Mitosis-specific MPM-2 phosphorylation of DNA topoisomerase IIα is regulated directly by protein phosphatase 2A

Abstract

Recent results suggest a role for topoIIα (topoisomerase IIα) in the fine-tuning of mitotic entry. Mitotic entry is accompanied by the formation of specific phosphoepitopes such as MPM-2 (mitotic protein monoclonal 2) that are believed to control mitotic processes. Surprisingly, the MPM-2 kinase of topoIIα was identified as protein kinase CK2, otherwise known as a constitutive interphase kinase. This suggested the existence of alternative pathways for the creation of mitotic phosphoepitopes, different from the classical pathway where the substrate is phosphorylated by a mitotic kinase. In the present paper, we report that topoIIα is co-localized with both CK2 and PP2A (protein phosphatase 2A) during interphase. Simultaneous incubation of purified topoIIα with CK2 and PP2A had minimal influence on the total phosphorylation levels of topoIIα, but resulted in complete disappearance of the MPM-2 phosphoepitope owing to opposite sequence preferences of CK2 and PP2A. Accordingly, short-term exposure of interphase cells to okadaic acid, a selective PP2A inhibitor, was accompanied by the specific appearance of the MPM-2 phosphoepitope on topoIIα. During early mitosis, PP2A was translocated from the nucleus, while CK2 remained in the nucleus until pro-metaphase thus permitting the formation of the MPM-2 phosphoepitope. These results underline the importance of protein phosphatases as an alternative way of creating cell-cycle-specific phosphoepitopes.

INTRODUCTION

Deregulation of cell cycle controls (checkpoints) is universal in human tumours and represents a leading cause of genetic instability [1–3]. The G₂ checkpoint controls mitotic entry of damaged cells and is triggered by a wide range of endogenous, environmental and pharmacological injuries resulting in genotoxic lesions [2,4,5]. Accordingly, abrogation of the G₂ checkpoint is accompanied by enhanced mitotic cell death and an increased incidence of genetic alterations among the surviving daughter cells [6,7]. Interestingly, unscheduled activation of mitotic kinases has also been associated with Alzheimer's disease, where the accumulation of mitotic phosphoepitopes such as MPM-2 (mitotic protein monoclonal 2) is a hallmark of neurofibrillary tangles [8,9].

Traditionally defined, mitosis is initiated with the first visible signs of chromosome condensation [10]. Chromosome condensation is associated with extensive phosphorylation of proteins involved in the regulation of chromatin structure on mitosis-specific phosphorylation sites [11–13]. Among these, the MPM-2 epitope appears to be particularly important, since its presence on mitotic chromosomes is closely associated with the condensed state [14]. The principal MPM-2 kinases include the mitotic kinases Cdk1 (Cdc2), plk (polo-like kinase) and NIMA (never in mitosis in Aspergillus nidulans) [15–20]. The major chromosomal protein recognized by the MPM-2 antibody has been identified as the α isoform of DNA topoisomerase II (topoIIα) [11]. This protein plays several essential roles during late G₂ and early mitosis (for recent reviews, see [21–23]), including pre-mitotic chromosome individualization, chromosome condensation and decatenation of intertwined sister chromatids [24–29].

Unexpectedly, a combination of in vitro and in vivo studies demonstrated unambiguously that the MPM-2 epitope on Ser-1469 of topoIIα was created by protein kinase CK2, otherwise known as a constitutive interphase kinase [30]. These findings were even more surprising since the major interphase phosphoepitope on Ser-1525 of topoIIα is also created by CK2 [30,31]. Together, these observations suggested the existence of alternative pathways for the creation of mitotic phosphoepitopes different from the classical pathway, where the substrate is phosphorylated by a mitotic kinase.

In the present paper, we report that the MPM-2 phosphorylation of topoIIα is regulated directly by PP2A (protein phosphatase 2A) which prevents the appearance of the Ser-1469 MPM-2 epitope, but not the Ser-1525 phosphorylation site, during interphase. These studies suggest a novel paradigm for the creation of cell-cycle-specific phosphorylation sites based on the sequence specificity of protein phosphatases toward their substrates.

