©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cell Cycle Phase-specific Phosphorylation of Human Topoisomerase II
EVIDENCE OF A ROLE FOR PROTEIN KINASE C (*)

(Received for publication, April 20, 1995; and in revised form, September 6, 1995 )

Nicholas J. Wells Andrew M. Fry (§) Fulvio Guano Chris Norbury Ian D. Hickson (¶)

From the Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Type II topoisomerases are essential for faithful cell division in all organisms. In human cells, the alpha 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 IIalpha 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 IIalpha in HeLa cells. Two-dimensional tryptic phosphopeptide mapping revealed that topoisomerase IIalpha protein immunoprecipitated from metabolically labeled HeLa cells was differentially phosphorylated during the G(2)/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 IIalpha protein were overexpressed in Escherichia coli and purified by affinity chromatography. Phosphorylation of a short fragment of the N-terminal ATPase domain of topoisomerase IIalpha by protein kinase C in vitro generated two phosphopeptides that co-migrated with prominent G(2)/M phase-specific phosphopeptides from the HeLa cell-derived topoisomerase IIalpha 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 IIalpha enzymatic activity in human cells.


INTRODUCTION

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 IIalpha (170-kDa form) and topoisomerase IIbeta (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) (^1)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 IIalpha and beta (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 IIalpha 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 IIalpha protein is hyperphosphorylated during the G(2) 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 IIalpha protein. We have identified a serine residue in the N-terminal ATPase domain of topoisomerase IIalpha protein, which is modified specifically during the G(2)/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 IIalpha protein purified from HeLa cells by PKC strongly stimulates enzymatic activity in vitro.


MATERIALS AND METHODS

Cell Lines

HeLa S3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 3 mML-glutamine, and antibiotics in a humidified atmosphere containing 5% CO(2) at 37 °C.

Purification of Human Topoisomerase IIalpha Protein

All procedures were carried out at 4 °C. Buffers contained the following protease and phosphatase inhibitors: 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 µg/ml chymostatin, 1 µg/ml soybean trypsin inhibitor, 1 mM benzamidine, 1 µg/ml antipain, 50 µg/ml L-1-chloro-3-(4-tosylamido)-7-amino-2-heptonone hydrochloride, 0.1 mM beta-glycerophosphate, 0.1 mMp-nitrophenylphosphate, 0.5 mM glucose-1-phosphate, and 10 mM 2-mercaptoethanol. 40 liters of exponentially growing HeLa cells in suspension (approximately 3 times 10 cells, provided by the Imperial Cancer Research Fund Cell Production Unit) were used as the source of topoisomerase IIalpha protein. A 1 M sodium chloride extract of purified nuclei was prepared and separated by hydroxylapatite and phosphocellulose chromatography according to the method of Miller et al.(1981). Peak topoisomerase II activity eluted from these columns at 400 mM potassium phosphate and 300 mM potassium phosphate, pH 6.8, respectively. This partially purified activity was further purified by chromatography on fast protein liquid chromatography monoQ, phenyl-superose, and mono-S columns, according to the methods of Drake et al.(1987) and Strausfeld and Richter(1989).

Peptide Synthesis

A 15-mer synthetic peptide representing residues Ala-24 through Thr-39 (Ala-Lys-Lys-Arg-Leu-Ser-Val-Glu-Arg-Ile-Tyr-Gln-Lys-Lys-Thr) of the human topoisomerase IIalpha protein sequence was supplied by Severn Biotech Ltd. (Kidderminster, UK).

Plasmid Relaxation Assays

These assays were performed essentially as described by Osheroff et al. (1983). Reaction mixtures (50 µl) contained 50 mM Tris-HCl, pH 7.5, 120 mM KCl, 10 mM MgCl(2), 0.5 mM dithiothreitol, 0.5 mM EDTA, 1.0 mM ATP, 30 µg/ml nuclease-free bovine serum albumin, and 500 ng X174 RFI DNA, together with topoisomerase IIalpha protein. Reactions were incubated at 30 °C for up to 30 min, after which 5 µl of 10 times DNA loading buffer (50% w/v sucrose, 50 mM EDTA, 0.01% SDS, 0.1% bromphenol blue) was added. The samples were electrophoresed on a 1% agarose gel in TBE running buffer (89 mM Tris-HCl, pH 8.0, 89 mM boric acid, and 2 mM EDTA). DNA was stained with ethidium bromide and viewed under UV light. For quantitation, the negatives of polaroid photographs were scanned on a BioImage Analyser (MilliGen/BioSearch).

