Keywords: DNA topoisomerase II, Protein Phosphatase PP2A, Mitosis, MPM-2

Recent results suggest a role for topoisomerase II alpha (topoIIalpha) in the finetuning of mitotic entry. Mitotic entry is accompanied by the formation of specific phosphoepitopes such as MPM-2 that are believed to control mitotic processes. Surprisingly, the MPM-2 kinase of topoIIalpha 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. We now report that topoIIalpha is colocalized with both CK2 and protein phosphatase 2A (PP2A) during interphase. Simultaneous incubation of purified topoIIalpha with CK2 and PP2A had minimal influence on the total phosphorylation levels of topoIIalpha, but resulted in complete disappearance of the MPM-2 phosphoepitope due 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 topoIIalpha. During early mitosis, PP2A was translocated from the nucleus while CK2 remained in the nucleus until prometaphase 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.

The mitosis-specific MPM -2 phosphorylation of DNA topoisomerase II alpha is directly regulated by protein phosphatase 2A Alexandre E. ESCARGUEIL and Annette K. LARSEN* Group of Cancer Biology and Therapeutics, INSERM U673 and Université Pierre et Marie Curie, Hôpital Saint-Antoine, Paris, France

INTRODUCTION

Deregulation of cell cycle controls (checkpoints) is universal in human tumors

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 p harmacological injuries resulting in genotoxic lesions [START_REF] Paulovich | When checkpoints fail[END_REF][START_REF] Boddy | Replication checkpoint enforced by kinases Cds1 and Chk1[END_REF][START_REF] Samuel | Linking DNA damage to cell cycle checkpoints[END_REF]. 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 [START_REF] Lau | Mechanism by which caffeine potentiates lethality of nitrogen mustard[END_REF]7]. Interesti ngly, unscheduled activation of mitotic kinases has also been associated with Alzheimer's disease where the accumulation of mitotic phosphoepitopes such as MPM-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][12][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), polo-like kinase (plk) and NIMA [START_REF] Westendorf | Cloning of cDNAs for Mphase phosphoproteins recognized by the MPM2 monoclonal antibody and determination of the phosphorylated epitope[END_REF][START_REF] Ye | The NIMA protein kinase is hyperphosphorylated and activated downstream of p34cdc2/cyclin B: coordination of two mitosis promoting kinases[END_REF][START_REF] Kumagai | Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts[END_REF][START_REF] Renzi | MPM-2 antibody-reactive phosphorylations can be created in detergent-extracted cells by kinetochore-bound and soluble kinases[END_REF][START_REF] Stukenberg | Systematic identification of mitotic phosphoproteins[END_REF][START_REF] Logarinho | The Drosophila POLO kinase localises to multiple compartments of the mitotic apparatus and is required for the phosphorylation of MPM2 reactive epitopes[END_REF]. 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 [START_REF] Larsen | From DNA damage to G2 arrest: the many roles of topoisomerase II[END_REF][22][23] including premitotic chromosome individualization, chromosome condensation and decatenation of intertwined sister chromatids [START_REF] Gimenez-Abian | Premitotic chromosome individualization in mammalian cells depends on topoisomerase II activity[END_REF][START_REF] Adachi | Chromosome assembly in vitro: topoisomerase II is required for condensation[END_REF][START_REF] Bojanowski | DNA topoisomerase II can drive changes in higher order chromosome architecture without enzymatically modifying DNA[END_REF][START_REF] Gimenez-Abian | A postprophase topoisomerase II-dependent chromatid core separation step in the formation of metaphase chromosomes[END_REF][28][START_REF] Losada | Cohesin release is required for sister chromatid resolution, but not for condensin-mediated compaction, at the onset of mitosis[END_REF].

Unexpectedly, a combination of in vitro and in vivo studies unambiguously demonstrated that the MPM-2 epitope on Ser-1469 of topoIIα was created by protein kinase CK2 (CK2), otherwise known as a constitutive interphase kinase [START_REF] Escargueil | Serine 1524 is a major site of phosphorylation on human topoisomerase II alpha protein in vivo and is a substrate for casein kinase II in vitro[END_REF]. These findings were even more surprising since the major interphase phosphoepitope on Ser-1525 of topoIIα is also created by CK2 [START_REF] Escargueil | Serine 1524 is a major site of phosphorylation on human topoisomerase II alpha protein in vivo and is a substrate for casein kinase II in vitro[END_REF]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.