EXPERIMENTAL

Purified enzymes

Human topoIIα was overexpressed in Saccharomyces cerevisiae and purified as described previously [30]. CK2 was kindly provided by Dr Claude Cochet (INSERM EMI 104 Département de Biologie Moléculaire et Structurale, CEA, Grenoble, France) and PP2A was purchased from Upstate Biotechnology.

Cell culture

HeLa and LLP-CK cells were grown in DMEM (Dulbecco's modified Eagle's Medium) (Sigma) supplemented with 10% (v/v) fetal calf serum (Gibco Life Technology), 60 μg/ml penicillin G and 100 μg/ml streptomycin sulfate. For mitotic arrest, cells were incubated overnight in the presence of 75 ng/ml nocodazole. For arrest in the G₁/S phase of the cell cycle, cells were incubated for 24 h in the presence of 5 μM aphidicolin or synchronized by a double thymidine block. Briefly, cells were incubated with 2 mM thymidine for 16 h followed by 8 h recovery and a second thymidine exposure for 16 h. The efficiency of synchronization was assessed by flow cytometry using a Epics XL/MCL flow cytometer equipped with an argon laser to give 488 nm light, and the cell cycle distribution was analysed by Multicycle software (Phoenix Flow Systems). Nocodazole increased the fraction of G₂/M cells from 8 to more than 80% compared with the unsynchronized cells. The mitotic index was also determined by microscopic analysis of propidium iodide-stained cells and ranged from 70 to 90% for nocodazole-blocked cells. Both aphidicolin and thymidine reduced the fraction of S-phase cells from 37 to approx. 20% compared with the unsynchronized cells. After aphidicolin or thymidine removal, the fraction of S-phase cells reached 40% by 1 h and more than 80% by 4 h. For in vivo immunoprecipitation of protein kinase CK2, HeLa S3 cells were released in fresh medium for 1 or 4 h after aphidicolin block.

Immunofluorescence

HeLa cells or LLP-CK cells expressing GFP (green fluorescent protein)-labelled topoIIα [32] were grown on coverslips and then fixed in 4% (w/v) paraformaldehyde. Antigens were revealed by immunolabelling [33] using primary goat antibody (1:25 dilution) directed against the α subunit of CK2 (Santa Cruz Biotechnology) or primary rabbit antibody (1:25 dilution) directed against PP2A (Santa Cruz Biotechnology). Secondary antibodies were donkey anti-goat or anti-rabbit IgGs tagged with Cy3 (1:200 dilution; Jackson ImmunoResearch Laboratories). Nuclear DNA was counterstained with To-Pro 3 (Molecular Probes) and the images were collected using a Radiance 2000 MP confocal microscope (Bio-Rad Laboratories).

CK2 immunoprecipitation and topoIIα phosphorylation assay

Mitotic- or G₁/S-synchronized HeLa cells were lysed in a buffer containing TEM (50 mM Tris/HCl, pH 7.5, 10 mM EGTA, 4 mM MgSO₄), 1% CHAPS, 200 mM NaCl, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 2 μg/ml pepstatin A, 200 nM microcystin-LR (Calbiochem) and 200 nM okadaic acid (Sigma). Cellular extracts (250 μg) were incubated for 3 h at 4 °C under rotation in the presence of a polyclonal antibody directed against the regulatory β subunit of CK2 (kindly provided by Dr Claude Cochet) diluted 200 times. Extracts were incubated with 40 μl of Protein A–Sepharose beads for 30 min at 4 °C. The beads were washed three times with 500 μl of lysis buffer. Then, 500 ng of purified human topoIIα diluted in TEM buffer containing 0.5 mM ATP was added to 15 μl of beads and incubated for 30 min at 37 °C. For the total phosphorylation assay, 5 μCi of [³²P]ATP was added to the unlabelled ATP. Reactions were stopped by adding 30 μl of 2× SDS/PAGE loading buffer. Samples were then subjected to electrophoresis, and the results were revealed by Western blot analysis or by autoradiography.

Influence of okadaic acid on the MPM-2 phosphorylation of topoIIα

HeLa cells were synchronized by a double thymidine block. After synchronization, cells were released for 1 h in fresh medium containing 0, 50 or 500 nM okadaic acid, and the cells were extracted with high salt buffer containing 750 mM NaCl, 2 mM EDTA, 0.75% Nonidet P40, 50 mM Tris/HCl, pH 8, 5 μg/ml protease inhibitors, 200 nM microcystin-LR and 200 nM okadaic acid. TopoIIα was immunoprecipitated as described previously [28]. The relative levels of MPM-2 phosphorylation were revealed by Western blot and densitometric analysis (PhosphorImager Storm 840, Molecular Dynamics).

