells as described under "Experimental Procedures." The purified topo II␣ fraction contained a single 170-kDa band (Fig. 1A) and purified casein kinase II fraction consisted of two major bands at 28 and 43 kDa (Fig. 1B). The purified topo II␣ was phosphorylated by protein kinase C, casein kinase II, or PKII. The labeled topo II␣ was separated by SDS-PAGE, digested by Achrombacter protease 1, and the phosphopeptides were mapped as described under "Experimental Procedures." Topo II␣ labeled by each kinase had a distinct phosphopeptide profile (Fig. 2, lanes 1-3), indicating that these kinases phosphorylate topo II␣ at different sites. By contrast, the phosphopeptide maps of topo II␣ phosphorylated by casein kinase II and that of topo II␣ labeled in intact cells, which were produced by digestion with Achromobacter protease 1, V8 protease, or endopeptidase Asp-N, were similar (Fig. 2, lanes 4-9). Thus it is likely that casein kinase II phosphorylates topo II␣ in FM3A cells.
The Effect of Phosphorylation by Casein Kinase II upon Topo II␣ Activity-Casein kinase II appeared to phosphorylate topo II␣ in intact cells. Thus, we examined the effect of phosphorylation by casein kinase II upon the activity of topo II␣. Fig. 3 shows the time course of topo II␣ phosphorylation by casein kinase II. At the maximal level, about 2 molecules of phosphate were incorporated into one molecule of topo II␣. Incubating topo II␣ with heat-inactivated casein kinase II or in the absence of the kinase resulted in no phosphate incorporation.
Topo II␣ was incubated with casein kinase II or heat-inactivated casein kinase II for 30 min under the same conditions as those of Fig. 3, then the activity of topo II␣ was measured. The levels of activity of topo II␣ incubated with casein kinase II or heat-inactivated casein kinase II were considerably higher than that of activity of topo II␣ without incubation when topo II activity was determined by the DNA relaxing assay (Fig. 4A) or the DNA unknotting assay (Fig. 4B). This indicated that the activity of topo II␣ was stimulated during the incubation independently of phosphorylation by casein kinase II, because the heat-inactivated casein kinase II did not phosphorylate topo II␣ (Fig. 3).
The Effect of Dephosphorylation on Topo II␣ Activity-To determine whether the purified topo II␣ had been sufficiently phosphorylated in the cells and that additional phosphorylation scarcely affected the activity of topo II␣, we incubated it with PAP. We first confirmed the dephosphorylating activity of PAP using topo II␣ labeled with 32 P in intact cells. The 32 Plabeled topo II␣ was incubated with PAP or heat-inactivated PAP. As shown in Fig. 5A, PAP removed almost all 32 P label from topo II␣ and heat-inactivated PAP had no effect upon phosphate removal (Fig. 5A, lane 3).
Topo II␣ was incubated with PAP or heat-inactivated PAP under the same conditions as those of Fig. 5A, and the activity of topo II␣ was assayed. Again, the activity of topo II␣ was stimulated by an incubation either with PAP or with heatinactivated PAP, when the activity was compared with that of topo II␣ without this incubation (Fig. 5, B and C). The levels of topo II␣ activity incubated with PAP and heat-inactivated PAP were similar in the DNA relaxing (Fig. 5B) or the DNA unknotting assays (Fig. 5C), indicating that dephosphorylated and phosphorylated topo II␣ retained the same level of activity. By contrast, incubation with calf intestine alkaline phosphatase markedly inhibited topo II␣ activity (compare Fig. 6A, a and b). In this case, ATP in the reaction mixture was almost completely degraded (Fig. 6B).
Incubation Itself Stimulates the Activity of Topo II␣-We determined whether the activity of topo II␣ was affected by the incubation itself. Topo II␣ was incubated in medium containing 20 mM Hepes, pH 7.4, 0.1 mM ATP, 150 mM NaCl, 1 mM MgCl 2 , 1 mM 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, 0.1 mg/ml bovine serum albumin, and 5% glycerol for the indicated periods, then its activity was assayed. Both DNA relaxing and unknotting activities of topo II␣ were stimulated more than 3-fold by a 30-min incubation in the buffer indicated above (Fig.  7, A and B). The stimulatory effect was evident after 5 min (compare Fig. 7A, a and b) and nearly saturated by 15 min (compare c and d).
Conditions for Stimulating Topo II␣ Activity-Purified topo II␣ was stored in a buffer containing 50% glycerol, whereas the concentration of glycerol in the buffer in which topo II␣ was preincubated was only 5%. Thus we examined the effect of glycerol concentration on the activation of topo II␣ activity. Topo II␣ was incubated in the buffer containing various concentrations of glycerol for 30 min. Then the activity of topo II␣ was measured by the DNA relaxing (Fig. 8A) or unknotting assays (Fig. 8B) in a reaction mixture containing glycerol of a concentration below 3.75%. Incubation of topo II␣ in the buffer containing 75 and 50% glycerol did not stimulate the activity of topo II␣, whereas the activity was enhanced during incubation in the buffer containing 5 or 20% glycerol.