We now report that the MPM-2 phosphorylation of topoIIα is directly regulated by protein phosphatase 2A (PP2A) that 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 Saccharormyces cerevisiae and purified as previously described [START_REF] Escargueil | Serine 1524 is a major site of phosphorylation on human topoisomerase II alpha protein in vivo and is a substrate for casein kinase II in vitro[END_REF], CK2 was kindly provided by Dr. Claude Cochet (Grenoble, France) while PP2A was purchased from Upstate Biotechnology (distributed by Euromedex, Mundolsheim, France).

Cell Culture

HeLa and LLP-CK cells were grown in Dulbecco's modified Eagle's Medium (Sigma) supplemented with 10% 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 analyzed by Multicycle software (Phoenix Flow Systems, San Diego, CA). Nocodazole increased the fraction of G2/M cells from 8 % to more than 80 % compared to 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 approximately 20 % compared to 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 green fluorescent labeled topoIIα [START_REF] Tavormina | Rapid exchange of mammalian topoisomerase II alpha at kinetochores and chromosome arms in mitosis[END_REF] were grown on coverslips and then fixed in 4% paraformaldehyde. Antigens were revealed by immunolabeling [START_REF] Iborra | Coupled transcription and translation within nuclei of mammalian cells[END_REF] using primary goat antibody (1:25 dilution) directed against the α subunit of CK2 (Santa Cruz Biotechnology, Santa Cruz, CA) 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

CK2 immunoprecipitation and topoIIα phosphorylation assay

Mitotic-or G1/S-synchronized HeLa cells were lysed in a buffer containing 50 mM Tris-HCl pH 7.5, 10 mM EGTA, 4 mM MgSO 4 (TEM), 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 beta subunit of CK2 (kindly provided by Dr. Claude Cochet, Grenoble, France) 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 32 P-ATP was added to the unlabeled ATP. Reactions were stopped by adding 30 µl of 2x 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% NP-40, 50 mM Tris-HCl pH 8, 5 µg/ml protease inhibitors, 200 nM microcystin-LR and 200 nM okadaic acid. TopoIIα was immunoprecipitated as previously described [28]. The relative levels of MPM-2 phosphorylation were revealed by Western blot and densitometry analysis (phosphoimager Storm 840, Molecular Dynamics).

Preparation of phosphorylated human topoIIα , K15F and R20A peptides MPM-2 and radiolabeled phosphorylated topoIIα were prepared by incubation of topoIIα with CK2 in the presence of 20 µM ATP containing 5 µCi 32 P-ATP. After phosphorylation, the ATP was separated from topoIIα using a Sephadex G -50 column (5'-3' Inc., Boulder, CO). The purified phosphorylated topoIIα was used as substrate for the dephosphorylation experiments. Radiolabeled phosphopeptides were prepared as previously described [START_REF] Escargueil | Serine 1524 is a major site of phosphorylation on human topoisomerase II alpha protein in vivo and is a substrate for casein kinase II in vitro[END_REF].

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% NP-40, 5 mM MgSO 4 , 50 mM Tris-HCl pH 7.5, 5 µg/ml aprotinin, 5 µg/ml leupeptin and 2 µ/ml pepstatin A. Cellular extracts from 10 4 cells were added to 300 ng of MPM-2 positive topoIIα in a buffer containing 20 mM Tris-HCl pH 7.5, 5 mM MgCl 2 , 0.1 mM MnCl 2 , 1 mM DTT, 50 ng/µl heparin, protease inhibitors and increasing amounts of protein inhibitor-2 (Upstate Biotechnolgy) or okadaic acid. Reactions were performed in a final volume of 20 µl for 30 min at 30°C and stopped by adding 2x SDS-PAGE loading buffer. Samples were then subjected to electrophoresis and the results revealed by Western blot analysis.