Preparation of phosphorylated human topoIIα, K15F and R20A peptides

MPM-2 and radiolabelled phosphorylated topoIIα were prepared by incubation of topoIIα with CK2 in the presence of 20 μM ATP containing 5 μCi of [³²P]ATP. After phosphorylation, the ATP was separated from topoIIα using a Sephadex G-50 column (Eppendorf-5Prime). The purified phosphorylated topoIIα was used as substrate for the dephosphorylation experiments. Radiolabelled phosphopeptides K15F (containing Lys-1517 to Phe-1531, including the major Ser-1525 phosphorylation site of topoIIα) and R20A (containing Arg-1466 to Ala-1485, including the MPM-2 Ser-1469 phosphorylation site of topoIIα) were prepared as described previously [30].

Dephosphorylation of the MPM-2 epitope by extracts from unsynchronized cells

Unsynchronized HeLa cells were extracted with a lysis buffer containing 100 mM KCl, 2 mM EGTA, 0.2% Nonidet P40, 5 mM MgSO₄, 50 mM Tris/HCl, pH 7.5, 5 μg/ml aprotinin, 5 μg/ml leupeptin and 2 μg/ml pepstatin A. Cellular extracts from 10⁴ cells were added to 300 ng of MPM-2-positive topoIIα in a buffer containing 20 mM Tris/HCl, pH 7.5, 5 mM MgCl₂, 0.1 mM MnCl₂, 1 mM DTT (dithiothreitol), 50 ng/μl heparin, protease inhibitors and increasing amounts of protein inhibitor-2 (Upstate Biotechnology) or okadaic acid. Reactions were performed in a final volume of 20 μl for 30 min at 30 °C and stopped by adding 2× SDS-PAGE loading buffer. Samples were then subjected to electrophoresis and the results were revealed by Western blot analysis.

Incubation of topoIIα in the presence of both CK2 and PP2A

TopoIIα (200 ng) was incubated in the presence of 5 ng of CK2 and 0.05 unit of PP2A (corresponding to 25 ng of protein) in a buffer containing 20 mM Tris/HCl, pH 7.8, 7 mM MgCl₂, 50 mM KCl, 1 mM DTT, 100 ng/μl BSA and 20 μM unlabelled ATP or GTP. For the total phosphorylation assay, 5 μCi of [³²P]ATP was added to the unlabelled ATP. To determine the influence of Mn²⁺, 0.1 mM MnCl₂ was added to the reaction mixture. Reactions were carried out and products were subjected to Western blot analysis as described above.

PP2A dephosphorylation of K15F and R20A phosphopeptides

Radiolabelled phosphopeptides (200 μM) were incubated with increasing amounts of PP2A in a buffer containing 20 mM Tris/HCl, pH 7.8, 7 mM MgCl₂, 50 mM KCl, 1 mM DTT and 100 ng/μl BSA (10 μl final volume). To determine the influence of Mn²⁺, 0.1 mM MnCl₂ was added. Reactions were performed for 30 min at 30 °C and stopped by addition of the Tris/Tricine gel loading buffer (Bio-Rad Laboratories). Peptides were subjected to Tris/Tricine gel electrophoresis, and the results were analysed by densitometry.

Peptide phosphorylation in the presence of CK2 and PP2A

The K15F or R20A peptides (200 μM) were incubated in the presence of 5 ng of CK2 and 0.05 unit of PP2A (corresponding to 25 ng of protein) in a buffer (10 μl final volume) containing 20 mM Tris/HCl, pH 7.8, 7 mM MgCl₂, 50 mM KCl, 1 mM DTT, 100 ng/μl BSA and 20 μM ATP including 5 μCi of [³²P]ATP. To determine the influence of Mn²⁺, 0.1 mM MnCl₂ was added. Reactions were performed and analysed by Tris/Tricine gel electrophoresis as described above.

