Casein Kinase II Stabilizes the Activity of Human Topoisomerase IIalpha in a Phosphorylation-independent Manner*

Charles Redwood, Sally L. Davies, Nicholas J. WellsDagger , Andrew M. Fry§, and Ian D. Hickson

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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Previous reports have indicated that topoisomerase II (topo II) co-purifies with and is a substrate for casein kinase II. We have carried out a detailed study of the effect that purified casein kinase II has on the activity of purified recombinant human topo IIalpha . Co-incubation of topo IIalpha and casein kinase II led to an apparent activation of the topo IIalpha ; however, in experiments in which topo IIalpha was preincubated at 37 °C with or without native casein kinase II prior to assaying for decatenation activity, it emerged that the kinase was exerting its "activating" function via a decrease in the rate of topo IIalpha enzyme inactivation during the incubation period. This stabilization of topo IIalpha by casein kinase II was ATP-independent and was observed in both mutated and truncated derivatives of topo IIalpha lacking the major casein kinase II phospho-acceptor sites, indicating the lack of a requirement for phosphorylation. Consistent with a nonenzymatic role for casein kinase II, stoichiometric quantities of kinase were required for topo IIalpha stabilization. These data indicate that casein kinase II plays a significant role in regulating human topo IIalpha protein action via stabilization against thermal inactivation.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

DNA topo1 II is a ubiquitous and structurally conserved enzyme required for many different aspects of DNA metabolism (reviewed in Refs. 1 an 2). Studies in bacterial and lower eukaryotic species have revealed that topo II is required for cell viability due to an essential role in the segregation of newly replicated chromosomes during cell division (3-7). During replication, sister DNA molecules become knotted and catenated as a consequence of the difficulties inherent in the unwinding and copying of a double-stranded, helical nucleic acid structure (reviewed in Ref. 8). In eukaryotes, topo II catalyzes the disentanglement of sister chromatids and hence permits their faithful segregation during mitosis. Studies on yeast temperature-sensitive top2 mutants have shown that both chromosome hypercondensation and sister chromatid segregation are dependent upon functional topo II, and that in the absence of topo II chromosomes break or nondisjoin as the mitotic spindle attempts to pull apart the still interlinked DNA molecules (3-8).

In mammalian cells, but not in yeasts, topo II exists as two closely related isoforms designated alpha  (170-kDa form) and beta  (180-kDa form) (9-13). The respective roles of the two isoforms are not known currently, although several differences in their activities/regulation have been noted. For example, the alpha  isoform is a strict proliferation marker in vitro and in vivo and is expressed at a high level in each cell cycle during the period just prior to cell division (14-17). This isoform is tightly associated with condensed chromatin during metaphase. In contrast, topo IIbeta is ubiquitously expressed in human tissues and is apparently excluded from chromosomes around the time of mitotic chromosome condensation, although it is found in the nucleoplasm during interphase (14-18).

Topo II is a phosphoprotein in cells from both higher and lower eukaryotes (reviewed in Ref. 1). In those species that have been analyzed, the extent of this phosphorylation is modulated as cells traverse the different phases of the cell division cycle. In particular, topo IIalpha in mammalian cells is hyperphosphorylated during mitosis as a result of the actions of at least two different protein kinases (19-23). However, the precise functional role of these cell cycle-specific modifications is not clear at this stage.

The kinase most closely associated with the regulation of topo II is casein kinase II. Topo II from Drosophila, budding and fission yeast, as well as from mammalian cells, is a high affinity substrate for casein kinase II in vitro (24-29). Several studies have confirmed that this kinase also phosphorylates topo II in intact cells, primarily on sites located within the poorly conserved, noncatalytic C-terminal domain (25, 28-30). The fission yeast topo II is also phosphorylated on a site in the N-terminal ATPase domain by casein kinase II (28). To date, three sites have been identified within the C-terminal domain of human topo IIalpha that are phosphorylated by casein kinase II in vivo (29, 31). None of these sites is modified differentially during cell cycle transit, and only one, serine 1524 in humans, is highly conserved in all of the mammalian topo IIalpha enzymes. This residue is a major site of phosphorylation in several different human cell lines studied (29). Of the other two sites, serine 1376 is conserved in mouse and rat topo IIalpha , but its flanking sequence is not, while threonine 1342 is not conserved, being replaced by an aspartate in the rodent enzymes (32, 33).