Incubation of topoIIα in the simultaneous presence of CK2 and PP2A

TopoIIα (200 ng) was incubated in the presence of 5 ng CK2 and 0.05 units PP2A (corresponding to 25 ng protein) in a buffer containing 20 mM Tris-HCl pH 7.8, 7 mM MgCl 2 , 50 mM KCl, 1 mM DTT, 100 ng/µl BSA and 20 µM unlabeled ATP or GTP. For the total phosphorylation assay, 5 µCi 32 P-ATP was added to the unlabeled ATP. To determine the influence of manganese, 0.1 mM MnCl 2 was added to the reaction mixture. Reactions were performed and subjected to Western blot analysis as described above.

PP2A dephosphorylation of K15F and R20A phosphopeptides

Radiolabeled phosphopeptides (200 µM) were incubated with increasing amounts of PP2A in a buffer containing 20 mM Tris-HCl pH 7.8, 7 mM MgCl 2 , 50 mM KCl, 1 mM DTT and 100 ng/µl BSA (10 µl final volume). To determine the influence of manganese, 0.1 mM MnCl 2 was added. Reactions were performed for 30 min at 30°C and stopped by addition of the Tris-Tricine gel loading buffer (Bio-Rad).

Peptides were subjected to Tris-Tricine gel electrophoresis and the results were analyzed 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 CK2 and 0.05 units PP2A (corresponding to 25 ng protein) in a buffer (10 µl final volume) containing 20 mM Tris-HCl, pH 7.8, 7 mM MgCl 2 , 50 mM KCl, 1 mM DTT, 100 ng/µl BSA and 20 µM ATP including 5 µCi 32 P-ATP. To determine the influence of manganese, 0.1 mM MnCl 2 was added. Reactions were performed and analyzed by Tris-Tricine gel electrophoresis as described above.

RESULTS

CK2 is colocalized 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 [START_REF] Bosc | Expression and regulation of protein kinase CK2 during the cell cycle[END_REF]. To explain the selective formation of the MPM-2 phosphoepitope on topoIIα during mitosis, we explored different hypothesis. First, the two enzymes might not colocalize during interphase. Although topoIIα is phosphorylated by CK2 during interphase [31,[START_REF] Ackerman | Phosphorylation of DNA topoisomerase II in vivo and in total homogenates of Drosophila Kc cells. The role of casein kinase II[END_REF][START_REF] Cardenas | Casein kinase II phosphorylates the eukaryote -specific C -terminal domain o f topoisomerase II in vivo[END_REF], and forms molecular complexes with CK2 [START_REF] Bojanowski | DNA topoisomerase II and casein kinase II associate in a molecular complex that is catalytically active[END_REF][START_REF] Redwood | Casein kinase II stabilizes the activity of human topoisomerase IIalpha in a phosphorylation-independent manner[END_REF], 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 green fluorescent-labeled topoIIα [START_REF] Tavormina | Rapid exchange of mammalian topoisomerase II alpha at kinetochores and chromosome arms in mitosis[END_REF]. The results (Fig. 1A) showed that both enzymes were predominantly nuclear in interphase cells (panels a and b). Furthermore, topoIIα and CK2 showed substantial colocalization (indicated by yellow in panel d). Similar results were obtained when both enzymes were localized by immunolabeling in HeLa cells (data not shown).