RESULTS

CK2 is co-localized with topoIIα during interphase and is intrinsically able to induce the MPM-2 phosphoepitope

CK2 is an ubiquitous kinase that is active throughout the cell cycle [34]. To explain the selective formation of the MPM-2 phosphoepitope on topoIIα during mitosis, we explored different hypotheses. First, the two enzymes might not co-localize during interphase. Although topoIIα is phosphorylated by CK2 during interphase [31,35,36], and forms molecular complexes with CK2 [37,38], it was still possible that this interaction concerned only a minor fraction of the two enzymes. The cellular localization of topoIIα and CK2 was determined in LLP-CK cells expressing a functional enhanced GFP-labelled topoIIα [32]. The results (Figure 1A) showed that both enzymes were predominantly nuclear in interphase cells (panels a and b). Furthermore, topoIIα and CK2 showed substantial co-localization (indicated by yellow in panel d). Similar results were obtained when both enzymes were localized by immunolabelling in HeLa cells (results not shown).

Figure 1. Interaction between CK2 and topoIIα in interphase cells.

(A) Immunofluorescence microscopy reveals partial co-localization (indicated by yellow in panel d) between enhanced GFP–topoIIα (indicated in green in panels a and d) and the catalytic CK2α subunit (indicated in red in panels b and d) during interphase. DNA counterstaining (indicated in blue in panels c and d) shows that the co-localization of the two enzymes occurs preferentially in low-condensed chromatin domains. (B) In vitro phosphorylation assays reveal that mitotic and non-mitotic forms of CK2 are equally able to phosphorylate topoIIα. Left-hand panel, total topoIIα phosphorylation; right-hand panel, MPM-2 phosphorylation of topoIIα. Control lanes 1 show the absence of topoIIα phosphorylation in an immunoprecipitate obtained with a non-immune antibody. Lanes 2 show topoIIα phosphorylation by CK2 immunoprecipitated from nocodazole-blocked HeLa cells (M-phase). Lanes 3 and 4 show topoIIα phosphorylation by CK2 immunoprecipitated from HeLa cells blocked by aphidicolin and released in fresh medium for 1 or 4 h (S-phase) respectively. (C) The MPM-2 epitope on topoIIα (lane 1) disappears in the presence of HeLa cell extracts (lane 2). Incubation with increasing amounts of okadaic acid (10, 20 and 50 nM in lanes 3, 4 and 5 respectively) completely inhibits the MPM-2 phosphatase activity in the cellular extracts, while incubation with increasing amounts of protein inhibitor-2 (50, 100 and 500 nM in lanes 6, 7 and 8 respectively) has no effect. (D) Immunofluorescence microscopy reveals partial co-localization (indicated by yellow in panel d) between enhanced GFP–topoIIα (indicated in green in panels a and d) and the catalytic PP2A subunit (indicated in red in panels b and d) during interphase. DNA counterstaining (indicated in blue in panels c and d) shows that the co-localization of the two enzymes preferentially occurs in low-condensed chromatin domains. (E) HeLa cells were synchronized by double thymidine block. After synchronization, cells were released for 1 h in fresh medium containing 0, 50 or 500 nM okadaic acid (lanes 1, 2 and 3 respectively). The upper panel shows MPM-2-positive topoIIα after specific immunoprecipitation, and the lower panel shows MPM-2-positive proteins in total cell extracts. (F) The relative intensities of MPM-2-positive topoIIα (left-hand panel) and MPM-2-positive proteins in total cell extracts (right-hand panel), obtained as described in (E), were quantified by densitometric analysis. Results are means±S.E.M. for three independent experiments. White columns, cells released in drug-free medium for 1 h; hatched columns, cells released for 1 h in medium with 50 nM okadaic acid; black columns, cells released for 1 h in medium with 500 nM okadaic acid. htopoIIα, human topoIIα.

Although a substantial part of topoIIα and CK2 co-localized during interphase, it was possible that only the mitotic form of the kinase was able to create the MPM-2 phosphoepitope. In support of this hypothesis, it has been reported that CK2 is phosphorylated on multiple sites by Cdk1 during mitosis resulting in substantial conformational and functional alterations [39,40] that might potentially influence its sequence specificity. To explore this possibility, CK2 was immunoprecipitated from HeLa cells synchronized in the G₁, S- or M-phase of the cell cycle and was incubated with purified human topoIIα in the presence of radiolabelled ATP or GTP. The results showed that CK2 isolated from interphase (Figure 1B, lanes 3 and 4) or mitotic cells (Figure 1B, lane 2) was equally able to phosphorylate topoIIα (Figure 1B, left-hand panel) and to induce the MPM-2 epitope (Figure 1B, right-hand panel). Similar levels of phosphorylation were observed in the presence of ATP and GTP, which is a hallmark of CK2 (results not shown). Together, these results indicate that interphase CK2 is intrinsically able to create the MPM-2 phosphoepitope on topoIIα.