In this study, we have investigated the role played by casein kinase II in regulating the activity of human topo IIalpha . We show that co-incubation of topo IIalpha with casein kinase II strongly influences the ability of topo IIalpha to decatenate kinetoplast DNA. We present evidence that casein kinase II is able to stabilize topo IIalpha against inactivation during incubation at 37 °C, and that this effect is not dependent upon phosphorylation of topo IIalpha .

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Site-directed Mutagenesis-- The topo IIalpha cDNA was mutated using the single-stranded oligonucleotide-directed mutagenesis method of Kunkel et al. (34), as described in Wells and Hickson (22). An AvrII/XhoI fragment containing the 3' end of the topo IIalpha cDNA (12) was subcloned into pBluescript and a single-stranded uracil-containing template generated using the Escherichia coli strain CJ236. For the mutation of serine 1376 to alanine (S1376A) the oligonucleotide 5'-TTCAAGGTCTGCCACGACACTTTT-3' was used; for the mutation of serine 1524 to alanine (S1524A) the oligonucleotide 5'-ACCAGTCTTGGGGCTGGTAAGATA-3' was used; for the truncation at amino acid 1178 the oligonucleotide 5'-TTAAACAGCCTACAATTCTTCAAT-3' was used. The double phosphorylation site mutant was generated using the S1376A and S1524A oligonucleotides simultaneously. Double-stranded mutated pBluescript constructs were verified by dideoxy DNA sequencing. AvrII/XhoI fragments containing the mutated phospho-acceptor residues were then subcloned into pYEpWob6 previously digested with the same enzymes.

Overexpression and Purification of Human Topo IIalpha Enzymes-- The expression construct pYEpWob6 (35) was the basis for the production of full-length and truncated human topo IIalpha proteins. Full-length, mutated, and truncated proteins were overexpressed in Saccharomyces cerevisiae and lysates were prepared by freeze/thawing and shearing cells with glass beads (35). Soluble proteins were then loaded onto an hydroxyapatite (Ultragel) column, and the topo IIalpha protein was eluted using a 200-600 mM NaCl gradient. Peak fractions were pooled and loaded onto a 1-ml HiTrapSP column (Pharmacia Biotech Inc.), and bound proteins were eluted using a 200 mM to 1 M NaCl gradient. Peak fractions, containing >95% pure topo IIalpha , as determined by Coomassie Blue staining of SDS-polyacrylamide gels, were adjusted to 50% glycerol and stored at -70 °C.

Purification of Casein Kinase II from Rabbit Skeletal Muscle-- All procedures were carried out at 4 °C. The tissue was homogenized in 2.5 volumes of 4 mM EDTA, 15 mM 2-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine at pH 7.0. Following centrifugation at 10,000 rpm for 10 min, the supernatant was collected and adjusted to pH 7.5 with 15 M NH4OH. Solid (NH4)2SO4 was added slowly with stirring to 33% saturation. The precipitate was then removed by centrifugation at 10,000 rpm for 20 min. Further (NH4)2SO4 was added to the supernatant to 55% saturation. After precipitation at 10,000 rpm for 10 min, the pellet was resuspended in buffer A (25 mM Tris-HCl, 1 mM EDTA, 15 mM 2-mercaptoethanol, pH 7.5) and dialyzed overnight against buffer A. The dialysate was applied to a phosphocellulose column (Whatman P-11) equilibrated with buffer A, the column was washed in buffer A containing 400 mM NaCl and eluted with buffer A containing M NaCl. The eluate was diluted with 4 volumes of buffer A and applied to a HiTrap Heparin column preequilibrated with buffer A. After washing with buffer A containing 400 mM NaCl, the bound proteins were eluted with buffer A containing 1 M NaCl. The casein kinase II-rich fractions were dialyzed against buffer A, before loading onto a fast protein liquid chromatography Mono Q column equilibrated in buffer A. The column was washed with buffer A plus 400 mM NaCl and eluted with a linear gradient of buffer A plus 1 M NaCl. Casein kinase II activity, as determined by SDS-PAGE and autophosphorylation assays, eluted from the column at 700 mM NaCl. The active fractions were pooled, dialyzed against 20 mM Tris-HCl, pH 7.5, 350 mM NaCl, 1 mM EDTA, and 15 mM 2-mercaptoethanol and stored in aliquots at -70 °C. The casein kinase II was judged to be >90% pure by Coomassie Blue-stained SDS-polyacrylamide gels.