Although a substantial part of topoIIα and CK2 colocalized 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 [START_REF] Bosc | Phosphorylation of casein kinase II by p34cdc2. Identification of phosphorylation sites using phosphorylation site mutants in vitro[END_REF][START_REF] Litchfield | Phosphorylation of casein kinase II by p34cdc2 in vitro and at mitosis[END_REF] that might potentially influence its sequence specificity. To explore this possibility, CK2 was i mmunoprecipitated from HeLa cells synchronized in the G1, S or M phase of the cell cycle, and incubated with purified human topoIIα in the presence of radiolabeled ATP or GTP. The results showed that CK2 isolated from interphase (Fig. 1B, lanes 3 and 4) or mitotic cells (Fig. 1B, lane 2) was equally able to phosphorylate topoIIα (left panel) and to induce the MPM-2 epitope (right 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 if 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 (Fig. 1C). In the absence of phosphatase inhibitors (lane 2) or in the presence of protein inhibitor-2, a specific inhibitor of protein phosphatase 1 (PP1, lanes 6 to 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 [START_REF] Bialojan | Inhibitory effect of a marine -sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics[END_REF]. The results showed that the MPM-2 phosphatase was completely inactive in the presence of okadaic acid (lanes 3-5) even at concentrations as low as 10 nM (lane 3). Further studies showed that the MPM-2 phosphatase was inhibited with an IC 50 of 1.5 nM, which is within the same dose range as previously reported for the inhibition of PP2A using the reference substrate, phosphorylated myosin light chain [START_REF] Bialojan | Inhibitory effect of a marine -sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics[END_REF] (results not shown).

PP2A is colocalized with topoIIα during interphase

To determine if PP2A might also influence the phosphorylation of topoIIα in living cells, the cellular localization of PP2A was determined by fluorescence microscopy (Fig. 1D). As previously reported, PP2A was predominantly nuclear duri ng interphase [START_REF] Turowski | Differential methylation and altered conformation of cytoplasmic and nuclear forms of protein phosphatase 2A during cell cycle progression[END_REF] and colocalized partly with topoIIα (Fig 1D panel d) and CK2 (data not shown).

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

To establish if 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 [START_REF] Yamashita | Okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, activates cdc2/H1 kinase and transiently induces a premature mitosis-like state in BHK21 cells[END_REF], 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 (data 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 (Fig. 1E, upper panel and Fig. 1F, left) without any detectable changes in the specific activity of CK2 (data not shown). In comparison, no major changes in the overall MPM-2 reactivity were observed for total cell extracts (Fig. 1 E, lower panel and Fig. 1 F,right). 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 [START_REF] Escargueil | Serine 1524 is a major site of phosphorylation on human topoisomerase II alpha protein in vivo and is a substrate for casein kinase II in vitro[END_REF]31]. To establish if PP2A shows a sequence preference toward one of these phosphoepitopes, topoIIα was phosphorylated by CK2 in the presence of radiolabeled ATP, and the ability of purified PP2A to dephosphorylate the labeled substrate was evaluated in the absence or presence of Mn 2+ , a potent phosphatase activator [START_REF] Cai | A metal-dependent form of protein phosphatase 2A[END_REF]. The results (Fig. 2A) showed that PP2A was only able to efficiently dephosphorylate topoIIα in the presence of Mn 2+ (Fig. 2A left panel, compare lanes 1 and 3). In marked contrast, PP2A completely dephosphorylated the MPM-2 phosphoepitope even at low Mn 2+ concentrations (Fig. 2A right panel, compare lanes 1 and 2). To further establish the sequence preference of PP2A, we selected two polypeptides; K15F, containing Lys-1517 to Phe -1531 that includes the major Ser-1525 phosphorylation site [START_REF] Escargueil | Serine 1524 is a major site of phosphorylation on human topoisomerase II alpha protein in vivo and is a substrate for casein kinase II in vitro[END_REF]31] and R20A, containing Arg-1466 to Ala-1485 that includes the MPM-2 Ser-1469 phosphorylation site of topoIIα [START_REF] Escargueil | Serine 1524 is a major site of phosphorylation on human topoisomerase II alpha protein in vivo and is a substrate for casein kinase II in vitro[END_REF]. The results (Fig. 2B) showed that Mn 2+ 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 Mn 2+ .

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 while previous studies have indicated that Ser-1525 is preferentially phosphorylated by CK2 [START_REF] Escargueil | Serine 1524 is a major site of phosphorylation on human topoisomerase II alpha protein in vivo and is a substrate for casein kinase II in vitro[END_REF]. 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 radiolabeled ATP in the absence or presence of PP2A (Fig. 2C). The protein phosphatase had hardly any influence on the overall phosphorylation of topoIIα (Fig. 2C, left panel). In marked contrast, the MPM-2 phosphoepitope was completely removed under the same conditions (Fig. 2C, right panel). Furthermore, the MPM-2 phosphoepitope was not formed in the absence of ATP or GTP, but was favored 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 (Fig. 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 Mn 2+ . In contrast, the MPM-2 epitope on R20A is clearly reduced even at low concentrations of Mn 2+ (Fig. 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 prometaphase

To determine when the MPM-2 phosphoepitope appears on topoIIα, the cellular localization of CK2 and PP2A was determined by fluorescence microscopy (Fig. 3).

