(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 (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) ()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-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 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
.
For phosphopeptide
mapping, purified topo II (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.
Figure 1:
SDS-PAGE of purified topo II 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.
Figure 2:
Phosphopeptide analysis of topo II
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.
Figure 3:
Time
course of phosphorylation of topo II 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
.
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).
Figure 4:
Effect of phosphorylation by casein kinase
II on the activity of topo II. 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.
Figure 5:
Effect of dephosphorylation by potato acid
phosphatase on the activity of topo II. 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.
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 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).
Figure 6:
Effect
of CIAP treatment and degradation of ATP. A, purified topo
II (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.
Figure 7:
Incubation itself stimulates the activity
of topo II. 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.
Figure 8:
Effect of the glycerol concentration upon
the activation of topo II 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.
When the
concentration of topo II 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).
Figure 9:
Effect of the topo II 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).
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 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. 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.
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
(
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) .
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 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. 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.
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.