When the concentration of topo II␣ was decreased to onetenth during the incubation for activation, topo II␣ was not activated by the incubation, indicating that the activation is dependent on the concentration of topo II␣ (Fig. 9). DISCUSSION Phosphorylation is an important means by which enzymatic activity and protein functions are regulated in the cells. DNA topoisomerase exists as a phosphoprotein in the cells of various species (16 -24). We reported that topo II is phosphorylated in mouse FM3A cells and that the phosphorylation of topo II␣ purified from the mouse cells by an unidentified protein kinase, PK II, stimulated the activity of topo II␣ (36).
To evaluate these results, we first tried to identify the protein kinase that phosphorylates topo II␣ in FM3A cells. The results shown in Fig. 1 1 (lanes 1-3, 6, and 7), V8 protease (lanes 4 and 5), or endoproteinase Asp-N (lanes 8 and 9), and resolved by Tris-Tricine SDS-PAGE as described under "Experimental Procedures." The 32 P-labeled phosphopeptides were detected with an image analyzer. Lane 1, phosphopeptide map of topo II␣ phosphorylated by protein kinase C; lanes 2, 4, 6, and 8, phosphorylated by casein kinase II; lane 3, phosphorylated by PKII; lanes 5, 7, and 9, phosphorylated in intact cells.  6). The reactions were terminated by adding SDS at final concentration of 1% and the DNA was resolved by agarose gel electrophoresis. B, topo II␣ was incubated, then assayed for topo II activity as described above except that knotted P4 phage DNA was used instead of pUC19 DNA. The position of unknotted DNA is indicated by the arrowhead.
in vitro, which are sites of modification in intact cells. Thus, casein kinase II is the major enzyme that phosphorylates topo II in various eukaryotic cells.
The activity of topo II from Drosophila and yeast cells is stimulated by casein kinase II phosphorylation. Thus we examined whether the phosphorylation of mouse topo II␣ by casein kinase II stimulated topo II␣ activity. Phosphorylation of topo II␣ (ϳ2 phosphates/enzyme) by casein kinase II had no effect on topo II␣ activity (Figs. 3 and 4). The inability to stimulate topo II␣ activity by phosphorylation may be due to the fact that the topo II␣ is sufficiently phosphorylated to exhibit enzyme activity. However, this possibility was excluded because the almost total dephosphorylation of topo II␣ by PAP did not decrease topo II␣ activity (Fig. 5). Thus phosphorylation of topo II␣ had no effect upon the enzyme activity under our experimental conditions. In this context, it is interesting that the C-terminal domain of topo II, which is the site of phosphorylation, is not required for the activity of Schizosaccharomyces pombe and Saccharomyces cerevisiae topo II (38,41,42).
The key finding in this study was that the incubation itself stimulates topo II␣ activity (Fig. 7). Frere et al. (43) have reported that the human topo II␣ 1013-1056 fragment associates into stable two-stranded ␣-helical coiled-coil structures through hydrophobic interactions. In addition, Lamhasni et al. (44) have reported that yeast topo II exists as a monomer-dimer equilibrium depending on both the enzyme concentration and salt concentration. Vassetzky et al. (45) indicated that multimerization of topo II␣ required its phosphorylation. Since topo II␣ was stimulated even by the incubation with PAP, multimerization of topo II␣ is not required for the stimulation. Thus it seems likely that mutual interaction of topo II␣ to form homodimers or a conformational change of topo II␣ dimers occurs  Fig. 4A. B, topo II␣ was incubated as described above for 0 (a) or 30 min (b), and DNA unknotting activity was assayed using knotted P4 phage DNA instead of pUC19DNA. during incubation, resulting in activation of topo II␣.
It must be noted that the stimulation by incubation was not observed with the topo II␣ after storage for long periods. In this case, the increase in the level of topo II activity was observed during the storage.
We reported that the activity of mouse topo II, which corresponded to topo II␣, was stimulated by an incubation with PKII (36). However, the degree of stimulation by PKII varied among preparations. The purified PKII fractions were stored in a medium containing 50% glycerol. It is likely that the purified PKII fractions contained inactive topo II␣, which could be activated by incubation in a medium containing a low concentration of glycerol and then, apparently stimulated topo II␣ activity, being independent of phosphorylation of topo II␣.
We also reported that treatment of topo II␣ with agarose bead-conjugated CIAP reduced topo II␣ activity. By contrast, in this study topo II␣ activity was not reduced after treatment with potato acid phosphatase, which removes almost all the phosphate groups from topo II␣. Fig. 6 shows that ATP in the reaction mixture for the assay of topo II activity degraded rapidly when topo II␣ was first incubated with CIAP. Although the cause of the inhibition of topo II activity in our previous study must be studied precisely, one possibility is that topo II␣ activity was inhibited by CIAP, which was released from agarose beads and carried over to topo II assay mixture, due not to the dephosphorylation of topo II␣ but to ATP degradation.
The present finding that phosphorylation has no effect on topo II␣ activity is incompatible with previous studies on Drosophila and S. cerevisiae topo II (27,29,30). This discrepancy must be analyzed in detail in future experiments. Thus, the conclusions of the present work do not at this time appear to be applicable to the regulation of topo II activity from lower eukaryotes.
This study showed that the activity of topo II␣ was stimulated by incubation itself. The stimulation was observed under specific conditions: a relatively low concentration of glycerol and high concentrations of topo II␣, which had not been stored for long periods. Thus, there is at present no evidence to suggest that the previously reported conclusion that the activity of topo II is modulated by its phosphorylated state is not valid. We emphasize that the studies on the effect of phosphorylation on the activity of topo II must be done and interpreted very carefully.