©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation-independent Stimulation of DNA Topoisomerase II Activity (*)

(Received for publication, May 11, 1995; and in revised form, January 31, 1996)

Keiji Kimura (1) (2) Masafumi Saijo (1)(§) Masato Tanaka (2) Takemi Enomoto (1)(¶)

From the  (1)Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo 113, Japan and the (2)Mitubishi Kasei Institute of Life Sciences, Machida, Tokyo 194, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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


INTRODUCTION

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, 10, 11, 12, 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, 17, 18, 19, 20, 21, 22, 23, 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, 27, 28, 29, 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 IIalpha and topo IIbeta, the latter having been discovered later(33) . Thus, most reports describing the phosphorylation of topo II of mammalian cells referred to topo IIalpha, 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 IIalpha. We reported that an unidentified protein kinase, PKII phosphorylated topo IIalpha, 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 IIalpha in mouse FM3A cells, which dominantly express topo IIalpha. We found that casein kinase II phosphorylated topo IIalpha in FM3A cells and that phosphorylation of topo IIalpha by casein kinase II had no effect on the activity of topo IIalpha under our experimental conditions. More importantly, we found that the incubation itself stimulated the activity of topo IIalpha.


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 µl) 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 µg/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 µl 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 IIalpha

All operations were performed at 0-4 °C. A total of 8 times 10^9 frozen FM3A cells were thawed, suspended in buffer 1 at a concentration of 1.25 times 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 times g. The supernatant was recovered as the crude extract and loaded onto a phosphocellulose column (30 ml) equilibrated with 0.45 M KCl in buffer 2. The column was washed with the same buffer and eluted with 0.6 M KCl in buffer 2. The eluted fractions were pooled and 5/12 volume of buffer 2 was added to bring the final concentration of KCl to 0.35 M and loaded onto a hydroxyapatite column (5 ml) equilibrated with 0.35 M KCl in buffer 2. The column was washed with 3 bed volumes of the same buffer and eluted with 10 bed volumes of a linear gradient of potassium phosphate buffer, pH 7.5, from 20 mM to 0.35 M in buffer 2 containing 0.35 M KCl. Topo IIalpha was detected by SDS-PAGE. Topo IIalpha was eluted from the column between 0.25 and 0.3 M potassium phosphate. The fractions containing topo IIalpha were pooled and dialyzed against 0.2 M KCl in buffer 2. The dialysate was loaded onto a Mono Q column (0.5 times 5 cm) equilibrated with 0.2 M KCl in buffer 2 at 0.5 ml/min. The column was washed with 5 bed volumes of the equilibration buffer and eluted with a 30-min gradient of 0.2-0.5 M KCl in buffer 2 at 0.5 ml/min. Topo IIalpha detected by SDS-PAGE was eluted at 0.3 M KCl. The peak fractions were pooled and dialyzed against 0.15 M KCl in buffer 3. The dialysate was stored at -20 °C.

Purification of Casein Kinase II

Cell extracts were prepared from 8 times 10^9 frozen FM3A cells as described above. To remove the nucleic acids, the extract was loaded onto a DEAE-cellulose column (70 ml) equilibrated with 0.3 M KCl in buffer 1. The flow-through fraction was dialyzed against 140 mM KCl in buffer 2 and applied to a second DEAE-cellulose column (40 ml) equilibrated with 140 mM KCl in buffer 2. The column was washed with 3 bed volumes of the same buffer and eluted with 3 bed volumes of 0.3 M KCl in buffer 2. The active fractions were pooled and loaded onto a phosphocellulose column (10 ml) equilibrated with 0.3 M KCl in buffer 2. The column was washed with 3 bed volumes of the equilibration buffer and eluted with a linear gradient of KCl from 0.3 to 1 M in buffer 2. Casein kinase activity was eluted from the column at 0.7 M KCl. The active fractions were pooled and dialyzed against 50 mM KCl in buffer 2. The dialysate was loaded onto a FPLC Mono Q column (0.5 times 5 cm) equilibrated with 50 mM KCl in buffer 2 at 0.5 ml/min. The column was washed with 5 bed volumes of the same buffer and eluted with a linear gradient of KCl from 50 mM to 0.6 M in buffer 2. Casein kinase II activity was eluted from the column between 0.4 and 0.45 M KCl. The active fractions were pooled, dialyzed against 0.15 M KCl in buffer 3, and stored at -20 °C.

Labeling Topo IIalpha 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 times 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 [P]orthophosphate (60 µCi/ml, 8500-9100 Ci/mmol) for 2 h at 37 °C. P-Labeled topo IIalpha was immunoprecipitated with anti-topo II antibody by the procedure of Saijo et al. (23) .