Phosphorylation of Topo IIalpha -- Phosphorylation of topo IIalpha was typically carried out at 30 °C for 15 min using 2 µg of topo IIalpha and 0.4 µg of casein kinase II in a final volume of 20 µl of kinase buffer (20 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2) supplemented with either 1 mM ATP or 110 µM [gamma -32P]ATP (final specific activity >23 TBq/mmol). PKI, the peptide inhibitor of protein kinase A, and H-7 were obtained from Sigma.

Determination of the Stoichiometry of Phosphorylation-- 12 µg of wild-type topo IIalpha were incubated with 2.4 µg of casein kinase II and 110 µM [gamma -32P]ATP in a total volume of 140 µl of kinase buffer at 30 °C. 20-µl aliquots were taken at 0, 2, 5, 10, 20, and 30 min, and the reaction was stopped by the addition of 10 µl of SDS gel loading buffer. The samples were separated by SDS-PAGE, and the amount of phosphate incorporated into the topo II band on the dried gel was quantitated using a Molecular Dynamics PhosphoImager model 425.

Phosphopeptide Mapping of Topo IIalpha -- This procedure was carried out as described by Wells et al. (29).

DNA Topo II Assays-- DNA topo II activity was assayed by measuring the decatenation of kDNA (Topogen). The standard mix for a 20-µl reaction consisted of 50 mM Tris-HCl, pH 7.9, 85 mM KCl, 10 mM MgCl2, 0.5 mM EDTA, 10 mM ATP, 1 mM dithiothreitol, 20 µg/ml bovine serum albumin, and 240 ng of kDNA. The reaction mixture was incubated at 30 °C, and at timed intervals the reaction was stopped by the addition of 5 µl of stop solution (5% SDS, 25% Ficoll, and 0.05% bromphenol blue). The samples were loaded onto a 1% agarose gel in Tris borate EDTA buffer. After electrophoresis, the gel was stained with ethidium bromide and photographed under UV illumination.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Co-purification of a Kinase with Topo IIalpha from HeLa Cell Nuclear Extracts-- Our initial premise was that because casein kinase II phosphorylates human topo IIalpha in vivo, it was very likely to play a role in the regulation of topo IIalpha function. Moreover, a kinase with properties very similar to those of casein kinase II has been reported to co-purify with topo II in extracts from Drosophila, mouse and yeast cells (26, 27, 36). To address whether this was also the case for human topo IIalpha , we tested the highly purified topo IIalpha preparations from HeLa cell nuclei that we have described previously (23) for kinase activity. Fig. 1 shows that a kinase capable of phosphorylating topo IIalpha (as well as casein; data not shown) co-purifies with topo IIalpha from HeLa nuclei. Next, we addressed whether this copurifying kinase displayed two of the features diagnostic of casein kinase II; sensitivity to heparin and an ability to utilize GTP, as well as ATP, as a phosphate donor (reviewed in Ref. 37). As shown in Fig. 1, the co-purifying kinase was inhibited by heparin, but not by a specific peptide inhibitor of protein kinase A, or by H-7, the inhibitor of protein kinase C, cyclic nucleotide-dependent kinases, and myosin light chain kinase. Moreover, this kinase could use GTP as a phosphate donor (data not shown), suggesting that it was either casein kinase II or a very closely related enzyme. These data, together with those of previous studies, suggest that topo IIalpha may be an important target for casein kinase II in human cells.


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Fig. 1.   Human topo IIalpha is a substrate for a co-purifying, heparin-sensitive kinase. Purified human topo IIalpha was incubated with [gamma -32P]ATP in the presence or absence of known protein kinase inhibitors and then run on a 7.5% SDS-polyacrylamide gel. The gel was dried and subjected to autoradiography. The positions of molecular mass standards (in kDa) are shown on the left, and the position of the topo IIalpha protein is indicated on the right. The reaction was carried out with no inhibitor (lane 1), 10 µg/ml heparin (lane 2), 10 µM protein kinase A inhibitor peptide (lane 3), 20 µM H-7 (lane 4), or all three inhibitors (lane 5).

