(Received for publication, April 20, 1995; and in revised form, September 6, 1995 )
From the
Type II topoisomerases are essential for faithful cell division
in all organisms. In human cells, the isozyme of topoisomerase II
has been implicated in catalyzing mitotic chromosome segregation via
its action as a DNA unlinking enzyme. Here, we have shown that the
enzymatic activity of topoisomerase II
protein purified from HeLa
cell nuclei was strongly enhanced following phosphorylation by protein
kinase C. We have investigated the possibility that this kinase is
involved in cell cycle phase-specific phosphorylation of topoisomerase
II
in HeLa cells. Two-dimensional tryptic phosphopeptide mapping
revealed that topoisomerase II
protein immunoprecipitated from
metabolically labeled HeLa cells was differentially phosphorylated
during the G
/M phases of the cell cycle. To identify sites
of phosphorylation and the kinase(s) responsible for this modification,
oligohistidine-tagged recombinant domains of topoisomerase II
protein were overexpressed in Escherichia coli and purified by
affinity chromatography. Phosphorylation of a short fragment of the
N-terminal ATPase domain of topoisomerase II
by protein kinase C in vitro generated two phosphopeptides that co-migrated with
prominent G
/M phase-specific phosphopeptides from the HeLa
cell-derived topoisomerase II
protein. Site-directed mutagenesis
studies indicated that phosphorylation of serine 29 generated both of
these phosphopeptides. Our results implicate protein kinase C in the
cell cycle phase-dependent modulation of topoisomerase II
enzymatic activity in human cells.
In order for chromosomes to be faithfully transmitted from
mother to daughter cells, DNA must be fully replicated and segregated
evenly. Chromosome segregation can be affected only when all covalent
DNA interlinks between replicated sister chromatids have been removed.
The enzyme that catalyzes the disentanglement of replicated chromosomes
via its ability to decatenate covalently interlinked duplex DNA
molecules is DNA topoisomerase II, a highly conserved, homodimeric
nuclear protein (see Wang(1985), Osheroff et al.(1991), Holm
(1994), and Watt and Hickson(1994) for reviews). Evidence of a role for
topoisomerase II in mitotic chromosome segregation has been derived
largely from studies in lower eukaryotes. Yeast mutants defective in
topoisomerase II activity fail to remove all chromosomal interlinks at
mitosis and subsequently incur chromosomal breakage as cell division is
attempted in the absence of proper segregation (DiNardo, et
al., 1984; Holm, et al., 1985; Uemura and Yanagida, 1986;
Uemura, et al., 1987; Holm, et al., 1989; Rose and
Holm, 1993; Spell and Holm, 1994). This defective segregation leads to
a rapid decline in cell viability (Goto and Wang, 1984; Uemura and
Yanagida, 1984). Because yeast cells contain a single topoisomerase II
gene, it has been possible to study topoisomerase II function using
conditional lethal mutants defective in topoisomerase II function at
the restrictive growth temperature. Similar studies in human cells have
been hampered both by a lack of suitable mutants deficient in
topoisomerase II activity and by the presence of two closely related
topoisomerase II isozymes. The human isozymes are termed topoisomerase
II (170-kDa form) and topoisomerase II
(180-kDa form)
(Tsai-Pflugfelder et al., 1988; Drake et al., 1989;
Chung et al., 1989; Jenkins et al., 1992; Austin et al., 1993) and are the products of distinct genes encoded
on different chromosomes (Tsai-Pflugfelder et al., 1988; Tan et al., 1992; Jenkins et al., 1992).