During interphase, PP2A was predominantly nuclear and colocalized partly with both topoIIα and CK2 (Fig. 1E). PP2A started to leave the nuclear compartment at mitotic onset (Fig. 3, series 2-4) and was in general not present in early mitotic nuclei (Fig. 3, series 2) or during late prophase (Fig. 3, series 3). During prometaphase, PP2A was diffused in the cytoplasm without any detectable association with mitotic chromosomes (Fig. 3, series 4).

In comparison, CK2 remained nuclear during early mitosis (Fig. 3, series 6-7) and only left the nuclear compartment at prometaphase when the nuclear membrane is disrupted (Fig. 3, compare series 7 (late prophase) and 8 (prometaphase)). 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 Fig. 4A, panels d andh).

Interestingly, CK2 never became chromosome-associated (as indicated by the absence of purple-pink on the overlay in Fig. 4A, panels d andh) and colocalized preferentially with the nucleoplasmic pool of topoIIα during prophase (as indicated by yellow on the overlay in Fig. 4 A,panel d). These findings suggest that CK2 predominantly induced the MPM-2 phosphoepitope on nucleoplasmic topoIIα which then associated with the mitotic chromosomes.

DISCUSSION

We here present an alternative model t o account for the regulation of a cellcycle-specific phosphorylation site, in this case the mitotic MPM-2 phosphoepitope on topoisomerase IIα, 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 10 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 [START_REF] Escargueil | Serine 1524 is a major site of phosphorylation on human topoisomerase II alpha protein in vivo and is a substrate for casein kinase II in vitro[END_REF]. Although the identity of the latter protein is not known, its molecular weight corresponds to another major CK2 substrate, nucleolin, which like topoIIα forms stable complexes with CK2 [START_REF] Li | Identification of proteins that associate with protein kinase CK2[END_REF] and undergoes mitosis-specific phosphorylation [START_REF] Gulli | Mitosis-specific phosphorylation of gar2, a fission yeast nucleolar protein structurally related to nucleolin[END_REF]. Therefore, the proposed model is likely 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 to this classical model, it is the final substrate, topoIIα, that is subject to competing kinase and phosphatase activities as outlined in Fig. 4B. During interphase, CK2 is colocalized with topoIIα in the nucleus and is intrinsically able to induce the formation of the MPM-2 phosphoepitope. However, the additional colocalization with PP2A ensures that the MPM-2 site on Ser-1469 is kept unphosphorylated due to a combination of strong PP2A activity and weak CK2 activity toward 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 (Fig 4B ) without marked change in their expression levels [START_REF] Bosc | Expression and regulation of protein kinase CK2 during the cell cycle[END_REF]47]. PP2A is rapidly excluded from the nucleus while CK2 remains nucl ear until nuclear membrane break-down in prometaphase. 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 t ransit in G2 and early mitosis [START_REF] Skladanowski | Down-regulation of DNA topoisomerase IIalpha leads to prolonged cell cycle transit in G2 and early M phases and increased survival to microtubule-interacting agents[END_REF] while RNAi-mediated topoIIα downregulation delays G2 and early mitosis [START_REF] Sakaguchi | Functional compatibility between isoform alpha and beta of type II DNA topoisomerase[END_REF]. 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 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. The peptidyl-prolyl-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 [START_REF] Escargueil | Serine 1524 is a major site of phosphorylation on human topoisomerase II alpha protein in vivo and is a substrate for casein kinase II in vitro[END_REF][START_REF] Shen | The essential mitotic peptidyl-prolyl isomerase Pin1 binds and regulates mitosisspecific phosphoproteins[END_REF]. Interestingly, Pin1 has also been shown to bind and modulate Cdc25, an other key regulator of mitotic entry [START_REF] Stukenberg | Pin1 acts catalytically to promote a conformational change in Cdc25[END_REF]. 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 tumors. TopoIIα is overexpressed in several tumor types including lung and breast cancer and high levels of topoIIα expression is generally associated with more aggressive tumor behavior [START_REF] Giaccone | Differential expression of DNA topoisomerases in non-small cell lung cancer and normal lung[END_REF][START_REF] Koren | Prognostic value of Topoisomerase II in female breast cancer[END_REF]. CK2 is also overexpressed in many tumors such as breast, lung, and malignant melanomas and is generally considered as a cellular survival factor [START_REF] Ahmed | Joining the cell survival squad: an emerging role for protein kinase CK2[END_REF]. Interestingly, genetic studies in yeast suggest that CK2 facilitate G2 checkpoint override following DNA damage [START_REF] Toczyski | CDC5 and CKII control adaptation to the yeast DNA damage checkpoint[END_REF]. In contrast, current evidence suggest that PP2A is a tumor suppressor. Loss-of-function mutations of the structural subunit of PP2A have been described in lung and breast cancer as well as in