Metabolic Labeling, Nuclear Extraction, Immunoprecipitations, and Phosphoamino Acid and Phosphopeptide Analyses

These procedures were performed as described by Wells et al.(1994). The isozyme-specific antibody used for analysis of the topoisomerase IIalpha protein was designated CRB and has been validated previously (Smith and Makinson, 1989; Wells et al., 1994).

In Vitro Phosphorylation Reactions

The PKC reaction buffer contained 50 mM Tris-HCl, pH 7.4, 0.25 mM EDTA, 0.75 mM CaCl(2), 12.5 mM MgCl(2), and 0.005% (v/v) Triton X-100, 200 µM ATP, and, where required, 1-5 µCi of [-P]ATP (3,000 Ci/mmol, Amersham Corp.). Reactions were initiated by the addition of 0.1 unit of specific PKC isotypes purified from bovine brain, kindly provided by Dr. P. J. Parker (Imperial Cancer Research Fund), in a reaction volume of 20 µl and were allowed to proceed for 10 min at 37 °C.

DNA Sequencing

Nucleotide sequencing was performed on double-stranded plasmid templates using the dideoxy chain termination method and Sequenase enzyme (U. S. Biochemical Corp.).

Purification of an N-terminal Recombinant Fragment of Topoisomerase IIalpha Protein

The region of the topoisomerase IIalpha cDNA (Jenkins et al., 1992) encoding residues Lys-25 to Lys-168 was amplified using the polymerase chain reaction with the following primers (both written 5` to 3`): 5` primer, AGAGAGCTCGAGAAGAAAAGACTGTCTGTTGAAAGA, and 3` primer, AGAGAGCTCGAGTTATTTGGCTCCATAGCCATTTCGA. The purified polymerase chain reaction product was digested with XhoI and ligated into XhoI-digested pET14b (Invitrogen), which contains the T7 promoter driving expression of fusion proteins linked to an oligohistidine leader peptide. The plasmid was transformed into Escherichia coli BL21 (DE3), and transformants were grown to A of 0.5. After the addition of isopropyl-1-thio-beta-D-galactopyranoside (0.4 mM) to induce expression from the T7 promoter in pET14b, bacteria were grown for another 4 h. The 18-kDa recombinant fragment was purified from crude cell lysates using affinity chromatography on a nickel chelate column as recommended by the suppliers (Invitrogen).

Site-directed Mutagenesis

The substitution of alanine for serine-29 was achieved by amplifying the region of the topoisomerase IIalpha cDNA identical to that described above, with the exception that the 5` primer (5`-AGAGAGCTCGAGAAGAAAAGACTGGCTGTTGAAAGA-3`) contained a GCT codon (encoding alanine) replacing the TCT codon (serine-29). Preparation of the mutant protein was as described above.

Western Blotting

Following protein gel electrophoresis, Western blotting was performed according to the method of Towbin et al.(1979). Electroblotting onto nitrocellulose filters (Hybond-C Super, Amersham Corp.) was performed at 30 V overnight in transfer buffer (50 mM Tris-HCl, pH 7.5, 380 mM glycine, 0.1% (w/v) SDS, and 20% (v/v) methanol) in a Bio-Rad transblot cell. The filters were incubated for 60 min in blocking buffer (20 mM Tris-HCl, pH 7.5, 0.9% (w/v) NaCl, 0.05% (v/v) Tween 20, and 1% (w/v) Marvel low-fat milk powder). Primary antibody reactions were carried out for 2 h in blocking buffer containing the CRB antibody at a dilution of 1:200. CRB is a rabbit polyclonal antiserum raised to a C-terminal peptide of topoisomerase IIalpha, supplied by Cambridge Research Biochemicals, and has been previously shown to specifically recognize the alpha isozyme of topoisomerase II (Smith and Makinson, 1989). The filters were washed in blocking buffer for 60 min with at least three changes of buffer before incubation with I-protein A (Amersham Corp.) for 60 min. Filters were then washed extensively with Tris-buffered saline and then analyzed by autoradiography.