Regulation of
mitotic events in mammalian cells may require the action of a number of
different protein kinases. The cyclin-dependent protein kinase,
p34, is regarded as the master controller of
mitotic events and phosphorylates a number of nuclear/nucleolar
proteins, including histone H1 and nucleolin (reviewed by Norbury and
Nurse(1992), Murray(1992), Nigg (1993), and Morgan(1995)). However,
recent studies have implicated both mitogen-activated protein (MAP) (
)kinase and protein kinase C (PKC) in the regulation of
certain mitosis-specific functions. The role of MAP kinase has not been
defined in detail, although this kinase is implicated in the generation
of the mitosis-specific phosphorylated epitope recognized by the MPM-2
antibody (Kuang and Ashorn, 1993; Westendorf et al., 1994).
This epitope is found in a number of nuclear proteins, including
topoisomerase II
and
(Taagepera et al., 1993). At
least one mitotic role for PKC, triggering the depolymerization of the
nuclear lamina, has been proposed (Goss et al., 1994).
Although little is known of the mechanisms by which the function of topoisomerase II is regulated in mammalian cells, a number of different protein kinases have been implicated in the modulation of topoisomerase II enzymatic activity. In general, dephosphorylation of eukaryotic topoisomerase II enzymes leads to loss of activity (Saijo, et al., 1990; Cardenas and Gasser, 1993), whereas phosphorylation by casein kinase II or PKC causes a mild stimulation of activity (Ackerman et al., 1985; Rottman et al., 1987; Ackerman et al., 1988; Cardenas et al., 1992; Corbett et al., 1993a, 1993b). Regulation by casein kinase II is particularly noteworthy in that Saccharomyces cerevisiae or mouse topoisomerase II proteins that have been inactivated by dephosphorylation can be reactivated by this kinase (Saijo et al., 1990; Cardenas and Gasser, 1993).
Several previous studies
have suggested that topoisomerase II protein from mammalian cells
is phosphorylated in vivo on multiple sites (Saijo et
al., 1990, 1992; Kroll and Rowe, 1991; Burden et al.,
1993; Ganapathi et al., 1993; Kimura et al., 1994;
Wells et al., 1994; Wells and Hickson, 1995). At least some of
these sites of phosphorylation correspond to recognition sequences for
casein kinase II (Wells et al., 1994). Moreover, topoisomerase
II
protein is hyperphosphorylated during the G
and/or
M phases of the cell cycle (Saijo et al., 1992; Burden et
al., 1993; Wells and Hickson, 1995). However, although casein
kinase II appears to phosphorylate yeast topoisomerase II protein in a
cell cycle phase-specific manner (Cardenas et al., 1992), no
evidence has been presented that this particular kinase is implicated
in the M phase-specific hyperphosphorylation of topoisomerase II
proteins from mammalian cells.
In this paper, we have studied the
cell cycle phase-specific phosphorylation of human topoisomerase
II protein. We have identified a serine residue in the N-terminal
ATPase domain of topoisomerase II
protein, which is modified
specifically during the G
/M phases of the HeLa cell cycle.
We have shown that this residue is a target in vitro for PKC
and that phosphorylation of topoisomerase II
protein purified from
HeLa cells by PKC strongly stimulates enzymatic activity in
vitro.
Figure 1:
Purification of
human topoisomerase II protein. Panel 1, the peak of
activity from the fast protein liquid chromatography MonoS column was
electrophoresed on a 7.5% polyacrylamide gel alongside molecular mass
standards (as indicated on the left) and stained with
Coomassie Blue (C-Blue). Panel 2, the purified
protein was electroblotted to Hybond-C Super and probed with the CRB
antiserum specific for topoisomerase II
(at 1:200 dilution). The
immunoreactive protein was detected with
I-protein A. The
position of the 170-kDa topoisomerase II
protein is indicated by
an arrow.
Figure 2:
The purified topoisomerase II protein
is a substrate for PKC in vitro. The topoisomerase II
protein was incubated in the absence(-) or the presence of PKC
,
, and
isotypes (as indicated above the lanes)
together with [
-
P]ATP. The position of the
phosphorylated topoisomerase II
protein is indicated on the right. The sizes (in kDa) of molecular mass standards run in
parallel are shown on the left.