Purification of Recombinant Topo IIalpha Protein-- Using this information, we set out to analyze the role of casein kinase II in regulating topo IIalpha enzyme activity in vitro. However, using human nuclear extracts as a source of enzyme, it proved very difficult to separate topo IIalpha from the co-purifying kinase. As a consequence, we adopted an alternative approach exploiting the recent description of an overexpression system for recombinant human topo IIalpha using S. cerevisiae as a host (35). With this system, we prepared homogeneous human topo IIalpha from yeast extracts (see Fig. 6 below) using a new purification protocol (see "Experimental Procedures"). In addition, we purified casein kinase II to apparent homogeneity from rabbit skeletal muscle using an established protocol. We first confirmed that the recombinant topo IIalpha preparation lacked intrinsic kinase activity and that the topo IIalpha was a substrate in vitro for the purified casein kinase II (Fig. 2a). Analysis of this reaction showed that the stoichiometry of phosphorylation was approximately 2 moles of phosphate per mole of topo II monomer, even after extended incubation periods (Fig. 2b). Thus, we conclude that this yeast-based expression system provides a means to analyze the effects of casein kinase II on topo IIalpha activity without the compounding influence of a co-purifying kinase.


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Fig. 2.   Phosphorylation of purified, recombinant topo IIalpha by casein kinase II. a, full-length, recombinant topo IIalpha was incubated in the presence of [gamma -32P]ATP at 30 °C for 15 min in the absence (lane 1) or presence (lane 2) of casein kinase II. Proteins were separated by SDS-PAGE, and the phosphorylated topo IIalpha protein (170 kDa) was detected by autoradiography. The positions of molecular mass standards (in kDa) are shown on the left. b, time course of incorporation of 32P into topo IIalpha catalyzed by casein kinase II. Recombinant topo IIalpha was incubated with casein kinase II in the presence of [gamma -32P]ATP at 30 °C. Aliquots were taken at the times indicated, and the reaction was stopped by the addition of SDS gel loading buffer. The samples were separated by SDS-PAGE, and the amount of phosphate incorporated into the topo IIalpha band on the dried gel was quantified using a PhosphoImager.

Effect of Casein Kinase II on the Activity of Recombinant Topo IIalpha Protein-- We next tested whether the purified rabbit casein kinase II could influence the activity of the recombinant topo IIalpha protein. Fig. 3 shows that the purified human topo IIalpha displayed significantly higher activity in a decatenation assay following preincubation with casein kinase II than it did following an otherwise identical incubation in buffer containing either a control protein (BSA) or heat-inactivated casein kinase II. Thus, native casein kinase II is required for this activation of topo IIalpha . As a control, we confirmed that the casein kinase II preparation contained no contaminating topoisomerase activities (data not shown).


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Fig. 3.   Topo IIalpha enzyme activity is apparently higher following preincubation with casein kinase II. a, recombinant topo IIalpha was preincubated for 15 min at 37 °C with either 0.2 µg of casein kinase II (square  and top panel of b), 0.2 µg of casein kinase II inactivated by heating to 70 °C for 20 min (open circle  and middle panel of b), or 0.6 µg of BSA (triangle  and bottom panel of b). Following preincubation, samples were added to a decatenation assay from which samples were removed at timed intervals for analysis on an agarose gel. a represents the quantification of data from the mean of three independent experiments. The percent DNA decatenated was determined by densitometric scanning of photographic negatives of ethidium bromide-stained agarose gels illuminated under UV light. 100% was the amount of DNA decatenated in 15 min at 30 °C, as shown in b, lane 1. Representative gels are shown in b, with the positions of the substrate (k) and the product (m) indicated on the right. Lane 1, positive control with no preincubation. Lanes 2-8, time course of decatenation activity following a 15-min preincubation at 37 °C. Assay times were: 0 min (lane 2), 0.5 min (lane 3), 1 min (lane 4), 2 min (lane 5), 4 min (lane 6), 6 min (lane 7), and 8 min (lane 8).