Phosphorylation of Topo IIalpha

Purified topo IIalpha (60 ng) was incubated with 50 ng of casein kinase II in the reaction mixture containing 20 mM Hepes, pH 7.4, 0.1 mM ATP or 10 µM [-P]ATP (0.1 Ci/mmol), 150 mM NaCl, 10 mM MgCl(2), 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, and 5% glycerol at 30 °C for the indicated periods.

For phosphopeptide mapping, purified topo IIalpha (1 µg) 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 µM [-P]ATP (1-5 Ci/mmol) at 30 °C for 30 min. Phosphorylation by PKII proceeded in a reaction mixture containing 20 mM Hepes, pH 7.4, 10 mM MgCl(2), 1 mM dithiothreitol, and 10 µM [-P]ATP (1-5 Ci/mmol) at 30 °C for 30 min. Protein kinase C phosphorylation proceeded in a reaction mixture containing 20 mM Hepes, pH 7.4, 3 mM MgCl(2), 10 µM [-P]ATP (1-5 Ci/mmol), 25 µg/ml phosphatidylserine, and 4 µg/ml dioleoylglycerol at 30 °C for 30 min.

Phosphopeptide Mapping

P-Labeled topo IIalpha was resolved by SDS-PAGE and stained with Coomassie Brilliant Blue R-250. The stained band of topo IIalpha 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 µl 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 times g, then the supernatant was recovered and dried. A solution (40 µl) 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 IIalpha

Topo IIalpha (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 IIalpha in FM3A Cells

We purified topo IIalpha and casein kinase II from mouse FM3A cells as described under ``Experimental Procedures.'' The purified topo IIalpha 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 IIalpha was phosphorylated by protein kinase C, casein kinase II, or PKII. The labeled topo IIalpha was separated by SDS-PAGE, digested by Achrombacter protease 1, and the phosphopeptides were mapped as described under ``Experimental Procedures.'' Topo IIalpha labeled by each kinase had a distinct phosphopeptide profile (Fig. 2, lanes 1-3), indicating that these kinases phosphorylate topo IIalpha at different sites. By contrast, the phosphopeptide maps of topo IIalpha phosphorylated by casein kinase II and that of topo IIalpha 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 IIalpha in FM3A cells.


Figure 1: SDS-PAGE of purified topo IIalpha and casein kinase II fractions. Purified topo IIalpha fraction (200 ng of protein) (A) and purified casein kinase II fraction (300 ng) (B) were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue.




Figure 2: Phosphopeptide analysis of topo IIalpha labeled in intact cells or in vitro with various kinases. Topo IIalpha was labeled in intact cells or in vitro with the indicated kinases, digested by Achrombacter protease 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 P-labeled phosphopeptides were detected with an image analyzer. Lane 1, phosphopeptide map of topo IIalpha 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.



The Effect of Phosphorylation by Casein Kinase II upon Topo IIalpha Activity

Casein kinase II appeared to phosphorylate topo IIalpha in intact cells. Thus, we examined the effect of phosphorylation by casein kinase II upon the activity of topo IIalpha. Fig. 3shows the time course of topo IIalpha phosphorylation by casein kinase II. At the maximal level, about 2 molecules of phosphate were incorporated into one molecule of topo IIalpha. Incubating topo IIalpha with heat-inactivated casein kinase II or in the absence of the kinase resulted in no phosphate incorporation.


Figure 3: Time course of phosphorylation of topo IIalpha by casein kinase II. Topo IIalpha (60 ng) was incubated with 50 ng of casein kinase II (bullet), 50 ng of heat-inactivated casein kinase II (circle), or without kinase (up triangle) for the indicated periods as described under ``Experimental Procedures.'' Levels of phosphorylation are expressed as molecules of phosphate incorporated per molecule of topo IIalpha.



Topo IIalpha 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 IIalpha was measured. The levels of activity of topo IIalpha incubated with casein kinase II or heat-inactivated casein kinase II were considerably higher than that of activity of topo IIalpha 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 IIalpha was stimulated during the incubation independently of phosphorylation by casein kinase II, because the heat-inactivated casein kinase II did not phosphorylate topo IIalpha (Fig. 3).


Figure 4: Effect of phosphorylation by casein kinase II on the activity of topo IIalpha. A, purified topo IIalpha (60 ng) was incubated with 50 ng of casein kinase II (b) or 50 ng of heat-inactivated casein kinase II (c) at 30 °C for 30 min in incubation medium (50 µl) containing 20 mM Hepes, pH 7.4, 0.1 mM ATP, 150 mM NaCl, 10 mM MgCl(2), 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, and 5% glycerol or the enzyme in the incubation medium was not incubated (a). The activity of the treated topo IIalpha (1.2 ng) was assayed in a reaction mixture (20 µl) containing supercoiled pUC19 DNA for 0 (lane 1), 5 (lane 2), 10 (lane 3), 15 (lane 4), 20 (lane 5), and 30 min (lane 6). The reactions were terminated by adding SDS at final concentration of 1% and the DNA was resolved by agarose gel electrophoresis. B, topo IIalpha 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.