Figure 3:
Activation of topoisomerase II
protein by PKC. Rate of plasmid relaxation catalyzed by purified
topoisomerase II
protein alone (A) or following
phosphorylation by PKC
,
, or
(B, C,
and D, respectively). The plasmids were run on a 1% agarose
gel, stained with ethidium bromide, and photographed under UV light.
The position of the gel wells (W), the supercoiled form I, and
relaxed form II DNA are indicated on the right. The time
course of the reaction (in minutes) is indicated above the
lanes. Lane HI contains heat-inactivated topoisomerase II
incubated with the relevant PKC isotype. The data presented are
representative of four independent
experiments.
Fig. 4shows a comparison
of tryptic phosphopeptide maps for topoisomerase II protein
derived either from an asynchronous culture of HeLa cells or from a
culture synchronized via a double thymidine block and released into
fresh thymidine-free medium for 8 h. Flow cytometric analysis revealed
that the culture released from the cell cycle blockade contained 91%
G
/M phase cells. A number of phosphopeptides that were
either specific for or greatly enriched within the G
/M
phase sample were evident (arrows in Fig. 4b).
Among these phosphopeptides are several that we have shown previously
to be dependent upon phosphorylation by a proline-directed kinase
(identified by open arrowheads in Fig. 4) and represent
phosphorylation of serine residues in the C-terminal regulatory domain
of topoisomerase II
protein (Wells and Hickson, 1995). However,
two of the most prominent G
/M phase-specific
phosphopeptides (indicated by arrows labeled A and B in Fig. 4b) have not been identified in
previous studies. Thus, we sought to identify the kinase(s) responsible
for phosphorylation of the serine or threonine residue(s) present in
phosphopeptides A and B. To determine the identity of the
phospho-acceptor residues, phosphopeptides A and B were excised from
the TLC plate and subjected to phosphoamino acid analysis. This
revealed, in each case, that serine was the sole phospho-acceptor
residue (not shown).
Figure 4:
Differential phosphorylation of
topoisomerase II protein during the G
/M phases of the
cell cycle. Two-dimensional tryptic phosphopeptide maps of
topoisomerase II
protein extracted from asynchronously growing
HeLa cells (a) or a culture enriched for G
/M phase
cells (b). Phosphopeptides were separated in the horizontal
dimension by electrophoresis at pH 1.9 (anode on left) and in
the vertical dimension by chromatography. The positions of the
G
/M phase-specific phosphopeptides identified in previous
studies (Wells and Hickson, 1995) are indicated by the open arrow
heads. The two G
/M phase-specific phosphopeptides
studied in this paper are indicated by solid arrows and
denoted A and B. The position of the origin (O) is indicated.
Figure 5:
The 18-kDa N-terminal domain of human
topoisomerase II protein is a substrate in vitro for PKC.
The E. coli-expressed 18-kDa fragment was electrophoresed on a
12% polyacrylamide gel and stained with Coomassie Blue (lane
C). The recombinant protein was then incubated in the presence (lane 1) or absence (lane 2) of PKC
I and
electrophoresed on a 12% gel, which was exposed to x-ray film after
drying. Lane 3 shows the PKC preparation in the absence of the
18-kDa topoisomerase II
fragment. The position of the
topoisomerase II
protein fragment is indicated by the arrows. The sizes (in kDa) of molecular mass standards run in
parallel are shown on the right.
Figure 6:
The G/M phase-specific
phosphopeptides A and B are derived from the N-terminal 18-kDa fragment
of topoisomerase II
protein. a, two-dimensional tryptic
phosphopeptide map of topoisomerase II
protein extracted from
G
/M phase-enriched HeLa cells. The positions of
phosphopeptides A and B are indicated. b, a map of the 18-kDa
N-terminal fragment phosphorylated in vitro by PKC
1. The
positions of three prominent phosphopeptides are shown. c, a
1:1 mix of the samples in a and b, indicating
co-migration of phosphopeptides A and B from a with
phosphopeptides 3 and 4 from b. Note the selective increase in
intensity of phosphopeptides A and B from the in vivo sample
due to co-migration with phosphopeptides from the in vitro sample. d, a mix of the sample in a with
phosphopeptide 3 extracted from a thin layer plate. Note the selective
increase in intensity in phosphopeptide A due to co-migration with
phosphopeptide 3.