( 1 :

 1 200 dilution; Jackson ImmunoResearch, Bar Harbor, ME). Nuclear DNA was counterstained with To-Pro 3 (Molecular Probes, Eugene, OR) and the images were collected using a Radiance 2000 MP confocal microscope (BioRad Laboratories, Hemel Hempstead, UK).

15 FIGURE LEGENDS Fig. 1 .Fig. 2 .Fig. 3 .Fig. 4 . 4 Biochemical

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Biochemical Journal Immediate Publication. Published on 10-Jan-2007 as manuscript BJ20061460 © The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

The authors thank Dr. Virginie Poindessous for careful reading of the manuscript. This work was supported by grants from Association pour la Recherche sur le Cancer (ARC) grant n o 4659 and Fondation Recherche Médicale (FRM). A. E. E. was a fellow of FRM and the Federation of European Biochemical Societies (FEBS).


Juliette Fortpied

Didier VERTOMMEN Rita Gemayel

Didier Vertommen

Emile Van

Emile Van Schaftingen
email: vanschaftingen@bchm.ucl.ac.

Identification of protein-ribulosamine-5-phosphatase as human lowmolecular-weight protein-tyrosine-phosphatase-A

Keywords: Dependent Phosphatase-1, pNPPase, p-nitrophenyl-phosphatase, RN5Pase, ribulosamine- glycation, ribose 5-phospate, ribulosamine 5-phosphate, protein-repair, deglycation, protein-phosphatase

published or not. The documents may come

Identification of protein-ribulosamine-5-phosphatase as human low-molecular-weight protein tyrosine phosphatase-A

INTRODUCTION

Sugars with a free carbonyl group spontaneously react with the amino groups of proteins or low-molecular-weight compounds to form Schiff bases, which slowly rearrange into Amadori products (ketoamines). This reaction, designated glycation, is known to take place in vivo with glucose, giving rise to fructosamines. It must also take place with other monosaccharides or sugar phosphates, in particular glucose 6-phosphate and ribose 5-phosphate, which, though usually present at lower concentrations than glucose, are intrinsically more reactive than this hexose [START_REF] Haney | Glycosylation of hemoglobin in affinity labeling of hemoglobin by glucose-6-phosphate[END_REF][START_REF] Stevens | Nonenzymatic glycosylation of hemoglobin[END_REF][START_REF] Sharma | Amadori product and age formation during nonenzymatic glycosylation of bovine serum albumin in vitro[END_REF][START_REF] Sandwick | Maillard reactions of ribose 5phosphate and amino acids[END_REF]. It should be noted, however, that the higher reactivity of ribose 5-phosphate has only been demonstrated by measuring the formation of advanced glycation end-products, and that this difference does not necessarily reflect a difference in the rate of early glycation.