PP2A potently dephosphorylates the MPM-2 site on topoIIα in vitro

The phosphorylation status of a protein is the result of a balance between kinase-mediated phosphorylation and phosphatase-mediated dephosphorylation. To establish whether the MPM-2 phosphoepitope on topoIIα was a preferred substrate for protein phosphatases during interphase, cellular extracts were prepared from interphase HeLa cells and their ability to dephosphorylate the MPM-2 phosphoepitope on purified human topoIIα was evaluated (Figure 1C). In the absence of phosphatase inhibitors (Figure 1C, lane 2) or in the presence of protein inhibitor-2, a specific inhibitor of PP1 (protein phosphatase 1) (Figure 1C, lanes 6–8), the cellular extracts completely removed the MPM-2 phosphoepitope. Similar studies were carried out in the presence of okadaic acid, a marine-sponge toxin that is a potent inhibitor of PP2A and, to a lesser degree, of PP1 [41]. The results showed that the MPM-2 phosphatase was completely inactive in the presence of okadaic acid (Figure 1C, lanes 3–5) even at concentrations as low as 10 nM (Figure 1C, lane 3). Further studies showed that the MPM-2 phosphatase was inhibited with an IC₅₀ of 1.5 nM, which is within the same dose range as reported previously for the inhibition of PP2A using the reference substrate, phosphorylated myosin light chain ([41], and results not shown).

PP2A is co-localized with topoIIα during interphase

To determine whether PP2A might also influence the phosphorylation of topoIIα in living cells, the cellular localization of PP2A was determined by fluorescence microscopy (Figure 1D). As reported previously, PP2A was predominantly nuclear during interphase [42] and co-localized partly with topoIIα (Figure 1D, panel d) and CK2 (results not shown).

Cellular exposure to okadaic acid is accompanied by specific induction of the MPM-2 epitope on topoIIα during interphase

To establish whether the MPM-2 phosphoepitope on topoIIα was also a substrate for PP2A in living cells, G₁/S synchronized HeLa cells were incubated with different concentrations of okadaic acid. Since prolonged treatment with okadaic acid may induce a premature mitotic state [43], incubation with okadaic acid was carried out at concentrations and exposure times where no significant alterations were observed with regard to cell cycle distribution, Cdk1 activity, DNA condensation or the overall formation of MPM-2 antigens as determined by flow cytometry (results not shown). Incubation of cells with low (50 nM) or high (500 nM) concentrations of okadaic acid for 1 h was accompanied by a dose-dependent increase in the proportion of MPM-2-reactive topoIIα as determined by Western blot analysis after immunoprecipitation with a topoIIα-directed antibody (Figures 1E, upper panel, and 1F, htopoIIα) without any detectable changes in the specific activity of CK2 (results not shown). In comparison, no major changes in the overall MPM-2 reactivity were observed for total cell extracts (Figures 1E, lower panel, and 1F, Total cell extract). The specific increase of the MPM-2 phosphoepitope on topoIIα strongly suggest that PP2A may play a direct role in the regulation of the MPM-2 epitope during interphase.