Cell Synchronization

HeLa cells were synchronized at the start of S phase by the use of a double thymidine block. Exponentially growing cells were treated with 2 mM thymidine for 14 h and then released into thymidine-free medium. After 11 h, 2 mM thymidine was again applied to the cells for a 15-h period before release into thymidine-free medium.

Flow Cytometry

Cells were fixed for 30 min in ice-cold 70% ethanol/30% phosphate-buffered saline collected by centrifugation and were treated with RNase A (100 µg/ml final concentration) and propidium iodide (40 µg/ml) in phosphate-buffered saline for 30 min at 37 °C. Cell cycle distribution was then determined using a Beckton Dickinson FACScan, and the data were analyzed using the Lysis II software.

Protein Gel Electrophoresis

Proteins were separated by the discontinuous SDS-polyacrylamide gel system described by Laemmli(1970).


RESULTS

Purification of Topoisomerase IIalpha Protein from HeLa Cells

It has been shown previously that the enzymatic activity of topoisomerase II proteins purified from lower eukaryotes can be stimulated by phosphorylation with PKC (Ackerman et al., 1985; Sahyoun et al., 1986; Rottman et al., 1987; DeVore et al., 1992; Corbett et al., 1993a, 1993b). To provide evidence that PKC is capable of influencing the enzymatic activity of human topoisomerase IIalpha, HeLa cell nuclear extracts were fractionated by conventional chromatography and fast protein liquid chromatography, and the topoisomerase IIalpha protein was purified to near homogeneity. This method of purification has been used previously to separate the alpha and beta isozymes of human topoisomerase II (Drake et al., 1987; Strausfeld and Richter, 1989). The purified topoisomerase II preparation contained a predominant 170-kDa protein that was recognized by the CRB antibody specific for topoisomerase IIalpha (Fig. 1). Several antibodies specific for the beta isozyme failed to detect any topoisomerase IIbeta in the purified protein preparation (data not shown).


Figure 1: Purification of human topoisomerase IIalpha 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 IIalpha (at 1:200 dilution). The immunoreactive protein was detected with I-protein A. The position of the 170-kDa topoisomerase IIalpha protein is indicated by an arrow.



Regulation of Topoisomerase IIalpha Activity by PKC

The purified topoisomerase IIalpha protein was tested as a substrate for PKC in vitro. Fig. 2shows that the purified toposimerase IIalpha preparation was free from contaminating kinases and had no intrinsic autophosphorylation activity. However, the 170 kDa topoisomerase IIalpha protein was a substrate in vitro for phosphorylation by 3 isoforms of PKC. To study the effects of phosphorylation by PKC, the enzymatic activity of the topoisomerase IIalpha protein was assayed using supercoiled plasmid DNA as a substrate. Fig. 3shows that the rate of plasmid relaxation catalyzed by the PKC-phosphorylated topoisomerase IIalpha protein was increased substantially over that catalyzed by the unmodified enzyme.


Figure 2: The purified topoisomerase IIalpha protein is a substrate for PKC in vitro. The topoisomerase IIalpha protein was incubated in the absence(-) or the presence of PKC alpha, beta, and isotypes (as indicated above the lanes) together with [-P]ATP. The position of the phosphorylated topoisomerase IIalpha 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 IIalpha protein by PKC. Rate of plasmid relaxation catalyzed by purified topoisomerase IIalpha protein alone (A) or following phosphorylation by PKC alpha, beta, 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 IIalpha incubated with the relevant PKC isotype. The data presented are representative of four independent experiments.



Topoisomerase IIalpha Protein Is Hyperphosphorylated During the G(2)/M Phases of the HeLa Cell Cycle

The alpha isozyme of topoisomerase II is a phosphoprotein in mammalian cells, and the level of its phosphorylation is regulated in a cell cycle phase-dependent manner. Studies with human and rodent cell lines have indicated that topoisomerase IIalpha protein is hyperphosphorylated during the G(2)/M phases of the mammalian cell cycle (Saijo, et al., 1992; Kroll and Rowe, 1991; Burden et al., 1993; Wells et al., 1994; Burden and Sullivan, 1994; Kimura et al., 1994; Wells and Hickson, 1995). In order to identify the G(2)/M phase-specific sites of phosphorylation on human topoisomerase IIalpha protein, as well as the kinases responsible for this modification, HeLa cells were labeled with [P]orthophosphate, and the topoisomerase IIalpha protein was immunoprecipitated with the isozyme-specific CRB antiserum. The topoisomerase IIalpha protein was then digested with trypsin, and the resulting phosphopeptides were separated in two dimensions on thin layer chromatography (TLC) plates.