We performed a number of experiments to define the mechanism by which casein kinase II could influence the activity of topo IIalpha during the short period of preincubation at 37 °C prior to performing the decatenation assay at 30 °C. During the course of these studies, we found that the activity of the topo IIalpha enzyme declined markedly during such a preincubation period in the absence of casein kinase II, but was largely unaffected when casein kinase II was included in the incubation mixture (Fig. 4a). However, it should be noted that in neither case did the level of topo IIalpha activity rise above that of enzyme that had not been preincubated at 37 °C prior to performing the decatenation assay. Moreover, the decline in activity seen following a period of preincubation at 37 °C was not observed if the preincubation temperature was lowered to 25 °C before performing a decatenation assay at 30 °C (Fig. 4b). Thus, the effect of casein kinase II was not to "activate" the topo IIalpha , but instead to maintain its activity at a relatively steady level during a period of incubation of 37 °C, presumably by preventing thermal inactivation. Hence, we will consider this to be stabilization rather than an activation of topo IIalpha by casein kinase II.


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Fig. 4.   Changes in topo IIalpha decatenation activity following preincubation. Relative decatenation activity of recombinant topo IIalpha (0.3 µg per reaction) following a preincubation at 37 °C (panel a) or 25 °C (panel b) in the presence of 0.2 µg of casein kinase II (open circle ) or in the presence of 0.6 µg BSA (square ) for the times indicated on the x axis. After preincubation, samples were assayed for decatenation activity at 30 °C for 15 min. Data represent the mean of three independent experiments

To address whether the effect of casein kinase II was to prevent inactivation of topo IIalpha during incubation at 37 °C, or to "renature" topo IIalpha that had already lost activity, we tested whether topo IIalpha inactivated by incubation at 37 °C in the absence of casein kinase II could be "reactivated" by subsequent addition of casein kinase II. Fig. 5 shows that, once inactivated, topo IIalpha could not be reactivated by casein kinase II, indicating that casein kinase II must be present during the preincubation period at 37 °C to exert its stabilizing effects.


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Fig. 5.   Casein kinase II cannot restore activity to heat-inactivated topo IIalpha . Recombinant topo IIalpha (0.3 µg per reaction) was preincubated at 37 °C for the times indicated. At intervals, aliquots were removed and added to a decatenation reaction either in the presence of 0.6 µg of BSA (square ) or in the presence of 0.2 µg casein kinase II (open circle ), and the incubation was continued for a further 15 min at 30 °C. Data represent the percent DNA decatenated as a function of time of preincubation at 37 °C.

Stabilization of Topo IIalpha by Casein Kinase II Is Independent of Phosphorylation and Requires Stoichiometric Amounts of Kinase-- Omission of ATP from the preincubation buffer had no effect on the ability of casein kinase II to stabilize topo IIalpha , suggesting a phosphorylation-independent mode of action (data not shown). Nevertheless, it was formally possible that the enzyme preparations themselves provided sufficient ATP to permit phosphorylation of topo IIalpha to take place during the preincubation period prior to performing the decatenation assay. However, to exclude a role for phosphorylation by casein kinase II, we studied mutated and truncated derivatives of recombinant topo IIalpha . We have shown previously that casein kinase II phosphorylates topo IIalpha in vitro primarily on two sites in the C-terminal domain (29). These sites, serine 1376 and serine 1524, are also phosphorylated in vivo (29). In addition, Ishida et al. (31) have shown recently that threonine 1342 is phosphorylated in human cell lines. Recombinant topo IIalpha proteins containing mutation of serine 1376 to alanine (S1376A), serine 1524 to alanine (S1524A), or a double mutant lacking both phospho-acceptor serine residues (designated topo IIalpha -SDM), were overexpressed in yeast and purified to apparent homogeneity (Fig. 6 and data not shown). In addition, a truncated derivative comprising residues 1-1178, which was designated topo IIalpha -CTT, was purified in a similar manner (Fig. 6). This truncated derivative lacks residues 1179-1530, which encompass the entire regulatory C-terminal domain of topo IIalpha and the three previously mapped phospho-acceptor sites discussed above.


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Fig. 6.   Purification of recombinant topo IIalpha proteins. a, a Coomassie Blue-stained SDS-polyacrylamide gel representing purified topo IIalpha -CTT (lane 1), full-length, wild-type topo IIalpha (lane 2), and the double mutant topo IIalpha -SDM (lane 3), as indicated on the right. The positions and sizes of the molecular mass standards (lane MWM) are shown on the left in kDa. b, diagrammatic representation of the recombinant proteins used in the study.