Figure 7: Phosphopeptides 3 and 4 are derived from phosphorylation of serine-29. A, two-dimensional phosphopeptide map of the 18-kDa N-terminal fragment phosphorylated in vitro by PKC. The positions of the four most prominent phosphopeptides are indicated by numbered arrows. B, two-dimensional phosphopeptide map of the 18-kDa fragment containing a single amino acid substitution of alanine for serine-29. Note the absence of phosphopeptides 3 and 4. C, a 1:1 mix of the samples in A and B. The position of the origin (O) is indicated in each panel.
To confirm that serine-29 is the only possible phospho-acceptor
residue in peptide 3 and, therefore, to prove it is this residue that
is modified in vivo, as well as in vitro, a synthetic
peptide representing residues 24-39 was synthesized. This
peptide, which contains only one serine residue (serine-29), was
phosphorylated in vitro by PKCI and then run on thin
layer plates after trypsin digestion. Fig. 8shows that a single
strong tryptic phosphopeptide was generated following complete
digestion, which was shown in mixing experiments to co-migrate with
peptide 3 from the trypsin-digested recombinant N-terminal domain. To
confirm the co-migration of these phosphopeptides, the level of
radioactivity in each phosphopeptide was also quantified by volume
integration using the ImageQuant program on a Molecular Dynamics
PhosphorImager. The values obtained were as follows: peptide alone,
39,900; phosphorylated N-terminal recombinant domain alone, 84,600
(peptide 3) and 84,900 (peptide 2); and a mix of synthetic peptide and
N-terminal recombinant domain, 126,000 (peptide 3) and 81,000 (peptide
2). These data show that the major phosphopeptide derived from the
synthetic peptide co-migrates with phosphopeptide 3 from the
recombinant N-terminal protein. This confirms that serine-29 is the
target residue for PKC in vitro and is the only possible
phospho-acceptor residue present in peptide A from the in vivo labeled topoisomerase II
protein.
Figure 8:
Serine-29 is the only serine residue in
phosphopeptide 3. a, two-dimensional tryptic phosphopeptide
map of a synthetic peptide representing residues 24-39
phosphorylated in vitro by PKC. b, the relevant
portion of a two-dimensional tryptic phosphopeptide map of the 18-kDa
N-terminal fragment phosphorylated in vitro by PKCI. c, a 1:1 mix of the samples in a and b. The
positions of phosphopeptides 2 and 3 are indicated by numbered
arrows.
We have identified serine-29 as a site of phosphorylation of
topoisomerase II protein from the human HeLa cell line and have
shown that this residue is a substrate for PKC in vitro.
Phosphorylation on serine-29 is increased greatly as HeLa cells
traverse the G
/M phases of the cell division cycle.
Further, we have shown that phosphorylation by PKC substantially
increases the catalytic activity of purified topoisomerase II
protein in vitro.