Fig. 4shows a comparison of tryptic phosphopeptide maps for topoisomerase IIalpha 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(2)/M phase cells. A number of phosphopeptides that were either specific for or greatly enriched within the G(2)/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 IIalpha protein (Wells and Hickson, 1995). However, two of the most prominent G(2)/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 IIalpha protein during the G(2)/M phases of the cell cycle. Two-dimensional tryptic phosphopeptide maps of topoisomerase IIalpha protein extracted from asynchronously growing HeLa cells (a) or a culture enriched for G(2)/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(2)/M phase-specific phosphopeptides identified in previous studies (Wells and Hickson, 1995) are indicated by the open arrow heads. The two G(2)/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.



Purification of Recombinant Domains of Human Topoisomerase IIalpha Protein

In order to map phospho-acceptor residues, oligohistidine-tagged recombinant domains of topoisomerase IIalpha protein were overexpressed in E. coli, purified by nickel chelate affinity chromatography, and phosphorylated in vitro by different protein kinases. Phosphorylation of the central breakage/reunion domain from residues Lys-701 to Arg-1217 and the C-terminal domain that commenced at Glu-1178 and terminated at the natural stop codon (prepared as described by Wells et al., 1994) by protein kinase A, PKC, casein kinase II, p34, and MAP kinase failed to yield phosphopeptides that co-migrated with phosphopeptides A and B identified in Fig. 4(data not shown). Consequently, a portion of the N-terminal ATPase domain of topoisomerase IIalpha protein, representing residues Lys-25 to Lys-168, was expressed in E. coli and purified to homogeneity. An SDS-polyacrylamide gel of the recombinant polypeptide is shown in Fig. 5. This 18-kDa polypeptide had no intrinsic autophosphorylation activity but was a substrate in vitro for PKC betaI isoform (Fig. 5) and for PKC alpha and (data not shown).


Figure 5: The 18-kDa N-terminal domain of human topoisomerase IIalpha 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 PKCbetaI 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 IIalpha fragment. The position of the topoisomerase IIalpha protein fragment is indicated by the arrows. The sizes (in kDa) of molecular mass standards run in parallel are shown on the right.



The ATPase Domain of Topoisomerase IIalpha Protein Is Phosphorylated in Vivo

A representative two-dimensional tryptic phosphopeptide map of the 18-kDa N-terminal fragment phosphorylated in vitro by PKC is shown in Fig. 6b. Four prominent phosphopeptides were evident, two of which (Fig. 6b, 3 and 4) had a mobility similar to that of phosphopeptides A and B from the HeLa cell-derived topoisomerase IIalpha protein. To determine whether the PKC-specific sites in the 18-kDa fragment and the G(2)/M phase-specific sites were identical, two approaches were undertaken. First, the entire in vitro and in vivo phosphopeptide samples were mixed, and the mixture was separated in two dimensions on TLC plates. Fig. 6(a-c) shows that phosphopeptides 3 and 4 from the in vitro sample appeared to co-migrate with phosphopeptides A and B from the HeLa cell-derived sample. To confirm this co-migration, single phosphopeptides from the in vitro sample were excised from the chromatography plate and mixed with the entire in vivo sample prior to separation of the mixtures on TLC plates as before. Fig. 6d shows that phosphopeptide 3 co-migrated with phosphopeptide A from the in vivo sample. The 18-kDa fragment was not phosphorylated efficiently in vitro by any of a range of other purified protein kinases tested, in particular those previously implicated in regulating topoisomerase II activity (Sahyoun et al., 1986; Ackerman et al., 1988; Saijo et al., 1990; Cardenas et al., 1992; Wells et al., 1994; Wells and Hickson, 1995), including casein kinase II, p34 kinase, MAP kinase, and Ca/calmodulin-dependent protein kinase (data not shown).


Figure 6: The G(2)/M phase-specific phosphopeptides A and B are derived from the N-terminal 18-kDa fragment of topoisomerase IIalpha protein. a, two-dimensional tryptic phosphopeptide map of topoisomerase IIalpha protein extracted from G(2)/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 PKCbeta1. 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.