To confirm that these proteins behaved as expected with respect to phosphorylation by casein kinase II, two-dimensional tryptic phosphopeptide mapping was performed. Fig. 7a shows that digestion of wild-type topo IIalpha following phosphorylation by casein kinase II generated two predominant phosphopeptides, designated 1 and 2, which have been identified previously as being specific for serine 1524 and serine 1376, respectively (29). As expected, two-dimensional maps derived from the S1424A mutant protein lacked phosphopeptide 1 (Fig. 7d), and those from the S1376A protein lacked phosphopeptide 2 (Fig. 7c). Moreover, the topo IIalpha -SDM double mutant protein was only very poorly phosphorylated (0.13 mol of phosphate/mol of topo IIalpha monomer), and any specific radiolabel (i.e. that which migrated away from the origin) was present at a very low level (Fig. 7b), consistent with the notion that serine 1376 and serine 1524 are the major in vitro casein kinase II phosphorylation sites on topo IIalpha . In agreement with this, the truncated derivative of topo IIalpha (topo IIalpha -CTT) lacking the C-terminal domain, could not be phosphorylated by casein kinase II in vitro to an extent that permitted meaningful two-dimensional mapping analysis to be undertaken (stoichiometry of 0.02 mol of phosphate/mol of topo IIalpha monomer). Taken together, these data are also consistent with the results shown in Fig. 2 indicating a stoichiometry of phosphorylation of 2 mol of phosphate/mol of wild-type topo IIalpha monomer.


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Fig. 7.   Two-dimensional tryptic phosphopeptide maps of recombinant topo IIalpha proteins phosphorylated in vitro by casein kinase II. a, full-length wild-type topo IIalpha ; b, topo IIalpha -SDM; c, topo IIalpha carrying a mutation of serine 1376 to alanine; and d, topo IIalpha containing a mutation of serine 1524 to alanine. Peptides were separated horizontally by electrophoresis at pH 1.9 and vertically by chromatography. The positions of phosphopeptides 1 (containing serine 1524) and phosphopeptide 2 (containing serine 1376), as well as the origin (o), are indicated.

To address whether casein kinase II was having a differential effect on the activity of the mutated/truncated versions of topo IIalpha in comparison with that of the wild-type enzyme, we analyzed the decatenation activity of these proteins following a 15-min preincubation period at 37 °C in the presence or absence of casein kinase II. As a preliminary step in this analysis, we determined the quantity of each of the topo II enzymes required to give an equivalent level of decatenation activity. Approximately equal levels of decatenation activity were exhibited by 0.34, 0.21, and 0.13 µg of the wild-type topo IIalpha , the topo IIalpha -SDM protein, and the truncated topo IIalpha -CTT protein, respectively. Following equalization of intrinsic decatenation activity, the effect of casein kinase II on the activity of these three topo IIalpha preparations was compared. Fig. 8 shows that casein kinase II was able to stabilize the activity of all three of these proteins, indicating strongly that phosphorylation of residues of the C-terminal domain of topo IIalpha (or indeed elsewhere in the protein, since the reaction was ATP-independent and the topo IIalpha -CTT protein was not a substrate for casein kinase II in vitro) is not required for this stabilization of topoisomerase activity. It should be noted that we consistently observed a greater degree of resistance to inactivation by the truncated topo IIalpha -CTT enzyme than by any of the full length versions of this protein. Nevertheless, it is clear that all of the mutated/truncated topo IIalpha derivatives behave qualitatively in a very similar fashion to that of the wild-type topo IIalpha .


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Fig. 8.   Stabilization of truncated and mutant topo IIalpha protein activities by preincubation with casein kinase II. Full-length recombinant topo IIalpha (a), topo IIalpha -SDM (b), and topo IIalpha -CTT (c), equalized for intrinsic decatenation activity, were incubated for 15 min at 37 °C with either 0.2 µg of casein kinase II (square ), 0.2 µg of casein kinase II inactivated by heating to 70 °C (open circle ), or 0.6 µg of BSA (triangle ). After this preincubation, the mixture was added to a decatenation assay, from which samples were removed at timed intervals, as indicated on the x axis, and the reaction products were analyzed on agarose gels.