Protein glycation is apparently detrimental and it is therefore not surprising that cells have enzymes that repair proteins by removing the accessible glycation adducts. The first deglycating enzyme that has been described is fructosamine-3-kinase (FN3K), which, by phosphorylating fructosamines on their third carbon, causes their destabilisation and shedding from proteins [START_REF] Delpierre | Identification, cloning, and heterologous expression of a mammalian fructosamine-3-kinase[END_REF][START_REF] Szwergold | Human fructosamine-3kinase: purification, sequencing, substrate specificity, and evidence of activity in vivo[END_REF]. The physiological occurrence of enzymatic deglycation was demonstrated by showing that FN3K deficiency leads to an increase in intracellular protein glycation [START_REF] Delpierre | Fructosamine 3kinase is involved in an intracellular deglycation pathway in human erythrocytes[END_REF][START_REF] Veiga-Da-Cunha | Increased protein glycation in fructosamine-3-kinase deficient mice[END_REF].

FN3K takes care also of the glycation adducts formed from glucose 6-phosphate, after these have been dephosphorylated by a 'fructosamine-6-phosphatase' (FN6Pase) recently identified as Magnesium-Dependent Phosphatase-1 (MDP-1) [START_REF] Fortpied | Magnesium-dependent phosphatase-1 is a protein-fructosamine-6-phosphatase potentially involved in glycation repair[END_REF]. FN3K is only found in higher vertebrates, such as mammals and birds. However, many species including mammals [START_REF] Collard | A mammalian protein homologous to fructosamine-3-kinase is a ketosamine-3-kinase acting on psicosamines and ribulosamines but not on fructosamines[END_REF], birds, fishes [11], plants [START_REF] Fortpied | Plant ribulosamine/erythrulosamine 3-kinase, a putative protein-repair enzyme[END_REF] and bacteria (R.

Gemayel, unpublished results), contain a fructosamine-3-kinase homologue (FN3K-RP) that does not act on fructosamines, but only on ketoamines with a hydroxyl group in the Dconfiguration on C3, most particularly ribulosamines. We previously postulated that ribulosamines are produced by enzymatic dephosphorylation of ribulosamine 5-phosphates (RN5Ps), which form spontaneously from ribose 5-phosphate. The phosphatase involved in this process is, however, unknown. In the present work we have quantified the rate of early glycation by ribose 5-phosphate and identified in human erythrocytes a phosphatase, distinct from FN6Pase/MDP-1, that uses protein-RN5Ps as its best substrate.

EXPERIMENTAL

Materials

Reagents, of analytical grade whenever possible, were from Sigma, Acros or Merck. DEAE-Sepharose, SP-Sepharose, Q-Sepharose, Sephacryl S-200, Superdex-200 10/300 GL column, were purchased from Amersham Biosciences; Biogel P2 and AG 50W-X4 (100-200 mesh) were from Bio-Rad. Dowex 1-X8-200 was purchased from Acros and Vivaspin-2 centrifugal concentrators from Vivascience. Radiolabelled compounds were purchased from Amersham Biosciences or American Radiolabelled Chemicals, or synthesised as described in [START_REF] Fortpied | Magnesium-dependent phosphatase-1 is a protein-fructosamine-6-phosphatase potentially involved in glycation repair[END_REF]. [1-14 C]Ribose 5-phosphate was prepared by phosphorylation of [1-14 C]ribose with 25 mU of human recombinant ribokinase; the product was purified as previously described for [ 14 C]glucose 6-phosphate [START_REF] Fortpied | Magnesium-dependent phosphatase-1 is a protein-fructosamine-6-phosphatase potentially involved in glycation repair[END_REF]. FN3K-RP and MDP-1 were prepared as previously described [START_REF] Collard | Fructosamine 3-kinase-related protein and deglycation in human erythrocytes[END_REF][START_REF] Fortpied | Magnesium-dependent phosphatase-1 is a protein-fructosamine-6-phosphatase potentially involved in glycation repair[END_REF].