Site-directed Mutagenesis of the cDNA Encoding the 18-kDa Fragment of Topoisomerase IIalpha Protein

The amino acid sequence of the 18-kDa protein contains a number of potential serine phospho-acceptor residues, although only one, serine-29, lies in a sequence context closely matching the consensus for a recognition site for PKC ((R/K)(R/K)XS). Site-directed mutagenesis of the cDNA encoding the 18-kDa protein was carried out to confirm that serine-29 was the phospho-acceptor residue that gave rise to phosphopeptides 3 and 4. Fig. 7shows that a two-dimensional tryptic phosphopeptide map generated by PKC-mediated phosphorylation of the purified 18-kDa protein containing a single amino acid substitution replacing serine-29 with alanine lacked both phosphopeptides 3 and 4.


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 PKCbetaI 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 IIalpha 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 PKCbetaI. c, a 1:1 mix of the samples in a and b. The positions of phosphopeptides 2 and 3 are indicated by numbered arrows.




DISCUSSION

We have identified serine-29 as a site of phosphorylation of topoisomerase IIalpha 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(2)/M phases of the cell division cycle. Further, we have shown that phosphorylation by PKC substantially increases the catalytic activity of purified topoisomerase IIalpha 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 betaII 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(2)/M phase transition in at least some cell types, and the expression of the betaII 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 betaII 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 IIalpha 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 IIalpha 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 IIalpha 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 IIalpha 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 IIalpha 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 IIalpha (Wells et al., 1994). The data presented here indicate both that the phosphorylation of serine-29 of human topoisomerase IIalpha 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 IIalpha protein might perform. Considering the G(2)/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 IIalpha protein. Indeed, the extent to which PKC can activate topoisomerase IIalpha 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(2) 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 IIalpha 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 IIalpha 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 IIalpha, serine-29 lies in a sequence context that comprises predominantly basic amino acids. This sequence motif is conserved in all mammalian topoisomerase IIalpha enzymes. Moreover, this motif is also conserved in the beta isozyme of human topoisomerase II. Whether this indicates that topoisomerase IIbeta 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 IIalpha and have identified a serine residue in the ATPase domain of the protein that is phosphorylated specifically during the G(2) 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 IIalpha protein and that appear to require the action of at least two distinct kinases (Wells and Hickson(1995) and this work).


FOOTNOTES

*
This work was supported by funds from the Imperial Cancer Research Fund (to N. J. W., A. M. F., F. G., and I. D. H.) and the Human Frontier Science Program (to C. N.). 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: Swiss Inst. for Experimental Cancer Research, CH-1066 Epalinges, Switzerland.

To whom correspondence should be addressed. Tel.: 44-1865-222417; Fax: 44-1865-222431; hickson@icrf.icnet.uk.

(^1)
The abbreviations used are: MAP, mitogen-activated protein; PKC, protein kinase C; TLC, thin layer chromatography.


ACKNOWLEDGEMENTS

We thank Dr. Peter Parker for the purified PKC proteins and Elizabeth Clemson and Ann-Marie Doherty for typing the manuscript.