PP2A preferentially dephosphorylates the MPM-2 epitope on human topoIIα

In addition to the MPM-2 phosphoepitope on Ser-1469, CK2 also creates the major interphase phosphorylation site on Ser-1525 of topoIIα [30,31]. To establish whether PP2A shows a sequence preference towards one of these phosphoepitopes, topoIIα was phosphorylated by CK2 in the presence of radiolabelled ATP, and the ability of purified PP2A to dephosphorylate the labelled substrate was evaluated in the absence or presence of Mn²⁺, a potent phosphatase activator [44]. The results (Figure 2A) show that PP2A was able to efficiently dephosphorylate topoIIα only in the presence of Mn²⁺ (Figure 2A, left-hand panel, compare lanes 1 and 3). In marked contrast, PP2A completely dephosphorylated the MPM-2 phosphoepitope even at low Mn²⁺ concentrations (Figure 2A, right-hand panel, compare lanes 1 and 2). To establish further the sequence preference of PP2A, we selected two polypeptides: K15F, containing Lys-1517 to Phe-1531 that includes the major Ser-1525 phosphorylation site [30,31] and R20A, containing Arg-1466 to Ala-1485 that includes the MPM-2 Ser-1469 phosphorylation site of topoIIα [30]. The results (Figure 2B) showed that Mn²⁺ is required for efficient dephosphorylation of the Ser-1525 phosphorylation site on peptide K15F. In clear contrast, PP2A efficiently dephosphorylated the Ser-1469 MPM-2 epitope on peptide R20A even without Mn²⁺.

Figure 2. PP2A shows high specificity towards the MPM-2 phosphoepitope on Ser-1469.

(A) Comparison of the ability of PP2A to dephosphorylate ³²P-labelled topoIIα (left-hand panel) and the topoIIα MPM-2 epitope (right-hand panel) reveals a strong preference of PP2A towards the Ser-1469 residue. Left-hand panel, PP2A is able to dephosphorylate topoIIα (lane 1) only in the presence of Mn²⁺ (lane 3), but not in the absence of Mn²⁺ (lane 2). Right-hand panel, PP2A completely dephosphorylates the MPM-2 phosphoepitope (lane 1) even in the absence of Mn²⁺ (lane 2). (B) Comparison of the capacity of PP2A to dephosphorylate ³²P-labelled peptides confirms the strong sequence preference towards Ser-1469 over Ser-1525. The R20A sequence corresponds to the region surrounding the MPM-2 phosphoepitope on Ser-1469, whereas the K15F sequence corresponds to the region surrounding the major phosphorylation site on Ser-1525. (C) CK2-mediated phosphorylation of topoIIα in the absence (lane 1) or presence (lane 2) of PP2A. Left-hand panel, total phosphorylation levels of topoIIα, as revealed by ³²P-labelling, are not affected by the presence of PP2A. Right-hand panel, the formation of the MPM-2 epitope on topoIIα, as revealed by Western blot analysis, is strongly inhibited when PP2A is present. Similar results were obtained in the presence and absence of Mn²⁺. (D) PP2A shows a high preference for Ser-1469 over Ser-1525. Lanes 1, peptide phosphorylation in the absence of PP2A; lanes 2, peptide phosphorylation in the presence of PP2A. CK2 phosphorylation of Ser-1469 on R20A is strongly inhibited in the presence of PP2A (right-hand bar), whereas CK2 phosphorylation of Ser-1525 on K15F is less inhibited by PP2A (left-hand bar) even in the presence of Mn²⁺. hTopoIIα, human topoIIα.

PP2A efficiently dephosphorylates the MPM-2 phosphoepitope on topoIIα even in the presence of CK2

The findings described above strongly suggest that the MPM-2 phosphoepitope is preferentially dephosphorylated by PP2A, whereas previous studies have indicated that Ser-1525 is preferentially phosphorylated by CK2 [30]. We therefore wished to establish how the simultaneous presence of catalytically active CK2 and PP2A influenced the phosphorylation pattern of topoIIα. For this, purified topoIIα was incubated with CK2 and radiolabelled ATP in the absence or presence of PP2A (Figure 2C). The protein phosphatase had hardly any influence on the overall phosphorylation of topoIIα (Figure 2C, left-hand panel). In marked contrast, the MPM-2 phosphoepitope was removed completely under the same conditions (Figure 2C, right-hand panel). Furthermore, the MPM-2 phosphoepitope was not formed in the absence of ATP or GTP, but was favoured in the presence of okadaic acid (results not shown). Therefore the observed effects are consistent with competing kinase and phosphatase activities, but not with physical blocking of the MPM-2 site by PP2A.

For further confirmation, the same experiment was carried out with the K15F and R20A peptides instead of full-length topoIIα (Figure 2D). The results showed that the K15F peptide containing the major Ser-1525 phosphorylation site was only influenced by PP2A in the presence of high concentrations of Mn²⁺. In contrast, the MPM-2 epitope on R20A is clearly reduced even at low concentrations of Mn²⁺ (Figure 2D). These results demonstrate how the opposing sequence preference of CK2 and PP2A can prevent the appearance of the MPM-2 epitope on topoIIα during interphase without notable influence on the other CK2 phosphorylation sites.