The Effect of Dephosphorylation on Topo IIalpha Activity

To determine whether the purified topo IIalpha had been sufficiently phosphorylated in the cells and that additional phosphorylation scarcely affected the activity of topo IIalpha, we incubated it with PAP. We first confirmed the dephosphorylating activity of PAP using topo IIalpha labeled with P in intact cells. The P-labeled topo IIalpha was incubated with PAP or heat-inactivated PAP. As shown in Fig. 5A, PAP removed almost all P label from topo IIalpha and heat-inactivated PAP had no effect upon phosphate removal (Fig. 5A, lane 3).


Figure 5: Effect of dephosphorylation by potato acid phosphatase on the activity of topo IIalpha. A, topo IIalpha labeled with [P]orthophosphate in intact cells was immunoprecipitated and incubated with 0.2 units of PAP (lane 2), heat-inactivated PAP (lane 3), or without PAP (lane 1) at 30 °C for 30 min. B, purified topo IIalpha (60 ng) was incubated with 0.2 units of PAP (b) or heat-inactivated PAP (c) at 30 °C for 30 min, or topo IIalpha in the incubation mixture was not incubated (a). Then an aliquot (1.2 ng) was assayed for topo II activity using pUC19 DNA for 0 (lane 1), 5 (lane 2), 10 (lane 3), 15 (lane 4), 20 (lane 5), and 30 min (lane 6) as described in the legend to Fig. 4A. C, topo IIalpha was incubated, then assayed for topo II activity as described in B except that knotted P4 phage DNA was used instead of pUC19 DNA.



Topo IIalpha was incubated with PAP or heat-inactivated PAP under the same conditions as those of Fig. 5A, and the activity of topo IIalpha was assayed. Again, the activity of topo IIalpha was stimulated by an incubation either with PAP or with heat-inactivated PAP, when the activity was compared with that of topo IIalpha without this incubation (Fig. 5, B and C). The levels of topo IIalpha 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 IIalpha retained the same level of activity. By contrast, incubation with calf intestine alkaline phosphatase markedly inhibited topo IIalpha activity (compare Fig. 6A, a and b). In this case, ATP in the reaction mixture was almost completely degraded (Fig. 6B).


Figure 6: Effect of CIAP treatment and degradation of ATP. A, purified topo IIalpha (60 ng) was incubated with 25 units of CIAP (b) or heat-inactivated CIAP (a) as described under ``Experimental Procedures.'' Then an aliquot (1.2 ng) was assayed for topo II activity using pUC19 DNA for 0 (lane 1), 5 (lane 2), 10 (lane 3), 20 (lane 4), and 30 min (lane 5). B, topo IIalpha (1.2 ng) incubated with 25 units of CIAP (lane 2) or heat-inactivated CIAP was incubated in the topo II assay mixture containing 0.1 mM [-P]ATP (1 Ci/mmol) at 30 °C for 30 min. An aliquot of the reaction mixture (1 µl) was spotted onto a polyethyleneimine-cellulose sheet and developed with 1 M LiCl, 1 M HCOOH. Radioactivity was visualized using an image analyzer.



Incubation Itself Stimulates the Activity of Topo IIalpha

We determined whether the activity of topo IIalpha was affected by the incubation itself. Topo IIalpha 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 IIalpha 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).


Figure 7: Incubation itself stimulates the activity of topo IIalpha. A, topo IIalpha (60 ng) was incubated as described under ``Experimental Procedures'' at 30 °C for 0 (a), 5 (b), 15 (c), or 30 min (d), then an aliquot (1.2 ng) was assayed for topo II activity using pUC19 DNA for 0 (lane 1), 5 (lane 2), 10 (lane 3), 15 (lane 4), 20 (lane 5), and 30 min (lane 6), as described in the legend to Fig. 4A. B, topo IIalpha 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.



Conditions for Stimulating Topo IIalpha Activity

Purified topo IIalpha was stored in a buffer containing 50% glycerol, whereas the concentration of glycerol in the buffer in which topo IIalpha was preincubated was only 5%. Thus we examined the effect of glycerol concentration on the activation of topo IIalpha activity. Topo IIalpha was incubated in the buffer containing various concentrations of glycerol for 30 min. Then the activity of topo IIalpha 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 IIalpha in the buffer containing 75 and 50% glycerol did not stimulate the activity of topo IIalpha, whereas the activity was enhanced during incubation in the buffer containing 5 or 20% glycerol.