REFERENCES

  1. Ackerman, P., Glover, C. V. C., and Osheroff, N. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3164-3168 [Abstract]
  2. Ackerman, P., Glover, C. V., and Osheroff, N. (1988) J. Biol. Chem. 263, 12653-12660 [Abstract/Free Full Text]
  3. Austin, C. A., Sng, J.-H., Patel, S., and Fisher, L. M. (1993) Biochim. Biophys. Acta 1172, 283-291 [Medline] [Order article via Infotrieve]
  4. Burden, D. A., and Sullivan, D. M. (1994) Biochemistry 33, 14651-14655 [Medline] [Order article via Infotrieve]
  5. Burden, D. A., Goldsmith, L. J., and Sullivan, D. M. (1993) Biochem. J. 293, 297-304 [Medline] [Order article via Infotrieve]
  6. Campbell, D. G., Hardie, D. G., and Vulliet, P. R. (1986) J. Biol. Chem. 261, 10489-10492 [Abstract/Free Full Text]
  7. Cardenas, M. E., and Gasser, S. M. (1993) J. Cell Sci. 104, 219-225 [Free Full Text]
  8. Cardenas, M. E., Dang, Q., Glover, C. V. C., and Gasser, S. M. (1992) EMBO J. 11, 1785-1796 [Abstract]
  9. Caron, P. R., and Wang, J. C. (1994) in Advances in Pharmacology: DNA Topoisomerases and Their Pharmacology , pp. 79-89, Academic Press, New York
  10. Chung, T. D. Y., Drake, F. H., Tan, K. B., Per, S. R., Crooke, S. T., and Mirabelli, C. K. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9431-9435 [Abstract]
  11. Clemens, M. J., Trayner, I., and Menaya, J. (1992) J. Cell Sci. 103, 881-887 [Free Full Text]
  12. Corbett, A. H., DeVore, R. F., and Osheroff, N. (1992) J. Biol. Chem. 267, 20513-20518 [Abstract/Free Full Text]
  13. Corbett, A. H., Hong, D., and Osheroff, N. (1993a) J. Biol. Chem. 268, 14394-14398 [Abstract/Free Full Text]
  14. Corbett, A. H., Fernald, A. W., and Osheroff, N. (1993b) Biochemistry 32, 2090-2097 [Medline] [Order article via Infotrieve]
  15. DeVore, R. F., Corbett, A. H., and Osheroff, N. (1992) Cancer Res. 52, 2156-2161 [Abstract]
  16. DiNardo, S., Voelkel, K., and Sternglanz, R. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 2616-2620 [Abstract]
  17. Drake, F. H., Zimmerman, J. P., McCabe, F. L., Bartus, H. F., Per, S. R., Sullivan, D. M., Ross, W. E., Mattern, M. R., Johnson, R. K., Crooke, S. T., and Mirabelli, C. K. (1987) J. Biol. Chem. 262, 16739-16747 [Abstract/Free Full Text]
  18. Drake, F. H., Hofmann, G. A., Bartus, H. F., Mattern, M. R., Crooke, S. T., and Mirabelli, C. K. (1989) Biochemistry 28, 8154-8160 [Medline] [Order article via Infotrieve]
  19. Ganapathi, R., Zwelling, L., Constantinou, A., Ford, J., and Grabowski, D. (1993) Biochem. Biophys. Res. Commun. 192, 1274-1280 [CrossRef][Medline] [Order article via Infotrieve]
  20. Goss, V. L., Hocevar, B. A., Thompson, L. J., Stratton, C. A., Burns, D. J., and Fields, A. P. (1994) J. Biol. Chem. 269, 19074-19080 [Abstract/Free Full Text]
  21. Goto, T., and Wang, J. C. (1984) Cell 36, 1073-1080 [Medline] [Order article via Infotrieve]
  22. Holm, C. (1994) Cell 77, 955-957 [Medline] [Order article via Infotrieve]
  23. Holm, C., Goto, T., Wang, J. C., and Botstein, D. (1985) Cell 41, 553-563 [Medline] [Order article via Infotrieve]
  24. Holm, C., Stearns, T., and Botstein, D. (1989) Mol. Cell. Biol. 9, 159-168 [Medline] [Order article via Infotrieve]
  25. Jenkins, J. R., Ayton, P., Jones, T., Davies, S. L., Simmons, D. L., Harris, A. L., Sheer, D., and Hickson, I. D. (1992) Nucleic Acids Res. 5587-5592
  26. Kimura, K., Nozaki, N., Saijo, M., Kikuchi, A., Ui, M., and Enomoto, T. (1994) J. Biol. Chem. 269, 24523-24526 [Abstract/Free Full Text]
  27. Kroll, D. J., and Rowe, T. C. (1991) J. Biol. Chem. 266, 7957-7961 [Abstract/Free Full Text]
  28. Kuang, J., and Ashorn, C. L. (1993) J. Cell Biol. 123, 859-868 [Abstract]