PP2A leaves the nucleus in early prophase, while CK2 remains nuclear until pro-metaphase

To determine when the MPM-2 phosphoepitope appears on topoIIα, the cellular localization of CK2 and PP2A was determined by fluorescence microscopy (Figure 3). During interphase, PP2A was predominantly nuclear and co-localized partly with both topoIIα and CK2 (Figure 1E). PP2A started to leave the nuclear compartment at mitotic onset (Figure 3, series 2–4) and was, in general, not present in early mitotic nuclei (Figure 3, series 2) or during late prophase (Figure 3, series 3). During pro-metaphase, PP2A was diffused in the cytoplasm without any detectable association with mitotic chromosomes (Figure 3, series 4).

Figure 3. PP2A is excluded from the nucleus in early prophase while CK2 remains nuclear until pro-metaphase.

Immunofluorescence reveals that PP2A (left-hand panels, red) disappears quickly from early mitotic nuclei while CK2 (right panels, red) stays associated with a non-chromosomal fraction until complete chromosome condensation and nuclear membrane breakdown. Series 1–4 show a rapid loss of PP2A during early prophase. Series 5–8 show gradual changes of CK2 localization until pro-metaphase. DNA counterstaining (left- and right-hand panels, blue) shows that neither PP2A or CK2 is associated with pre-condensed or condensed chromatin and that CK2 tends to surround the chromosomes.

In comparison, CK2 remained nuclear during early mitosis (Figure 3, series 6–7) and only left the nuclear compartment at prometaphase when the nuclear membrane is disrupted [Figure 3, compare series 7 (late prophase) and 8 (pro-metaphase)]. These results indicate that CK2 can phosphorylate topoIIα during early mitosis in the absence of PP2A resulting in the formation of the mitotic MPM-2 phosphoepitope. At the same time, topoIIα became progressively associated with the condensing chromosomes (as indicated by turquoise on the overlay in Figure 4A, panels d and h). Interestingly, CK2 never became chromosome-associated (as indicated by the absence of purple-pink on the overlay in Figure 4A, panels d and h) and co-localized preferentially with the nucleoplasmic pool of topoIIα during prophase (as indicated by yellow on the overlay in Figure 4A, panel d). These findings suggest that CK2 predominantly induced the MPM-2 phosphoepitope on nucleoplasmic topoIIα which then associated with the mitotic chromosomes.

Figure 4. Localization of CK2 and PP2A in early mitosis.

(A) Immunofluorescence reveals co-localization (yellow, panel d) between GFP–topoIIα (green, panels a and d) and the catalytic CK2α subunit (red, panels b and d) in early mitosis (upper panels). Co-localization was observed for the non-chromosomal fraction of GFP–topoIIα as revealed by DNA counterstaining (blue, panel c). In pro-metaphase (lower panels), topoIIα (green, panel e) is fully associated with the condensed chromosomes (blue, panel g), whereas CK2 (red, panel f) shows a diffused pattern surrounding condensed chromosomes. No yellow is seen after superimposition (panel g). (B) Model for the cell-cycle-dependent interaction of CK2 with topoIIα. The CK2-mediated MPM-2 phosphorylation of topoIIα is regulated directly by PP2A during interphase. The translocation of PP2A from the nuclear compartment during early mitosis allows CK2 to phosphorylate the Ser-1469 MPM2 site. Solid lines represent preferential residues targeted by either CK2 or PP2A. Broken lines represent weaker substrates targeted by either CK2 or PP2A.

DISCUSSION

In the present paper, we describe an alternative model to account for the regulation of a cell-cycle-specific phosphorylation site, in this case the mitotic MPM-2 phosphoepitope on topoIIα, a major regulator of mitotic chromosome structure. Although topoIIα was the only chromosome-associated protein that served as substrate for the MPM-2 kinase activity of CK2, up to ten potential substrates were identified in total mitotic extracts. The two proteins which most consistently served as substrates for the MPM-2 kinase activity of CK2 were topoIIα and a protein with a molecular mass of ∼110 kDa [30]. Although the identity of the latter protein is not known, its molecular mass corresponds to another major CK2 substrate, nucleolin, which like topoIIα forms stable complexes with CK2 [45] and undergoes mitosis-specific phosphorylation [46]. Therefore the proposed model is likely to be relevant for several proteins besides topoIIα.