Figure 8: Effect of the glycerol concentration upon the activation of topo IIalpha activity. A, topo IIalpha (60 ng) was incubated at 30 °C for 30 min in medium (50 µl) containing various concentrations of glycerol, 5, 20, 50, and 75%, then 1.2 ng of topo IIalpha (1 µl) was incubated in the reaction mixture (20 µl) containing pUC19 DNA for 0 (lane 1), 5 (lane 2), 10 (lane 3), 15 (lane 4), 20 (lane 5), and 30 min (lane 6). The final concentration of glycerol was 3.75%. B, topo IIalpha was incubated in medium containing 5 or 50% glycerol as described above, and DNA unknotting activity was assayed using knotted P4 phage DNA instead of pUC19DNA.



When the concentration of topo IIalpha was decreased to one-tenth during the incubation for activation, topo IIalpha was not activated by the incubation, indicating that the activation is dependent on the concentration of topo IIalpha (Fig. 9).


Figure 9: Effect of the topo IIalpha concentration on the activation of its activity. Topo IIalpha (60 ng) was incubated at 30 °C for 0 min (a) or 30 min (b), and 10-fold less topo IIalpha (6 ng) was incubated at 30 °C for 30 min (c) in the reaction mixture (50 ml) containing 5% glycerol, then 1.2 ng of thus treated topo IIalpha was assayed for topo II activity using pUC19 DNA for 0 (lane 1), 5 (lane 2), 10 (lane 3), 15 (lane 4), 20 (lane 5), and 30 min (lane 6).




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, 17, 18, 19, 20, 21, 22, 23, 24) . We reported that topo II is phosphorylated in mouse FM3A cells and that the phosphorylation of topo IIalpha purified from the mouse cells by an unidentified protein kinase, PK II, stimulated the activity of topo IIalpha(36) .

To evaluate these results, we first tried to identify the protein kinase that phosphorylates topo IIalpha in FM3A cells. The results shown in Fig. 1indicate that casein kinase II phosphorylates topo IIalpha in mouse cells. Topo II in Drosophila and yeast cells are phosphorylated by casein kinase II(27, 30) . Wells et al.(40) have reported that casein kinase II phosphorylates the C-terminal domain of topo IIalpha, primarily, the 2 serine residues 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 IIalpha by casein kinase II stimulated topo IIalpha activity. Phosphorylation of topo IIalpha (2 phosphates/enzyme) by casein kinase II had no effect on topo IIalpha activity ( Fig. 3and Fig. 4). The inability to stimulate topo IIalpha activity by phosphorylation may be due to the fact that the topo IIalpha is sufficiently phosphorylated to exhibit enzyme activity. However, this possibility was excluded because the almost total dephosphorylation of topo IIalpha by PAP did not decrease topo IIalpha activity (Fig. 5). Thus phosphorylation of topo IIalpha 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 IIalpha activity (Fig. 7). Frere et al. (43) have reported that the human topo IIalpha 1013-1056 fragment associates into stable two-stranded alpha-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 IIalpha required its phosphorylation. Since topo IIalpha was stimulated even by the incubation with PAP, multimerization of topo IIalpha is not required for the stimulation. Thus it seems likely that mutual interaction of topo IIalpha to form homodimers or a conformational change of topo IIalpha dimers occurs during incubation, resulting in activation of topo IIalpha.

It must be noted that the stimulation by incubation was not observed with the topo IIalpha 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 IIalpha, 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 IIalpha, which could be activated by incubation in a medium containing a low concentration of glycerol and then, apparently stimulated topo IIalpha activity, being independent of phosphorylation of topo IIalpha.

We also reported that treatment of topo IIalpha with agarose bead-conjugated CIAP reduced topo IIalpha activity. By contrast, in this study topo IIalpha activity was not reduced after treatment with potato acid phosphatase, which removes almost all the phosphate groups from topo IIalpha. Fig. 6shows that ATP in the reaction mixture for the assay of topo II activity degraded rapidly when topo IIalpha 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 IIalpha 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 IIalpha but to ATP degradation.

The present finding that phosphorylation has no effect on topo IIalpha 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 IIalpha was stimulated by incubation itself. The stimulation was observed under specific conditions: a relatively low concentration of glycerol and high concentrations of topo IIalpha, 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.


FOOTNOTES

*
This work was supported by Grants-in Aid for Scientific Research and for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Institute for Molecular and Cellular Biology, Osaka University, Suita, Osaka 565, Japan.

To whom correspondence should be addressed. Present address: Dept. of Molecular Cell Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai, Miyagi 980-77, Japan.

(^1)
The abbreviations used are: topo II, topoisomerase II; CIAP, calf intestine alkaline phosphatase; PAGE, polyacrylamide gel electrophoresis; PAP, potato acid phosphatase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.


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