Since we were unable to identify any role for phosphorylation by casein kinase II in the stabilization phenomenon, we considered whether the kinase might be acting in a stoichiometric manner rather than enzymatically. Fig. 9 shows that detectable stabilization was observed only when the molar ratio of casein kinase II to topo II rose to at least 0.3 and that full stabilization required equimolar levels of kinase and topoisomerase.


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Fig. 9.   Casein kinase II is required in stoichiometric amounts to exert its stabilizing effects. Full-length recombinant topo IIalpha (0.34 µg per reaction) was preincubated for 15 min at 37 °C, together with different concentrations of casein kinase II, prior to performing a 15-min decatenation assay at 30 °C. A molar protein ratio of 1:1 represents 0.34 µg of homodimeric topo IIalpha and 0.2 µg of heterotetrameric casein kinase II.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We have shown that casein kinase II co-purifies with and strongly influences the activity of human topo IIalpha . This activation of topo IIalpha is apparently independent of any phosphorylation and does not require sequences present in the regulatory C-terminal domain of the enzyme. Although casein kinase II treatment increases the activity of topo IIalpha to a level above that of mock-treated enzyme, this does not represent true activation, in that the role of the kinase appears to be to stabilize topo IIalpha and hence prevent a decline in activity that normally occurs during a period of incubation at 37 °C. We would suggest that this effect on enzyme activity might be widespread among nuclear enzymes and therefore important for chromosome function, since casein kinase II has been found in association with a wide variety of other enzymes important for different aspects of DNA metabolism (see below). Of possible significance in this regard is the observation that that the beta  subunit of casein kinase II confers thermal stability upon casein kinase IIalpha subunits, in which the catalytic activity resides (38).

How might casein kinase II effect its role as an enzyme stabilizer? The most obvious mechanism is via direct association with topo IIalpha . Our data showing the co-purification of topo IIalpha with casein kinase II and the requirement for stoichiometric amounts of casein kinase II to effect stabilization of topo IIalpha enzyme activity, are consistent with this suggestion. Moreover, Bojanowski et al. (39) have shown that the beta  subunit of casein kinase II interacts with topo II from budding yeast. However, despite extensive efforts, utilizing a variety of different methodologies including immunoprecipitation and chemical cross-linking, we have been unable to "trap" a stable complex containing pure human topo IIalpha with casein kinase II. This does not rule out the possibility that such an association occurs under certain conditions, or that the association is transient in nature. Casein kinase II is known to associate with a number of its other substrates, including topo I, p53, and protein phosphatase 2A (40-42). In addition, casein kinase II has been shown to stimulate topo I activity via a direct physical association (40).

Recently, Kimura et al. (43) reported that casein kinase II has no effect on the activity of mouse topo IIalpha . Further, they concluded that their previously reported results (27) showing a stimulation of topo IIalpha activity by casein kinase II could not be attributed to the kinase, but to the composition of the incubation buffer. Specifically, incubation in buffers containing low, but not high, concentrations of glycerol was stimulatory, and this effect was dependent upon topo IIalpha being present at a high concentration. Our results are partially in agreement with those of Kimura et al. (43) in that we could not identify a role for phosphorylation per se in the regulation of topo IIalpha activity by casein kinase II. However, our findings that native casein kinase II is a necessary component of the buffer, and that denatured casein kinase II is not active in this regard, are not consistent with their conclusions. Moreover, we have been unable to reproduce the stimulatory effects of manipulating the glycerol concentration alone, reported by Kimura et al. (43). The reason(s) for these discrepancies is not apparent at this stage.

We have shown that the C-terminal domain of topo IIalpha is not important for catalytic activity in vitro, and that phosphorylation of target residues within this domain is not required for the regulation of that activity by casein kinase II. It would be predicted, therefore, that a failure to phosphorylate these residues would not alter the known ability of the topo IIalpha cDNA to rescue temperature-sensitive yeast top2-4 mutants at the restrictive temperature (35, 44). Consistent with this prediction, we have shown that cDNAs carrying mutations of serine 1524 and/or serine 1376 to alanine will fully complement top2-4 strains.2 It is conceivable that the role of phosphorylation of these residues is normally not to regulate catalytic activity, but instead to influence subcellular or subnuclear localization in human cells, as has been shown for fission yeast topo II (28). Our data showing that residues 1179-1530 are dispensible for the catalytic activity of human topo IIalpha in vitro are consistent with previous analyses on truncated versions of the S. cerevisiae and Drosophila topo II proteins (45, 46). The possibility that phosphorylation of sites outside the C-terminal domain may influence the stability of the enzyme can be discounted, since the truncated protein, which was shown to incorporate only 0.02 mol of phosphate/mol of topo IIalpha monomer, is stabilized by casein kinase II in a fashion similar to that of the wild-type enzyme.