Many mitosis-specific events in mammalian
cells are regulated by the action of protein kinases. The most
extensively characterized of the mitosis-activating kinases is the
p34-cyclin B complex. Phosphorylation of target proteins
by p34
-cyclin B initiates many of the hallmark events in
mitosis, such as nucleolar disassembly and chromatin condensation
(reviewed by Norbury and Nurse, 1992; Murray, 1992; Nigg, 1993; Morgan,
1995). However, recent studies have indicated that kinases other than
p34
-cyclin B are intimately involved in the regulation
of mitotic events. For example, MAP kinase appears to be at least one
of the kinases responsible for the generation of the mitosis-specific
phosphorylated epitope recognized by the MPM-2 antibody (Kuang and
Asham, 1993; Westendorf et al., 1994). Moreover, although many
isoforms of PKC are cell membrane-associated and unlikely, therefore,
to be responsible for modulating nuclear events, certain PKC isoforms
are clearly critical for cell cycle traverse and are strongly
implicated in affecting an essential nuclear mitotic function (reviewed
by Clemens et al.(1992)). Current evidence suggests that the
translocation of the
II isoform from the cytoplasmic membrane to
the nucleus and the subsequent phosphorylation of nuclear lamins at the
G2/M phase transition are necessary for the depolymerization of the
nuclear lamina (Goss et al., 1994). This was a role previously
assigned to p34
-cyclin B. Indeed, PKC is known to be
required for the G
/M phase transition in at least some cell
types, and the expression of the
II isoform has been shown to be
essential for proliferation in a human leukaemic cell line (Usui et
al., 1991; Levin et al., 1990; Murray et al.,
1993). It is not unreasonable to assume, therefore, that PKC
II or
another isoform of PKC that is located in the nucleus, such as
PKC
, is involved in the regulation of other nuclear factors that
are required during mitosis. Our data are consistent with the proposal
that activation of topoisomerase II
during mitosis is mediated, at
least in part, by PKC. However, we cannot rule out the possibility that
kinases other than or in addition to PKC modify serine-29 of
topoisomerase II
in mitotic cells. Based upon the sequence context
in which serine-29 lies and the data presented in this paper, it seems
highly unlikely that any of the kinases previously implicated in
modifying either lower eukaryotic topoisomerase II proteins in vivo (casein kinase II and p34
kinase; Ackerman et
al., 1988; Cardenas et al., 1992; Shiozaki and Yanagida,
1992), or the human topoisomerase II
protein in vivo (casein kinase II and a proline-directed kinase; Wells et
al., 1994; Wells and Hickson, 1995) are directly responsible for
the modification of serine-29.
A number of previous studies have
suggested that PKC may be involved in the regulation of topoisomerase
II functions. The most extensively characterized system for an analysis
of the effects of phosphorylation on topoisomerase II activity to date
has been Drosophila. Osheroff and colleagues have shown that
phosphorylation by PKC enhances the activity of Drosophila topoisomerase II approximately 2.5-fold and that this activation
is mediated via an enhancement in the rate of ATP hydrolysis (Corbett et al., 1993a, 1993b). Moreover, topoisomerase II
phosphorylated by PKC has an altered susceptibility to inhibition by
certain antineoplastic agents that interfere with the catalytic cycle
of topoisomerase II (DeVore et al., 1992). Similarly, Rottmann et al.(1987) showed that PKC phosphorylates topoisomerase II
from the sponge, Geodia cydonium, in vivo and
increases its activity around 2.5-fold in vitro. Our data on
human topoisomerase II protein are in agreement with those
obtained with the Geodia and Drosophila topoisomerase
II proteins. Moreover, the location of the PKC target serine residue in
the N-terminal ATPase domain of human topoisomerase II
protein
suggests an interesting possibility that this phosphorylation event is
linked directly to alterations in ATP binding/hydrolysis. However, it
should be noted that phosphorylation of Drosophila topoisomerase II by either casein kinase II or PKC (both of which
apparently target the C-terminal domain) causes an increase in the rate
of ATP hydrolysis (Ackerman et al., 1988; Corbett et
al., 1992, 1993a, 1993b). Indeed, Ackerman et al.(1988)
have provided evidence that casein kinase II is the predominate
activity responsible for phosphorylating topoisomerase II in cultured Drosophila cells. Similarly, our previous data implicate
casein kinase II as a key regulator of human topoisomerase II
(Wells et al., 1994). The data presented here indicate both
that the phosphorylation of serine-29 of human topoisomerase II
is
almost certainly not mediated by casein kinase II and that this
strongly cell cycle-regulated modification would not necessarily have
been detected in previous studies in which asynchronously growing cell
cultures were employed.
