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(Received for publication, May 11, 1995; and in revised form, January 31, 1996) From the
It has been suggested that casein kinase II phosphorylates DNA
topoisomerase II DNA topoisomerase II (topo II) ( 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 In vertebrate organisms, two
isoforms of topo II have been identified, which have been designated
topo II 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
For phosphopeptide
mapping, purified topo II
Figure 1:
SDS-PAGE of purified topo II
Figure 2:
Phosphopeptide analysis of topo II
Figure 3:
Time
course of phosphorylation of topo II
Topo II
Figure 4:
Effect of phosphorylation by casein kinase
II on the activity of topo II
Figure 5:
Effect of dephosphorylation by potato acid
phosphatase on the activity of topo II
Topo II
Figure 6:
Effect
of CIAP treatment and degradation of ATP. A, purified topo
II
Figure 7:
Incubation itself stimulates the activity
of topo II
Figure 8:
Effect of the glycerol concentration upon
the activation of topo II
When the
concentration of topo II
Figure 9:
Effect of the topo II
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 II To evaluate these results, we first tried to
identify the protein kinase that phosphorylates topo II 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 The key finding in this
study was that the incubation itself stimulates topo II It must be noted that the
stimulation by incubation was not observed with the topo II We reported that
the activity of mouse topo II, which corresponded to topo II We
also reported that treatment of topo II The present finding that phosphorylation has no effect
on topo II This study
showed that the activity of topo II
Volume 271,
Number 18,
Issue of May 3, 1996 pp. 10990-10995
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Activity (*)
(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.
)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) .
-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. 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 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.
Buffers
Buffer 1 contained 20 mM potassium phosphate buffer, pH 7.5, 0.1 mM Na
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, 1 mM ATP, 0.5 mM dithiothreitol, 0.5 mM NaEDTA, 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 II
All operations
were performed at 0-4 °C. A total of 8 
10
frozen FM3A cells were thawed, suspended in buffer 1 at a
concentration of 1.25 10 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 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
II was detected by SDS-PAGE. Topo II was eluted from the
column between 0.25 and 0.3 M potassium phosphate. The
fractions containing topo II were pooled and dialyzed against 0.2 M KCl in buffer 2. The dialysate was loaded onto a Mono Q
column (0.5 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 II 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 10 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 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 II
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 in Intact
Cells 10
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 II was immunoprecipitated with anti-topo II antibody by the
procedure of Saijo et al. (23) .Phosphorylation of Topo II
Purified topo
II (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, 1
mM dithiothreitol, 0.1 mg/ml bovine serum albumin, and 5%
glycerol at 30 °C for the indicated periods. (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, 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, 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, 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
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 µl of medium containing 0.1 mg/ml Achrombacter protease 1 and 50 mM NH
HCO, pH 9.0, or 0.1 mg/ml V8 protease
and 50 mM NHHCO, 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 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 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.
The Kinase Responsible for Phosphorylating Topo II
We purified topo II
in FM3A Cells 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.
and
casein kinase II fractions. Purified topo II 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.
labeled in intact cells or in vitro with various kinases. Topo
II 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 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.
The Effect of Phosphorylation by Casein Kinase II upon
Topo II
Casein kinase II appeared to phosphorylate
topo II Activity in intact cells. Thus, we examined the effect of
phosphorylation by casein kinase II upon the activity of topo II. Fig. 3shows 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.
by casein kinase II. Topo
II (60 ng) was incubated with 50 ng of casein kinase II (
),
50 ng of heat-inactivated casein kinase II (
), or without kinase
(
) for the indicated periods as described under
``Experimental Procedures.'' Levels of phosphorylation are
expressed as molecules of phosphate incorporated per molecule of 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).
. A, purified topo II
(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, 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
II (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 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.
The Effect of Dephosphorylation on Topo II
To determine whether the purified topo II
Activity 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 P in
intact cells. The P-labeled topo II was incubated
with PAP or heat-inactivated PAP. As shown in Fig. 5A,
PAP removed almost all P label from topo II and
heat-inactivated PAP had no effect upon phosphate removal (Fig. 5A, lane 3).
. A, topo II
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
II (60 ng) was incubated with 0.2 units of PAP (b) or
heat-inactivated PAP (c) at 30 °C for 30 min, or topo
II 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 II
was incubated, then assayed for topo II activity as described in B except that knotted P4 phage DNA was used instead of pUC19
DNA.
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 heat-inactivated 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).
(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 II (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
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, 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).
. A, topo II (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 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.
Conditions for Stimulating Topo II
Purified topo II
Activity 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.
activity. A, topo II (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 II (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 II 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.
was decreased to one-tenth 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).
concentration
on the activation of its activity. Topo II (60 ng) was incubated
at 30 °C for 0 min (a) or 30 min (b), and 10-fold
less topo II (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 II 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).
purified from the mouse cells by an
unidentified protein kinase, PK II, stimulated the activity of topo
II(36) . in FM3A
cells. The results shown in Fig. 1indicate that casein kinase
II phosphorylates topo II 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 II, 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. by casein
kinase II stimulated topo II activity. Phosphorylation of topo
II (
2 phosphates/enzyme) by casein kinase II had no effect on
topo II activity ( Fig. 3and Fig. 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) . 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 during incubation,
resulting in activation of topo II. after
storage for long periods. In this case, the increase in the level of
topo II activity was observed during the storage., 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. 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. 6shows 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. 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. 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.
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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 P. S. Shapiro, A. M. Whalen, N. S. Tolwinski, J. Wilsbacher, S. J. Froelich-Ammon, M. Garcia, N. Osheroff, and N. G. Ahn Extracellular Signal-Regulated Kinase Activates Topoisomerase IIalpha through a Mechanism Independent of Phosphorylation Mol. Cell. Biol., May 1, 1999; 19(5): 3551 - 3560. [Abstract] [Full Text] [PDF]
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J. R. Daum and G. J. Gorbsky Casein Kinase II Catalyzes a Mitotic Phosphorylation on Threonine 1342 of Human DNA Topoisomerase IIalpha , Which Is Recognized by the 3F3/2 Phosphoepitope Antibody J. Biol. Chem., November 13, 1998; 273(46): 30622 - 30629. [Abstract] [Full Text] [PDF]
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C. Redwood, S. L. Davies, N. J. Wells, A. M. Fry, and I. D. Hickson Casein Kinase II Stabilizes the Activity of Human Topoisomerase IIalpha in a Phosphorylation-independent Manner J. Biol. Chem., February 6, 1998; 273(6): 3635 - 3642. [Abstract] [Full Text] [PDF]
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T. R. Hammonds and A. Maxwell The DNA Dependence of the ATPase Activity of Human DNA Topoisomerase IIalpha J. Biol. Chem., December 19, 1997; 272(51): 32696 - 32703. [Abstract] [Full Text] [PDF]
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K. Kimura, N. Nozaki, T. Enomoto, M. Tanaka, and A. Kikuchi Analysis of M Phase-specific Phosphorylation of DNA Topoisomerase II J. Biol. Chem., August 30, 1996; 271(35): 21439 - 21445. [Abstract] [Full Text] [PDF]
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A. E. Escargueil, S. Y. Plisov, O. Filhol, C. Cochet, and A. K. Larsen Mitotic Phosphorylation of DNA Topoisomerase II alpha by Protein Kinase CK2 Creates the MPM-2 Phosphoepitope on Ser-1469 J. Biol. Chem., October 27, 2000; 275(44): 34710 - 34718. [Abstract] [Full Text] [PDF]
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ABSTRACT