  29. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  30. Levin, D. E., Fields, F. O., Kunisawa, R., Bishop, J. M., and Thorner, J. (1990) Cell 62, 213-224 [Medline] [Order article via Infotrieve]
  31. Miller, K. G., Liu, L. F., and Englund, P. T. (1981) J. Biol. Chem. 256, 9334-9339 [Abstract/Free Full Text]
  32. Morgan, D. O. (1995) Nature 374, 131-134 [CrossRef][Medline] [Order article via Infotrieve]
  33. Murray, A. W. (1992) Nature 359, 599-604 [CrossRef][Medline] [Order article via Infotrieve]
  34. Murray, N. R., Baumgardner, G. P., Burns, D. J., and Fields, A. P. (1993) J. Biol. Chem. 268, 15847-15853 [Abstract/Free Full Text]
  35. Nigg, E. A. (1993) Trends Cell Biol. 3, 296-301 [CrossRef]
  36. Norbury, C., and Nurse, P. (1992) Annu. Rev. Biochem. 61, 441-470 [CrossRef][Medline] [Order article via Infotrieve]
  37. Osheroff, N., Shelton, E. R., and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9536-9543 [Abstract/Free Full Text]
  38. Osheroff, N., Zechiedrich, E. L., and Gale, K. C. (1991) Bioessays 13, 269-275 [Medline] [Order article via Infotrieve]
  39. Rose, D., and Holm, C. (1993) Mol. Cell. Biol. 13, 3445-3455 [Abstract]
  40. Rottman, M., Schroder, H. C., Gramzow, M., Renneisen, K., Kurelec, B., Dorn, A., Friese, U., and Muller, W. E. G. (1987) EMBO J. 6, 3939-3944 [Abstract]
  41. Sahyoun, N., Wolf, M., Besterman, J., Hsieh, T.-S., Sander, M., LeVine, H., III, Chang, K.-J., and Cuatrecasas, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1603-1607 [Abstract]
  42. Saijo, M., Enomoto, T., Hanaoka, F., and Ui, M. (1990) Biochemistry 29, 583-90 [Medline] [Order article via Infotrieve]
  43. Saijo, M., Ui, M., and Enomoto, T. (1992) Biochemistry 31, 359-363 [Medline] [Order article via Infotrieve]
  44. Shiozaki, K and Yanagida, M. (1992) J. Cell Biol. 119, 1023-1036 [Abstract]
  45. Smith, P. J., and Makinson, T. A. (1989) Cancer Res. 49, 1118-1124 [Abstract]
  46. Spell, R. M., and Holm, C. (1994) Mol. Cell. Biol. 14, 1465-1476 [Abstract]
  47. Strausfeld, U., and Richter, A. (1989) Prep. Biochem. 19, 37-48 [Medline] [Order article via Infotrieve]
  48. Taagepera, S., Rao, P. N., Drake, F. H., and Gorbsky, G. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8407-8411 [Abstract/Free Full Text]
  49. Tan, K. B., Dorman, T. E., Falls, K. M., Chung, T. D. Y., Mirabelli, C. K., Crooke, S. T., and Mao, J. (1992) Cancer Res. 52, 231-234 [Abstract]
  50. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354 [Abstract]
  51. Tsai-Pflugfelder, M., Liu, L. F., Liu, A. A., Tewey, K. M., Whang-Peng, J., Knutsen, T., Huebner, K., Croce, C. M., and Wang, J. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7177-7181 [Abstract]
  52. Uemura, T., and Yanagida, M. (1984) EMBO J. 3, 1737-1744 [Abstract]
  53. Uemura, T., and Yanagida, M. (1986) EMBO J. 5, 1003-1010
  54. Uemura, T., Ohkura, H., Adachi, Y., Morino, K., Shiozaki, K., and Yanagida, M. (1987) Cell 50, 917-925 [Medline] [Order article via Infotrieve]
  55. Usui, T., Yoshida, M., Abe, K., Osada, H., Isono, K., and Beppu, T. (1991) J. Cell Biol. 115, 1275-1282 [Abstract]
  56. Wang, J. C. (1985) Annu. Rev. Biochem. 54, 665-697 [CrossRef][Medline] [Order article via Infotrieve]
  57. Watt, P., and Hickson, I. D. (1994) Biochem. J. 303, 681-695 [Medline] [Order article via Infotrieve]
  58. Wells, N. J., and Hickson, I. D. (1995) Eur. J. Biochem. 231, 491-497 [Abstract]
  59. Wells, N. J., Addison, C. M., Fry, A. M., Ganapathi, R., and Hickson, I. D. (1994) J. Biol. Chem 269, 29746-29751 [Abstract/Free Full Text]
  60. Westendorf, J. M., Rao, P. N., and Gerace, L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 714-718 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.