There are a number of possible functions
that phosphorylation of human topoisomerase II protein might
perform. Considering the G
/M phase-specific nature of the
modification of serine-29, it would seem likely that this modification
is required to affect a mitosis-specific function. Our data indicate
that phosphorylation by PKC can substantially increase the catalytic
activity of purified human topoisomerase II
protein. Indeed, the
extent to which PKC can activate topoisomerase II
in vitro may be a significant underestimate of the true effect of PKC on
activity in vivo, because the purified enzyme was likely to be
already in at least a partially phosphorylated state. It is possible
that this activation may be linked to a requirement for a highly
efficient catalytic activity throughout the short time period in the
G
and/or M phases in which chromosome segregation must be
affected by topoisomerase II.
The data presented here indicate that
phosphorylation of serine-29 gives rise to two tryptic phosphopeptides.
We would suggest that this occurs via differential digestion of the
protein by trypsin, which is known to occur at sites of adjacent lysine
and arginine residues (Campbell et al., 1986). Indeed,
serine-29 lies in a sequence that contains three consecutive target
residues for trypsin (KKRLS; in the one-letter amino acid
code), which would appear to provide the opportunity for differential
digestion to occur.
Few of the previous studies on topoisomerase II
phosphorylation in lower or higher eukaryotic cells have analyzed the
location of phospho-acceptor residues. In those studies where sites
have been mapped definitively or predicted from the mobility of
phosphopeptides on TLC plates, it is the C-terminal domain that has
been implicated as the major target for kinases. In budding yeast
topoisomerase II protein, the C-terminal domain is proposed to be a
target for multiple phosphorylations by casein kinase II (Cardenas et al., 1992). Moreover, several of these C-terminal sites
appear to be hyperphosphorylated at mitosis. Similarly, we have shown
previously that human topoisomerase II protein is phosphorylated in vivo on two serine residues in the C-terminal domain by
casein kinase II (Wells et al., 1994). The only previous data
implicating the N-terminal ATPase domain of topoisomerase II as a
target for phosphorylation have come from the work of Shiozaki and
Yanagida(1992) on the fission yeast topoisomerase II enzyme. Their
study indicated that phosphorylation was implicated in controlling
nuclear localization. Clearly, therefore, PKC-mediated phosphorylation
of the N-terminal domain of human topoisomerase II
protein could
regulate enzymatic activity and/or nuclear localization. Studies are in
hand to address these possibilities.
We have examined the
possibility that an N-terminal site for PKC-mediated phosphorylation is
conserved in other eukaryotic topoisomerase II enzymes (see Caron and
Wang(1994) for a review of sequence homologies). In human topoisomerase
II, serine-29 lies in a sequence context that comprises
predominantly basic amino acids. This sequence motif is conserved in
all mammalian topoisomerase II
enzymes. Moreover, this motif is
also conserved in the
isozyme of human topoisomerase II. Whether
this indicates that topoisomerase II
protein is also a target for
PKC in vivo will require further studies. Although accurate
alignment of the human and lower eukaryotic topoisomerase II sequences
is difficult due to general sequence divergence, there are potential
target serine residues for PKC near to the N terminus of the yeast and Drosophila topoisomerase II proteins.
In summary, we have
shown that PKC can modulate the enzymatic activity of human
topoisomerase II and have identified a serine residue in the
ATPase domain of the protein that is phosphorylated specifically during
the G
and/or M phases of the HeLa cell cycle. The challenge
is now to identify the precise role of the cell cycle-regulated
phosphorylations that occur on topoisomerase II
protein and that
appear to require the action of at least two distinct kinases (Wells
and Hickson(1995) and this work).