The onset of mitotic chromosome condensation is accompanied by a burst of phosphorylation of chromatin-associated proteins. In most cases, the formation of the phosphorylation sites can be defined by a sequential model where competing kinases and phosphatases directly regulate the activation state of a mitotic kinase (such as Cdk1) that, once activated at the G₂/M interphase, will act on its substrates (such as the condensins). In contrast with this classical model, it is the final substrate, topoIIα, that is subject to competing kinase and phosphatase activities as outlined in Figure 4(B). During interphase, CK2 is co-localized with topoIIα in the nucleus and is intrinsically able to induce the formation of the MPM-2 phosphoepitope. However, the additional co-localization with PP2A ensures that the MPM-2 site on Ser-1469 is kept non-phosphorylated owing to a combination of strong PP2A activity and weak CK2 activity towards this residue. Simultaneously, topoIIα is phosphorylated by CK2 on Ser-1525 because of strong CK2 activity combined with weak PP2A activity. At mitotic onset, the subcellular distribution of CK2 and PP2A is altered (Figure 4B) without marked change in their expression levels [34,47]. PP2A is rapidly excluded from the nucleus, while CK2 remains nuclear until nuclear membrane breakdown in pro-metaphase. Consequently, the existing equilibrium between the MPM-2 kinase and MPM-2 phosphatase activities is disrupted, resulting in extensive MPM-2 phosphorylation of topoIIα.

A major question is why the MPM-2 epitope on topoIIα is subject to such complex regulation. TopoIIα seems to play a role in the fine-tuning of mitotic timing. Recent findings show that overexpression of topoIIα is accompanied by accelerated cell cycle transit in G₂ and early mitosis [48], while RNAi (RNA interference)-mediated topoIIα down-regulation delays G₂ and early mitosis [49]. A possible explanation for this activity is the capacity of topoIIα to recruit the major mitotic kinase Cdk1 to nuclear chromatin thus providing the kinase access to chromosomal proteins that are essential for further chromosome condensation. This is important, since Cdk1 by itself is not a DNA-binding protein, and is thus not able to associate with nuclear chromatin in the absence of topoIIα [28].

A second question concerns the biological function of the MPM-2 phosphoepitope. Although it is clearly established that the MPM-2 phosphoepitope is formed on numerous proteins during mitosis and that this is causally linked to different mitotic functions, the exact role of the MPM-2 modification is not well understood. The MPM-2 phosphoepitope is formed on a serine or threonine residue next to a proline residue. The peptidylprolyl cis–trans isomerase Pin1 shows almost the same substrate specificity as the MPM-2 antibody and has been shown to bind to the mitotic form of topoIIα [30,50]. Interestingly, Pin1 has also been shown to bind and modulate Cdc25, another key regulator of mitotic entry [51]. The findings for Cdc25C suggests that binding of Pin1 to the MPM-2 site of topoIIα might alter its structure, function and/or stability, thereby regulating the mitotic functions of topoIIα.

It is intriguing that the expression of topoIIα, CK2 and PP2A is frequently altered in human tumours. TopoIIα is overexpressed in several tumour types, including lung and breast cancer, and high levels of topoIIα expression is generally associated with more aggressive tumour behaviour [52,53]. CK2 is also overexpressed in many tumours, such as breast, lung and malignant melanomas, and is generally considered to be a cellular survival factor [54]. Interestingly, genetic studies in yeast suggest that CK2 facilitate G₂ checkpoint override following DNA damage [55]. In contrast, current evidence suggest that PP2A is a tumour suppressor. Loss-of-function mutations of the structural subunit of PP2A have been described in lung and breast cancer as well as in melanoma [56], and the PP2A inhibitor okadaic acid acts as a tumour promoter. Recent results indicate that down-regulation of PP2A activity might be a requirement for the G₂–M transition [47].

Taken together, our findings document the functional and spatial interaction between topoIIα, CK2 and PP2A during the different phases of the cell cycle. The direct regulation of the MPM-2 phosphoepitope on topoIIα by PP2A underlines the importance of protein phosphatases in the regulation of key cellular functions.