Our work and that of Kimura et al. (43) on mouse topo IIalpha have shown no direct effect on topo IIalpha activity of phosphorylation by casein kinase II. In addition, in both studies stoichiometric quantities of kinase were necessary to give efficient phosphorylation. This is in contrast to reports of the effect of casein kinase II phosphorylation on topo II activity from lower eukaryotes. Topo II from Drosophila is a high affinity substrate for casein kinase II both in vitro and in vivo and is fully phosphorylated using substoichiometric quantities of kinase (24, 30). The phosphorylated enzyme has an enhanced rate of ATP hydrolysis and a 3-fold higher plasmid relaxation activity compared with that of the dephosphorylated enzyme, effects that are fully reversed by dephosphorylation of the enzyme (24, 30, 47). Similarly, S. cerevisiae topo II is readily phosphorylated by casein kinase II, resulting in a greater than 10-fold stimulation of decatenation activity (36). In Drosophila and yeast there is a single gene encoding topo II, whereas in mammalian cells there are two closely related, but genetically distinct, isoforms. Thus it is possible that higher eukaryotes have evolved a mechanism for differentially regulating the two topo II isoforms.

Gasser and co-workers (36, 48) have reported that casein kinase II not only reactivates dephosphorylated yeast topo IIalpha , but also influences the extent of enzyme multimerization. Although it remains to be confirmed that higher order (greater than dimeric) forms of topo II have any functional significance in eukaryotic cells, it is formally possible that the regulatory phenomenon that we have observed reflects an ability of casein kinase II to prevent topo IIalpha from adopting a subunit composition that exhibits little or no catalytic activity. Further work is required to ascertain whether or not this suggestion is true.

The primary role of topo IIalpha in human cells is almost certainly to act during mitosis. It would appear that the level and activity of this enzyme is very tightly controlled by a combination of features acting at different levels. Thus, the mRNA is virtually absent from G0/G1 cells and only accumulates to high levels during late S phase (14-16, 49). The topo IIalpha protein peaks later, in G2/M, and is phosphorylated by at least two different kinases during mitosis (14, 22, 23). Indeed, it would appear that topo IIalpha activity is strongly activated only during a narrow time window when its decatenase function is required for mitotic chromosome condensation/segregation. Casein kinase II might contribute to this tight regulation at (at least) two levels; either via modulation of intrinsic topo IIalpha enzyme stability, or via its known ability to modulate the action of other kinases/phosphatases required for mitosis, including protein phosphatase 2A and the universal mitotic controller, p34cdc2 (42) (reviewed in Ref. 37). It might be significant that both casein kinase II and topo IIalpha are among the proteins that are phosphorylated during mitosis, leading to the generation of the phosphoepitope recognized by the MPM-2 antibody (50, 51).

In summary, we have shown that the activity of topo IIalpha is maintained during an incubation at 37 °C by the action of casein kinase II in a manner that is independent of phosphorylation. The challenge is now to delineate the functional significance of this regulation in vivo and to address whether casein kinase II plays a widespread role in regulating the stability of other important nuclear enzymes.

    ACKNOWLEDGEMENTS

We thank members of the ICRF Molecular Oncology Laboratories for helpful discussions, Dr. J. C. Wang for pYEpWob6, and Dr. C. Norbury for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by the Imperial Cancer Research Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Current address: Molecular Biology and Virology Laboratory, The Salk Institute, San Diego, CA 92138.

§ Current address: Dept. of Molecular Biology, University of Geneva, CH-1211 Geneva 4, Switzerland.

To whom correspondence should be addressed. Tel.: 44 1865 222417; Fax: 44 1865 222431; E-mail: hickson{at}icrf.icnet.uk.

1 The abbreviations used are: topo, topoisomerase; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin.

2 C. Redwood, S. L. Davies, N. J. Wells, A. M. Fry, and I. D. Hickson, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
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