Preparation of ribulosamine 5-phosphates

Lysozyme glycated with [ 32 P]ribose 5-phosphate was prepared as previously described for lysozyme glycated with [ 32 P]glucose 6-phosphate [START_REF] Fortpied | Magnesium-dependent phosphatase-1 is a protein-fructosamine-6-phosphatase potentially involved in glycation repair[END_REF], but the incubation time was reduced to 4 h and Tris was used instead of Hepes. For the preparation of lysozyme glycated with unlabelled ribose 5-phosphate, hen egg lysozyme (60 mg/ml) was incubated for 16 h at 50°C with 20 mM ribose 5-phosphate in a medium containing 25 mM Tris, pH 7.1, 1 mM EGTA and 0.5 mM AlF 3 . The samples were purified on Dowex 1-X8 and Biogel P2 [START_REF] Fortpied | Magnesium-dependent phosphatase-1 is a protein-fructosamine-6-phosphatase potentially involved in glycation repair[END_REF] and the concentration of lysozyme was determined [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF].

Ribuloselysine 5-phosphate was prepared by incubating a solution (3 ml) containing 200 mM N-α-t-Boc-lysine and 100 mM ribose 5-phosphate for 4 h at 50°C. The t-Boc group was removed by overnight incubation in 1 N HCl at room temperature. The sample was diluted in 12 ml water and loaded on an AG 50W-X4 column (H + form, 3 cm 3 ) equilibrated with water. The column was washed with 12 ml water and 36 ml 0.5 M NaCl. Fractions of 3 ml were collected, and those containing ribuloselysine 5-phosphate (assayed by the inorganic phosphate released by alkaline phosphatase, in the elution fractions) were concentrated under vacuum and desalted on Biogel P2 equilibrated with water.

Preparation of tissue extracts and purification of ribulosamine-5-phosphatase

Tissues were collected from anaesthetized male Wistar rats. They were quick-frozen between Wollenberger clamps cooled in liquid nitrogen and kept at -70°C until used. The frozen tissues were homogenized in a Potter-Elvehjem device with 2 ml/g tissue of a buffer containing 25 mM Tris, pH 7.1, 1 mM dithiothreitol (DTT), 1 µg/ml leupeptin, 1 µg/ml antipain and 10 mM KCl (buffer A). Rat packed erythrocytes were washed twice with a solution containing 150 mM NaCl and 0.5 mM EDTA and once with 150 mM NaCl. 1 volume of washed erythrocytes was diluted in 10 volumes of buffer A. The homogenates and haemolysates were centrifuged for 20 min at 11 000 g and the resulting supernatants were used for enzyme assay. Protein concentration in tissue extracts was determined as described in [14] using bovine γ-globulin as a standard; haemoglobin concentration in erythrocyte extracts was determined by the method of Drabkin and Austie [START_REF] Drabkin | Spectrophotometric studies. Preparations washed blood cells; nitric oxide hemoglobin and sulfhemoglobin[END_REF].

For the purification of human ribulosamine-5-phosphatase (RN5Pase), 80 ml of packed erythrocytes were washed three times with 150 mM NaCl and diluted in 400 ml of a buffer containing 10 mM Tris, pH 8, 1 mM DTT, 1 µg/ml leupeptin and 1 µg/ml antipain.

The hemolysate was centrifuged for 20 min at 11 000 g. The supernatant (300 ml) was diluted twice in buffer B (20 mM Tris, pH 8, 2 mM DTT, 1 µg/ml leupeptin and 1 µg/ml antipain) and applied to a DEAE-Sepharose column (200 cm 3 ) equilibrated with the same buffer. The column was washed with 400 ml of buffer B and protein was eluted with a 0-0.25 M NaCl gradient in 1000 ml of buffer B. The most active fractions were pooled (55 ml), brought to 200 ml with buffer C (20 mM MES, pH 6, 2 mM DTT, 1 µg/ml leupeptin and 1 µg/ml antipain), and applied to an SP-Sepharose column (20 cm 3 ) equilibrated with buffer C. The column was washed with 50 ml of equilibration buffer and protein was eluted with a 0-0.75 M NaCl gradient in 200 ml of buffer C. Fractions of 3 ml were collected. Three peaks of RN5Pase were found, each co-eluting with p-nitrophenyl-phosphatase (pNPPase) activity, one in the flow-through and washing fractions ("peak I"), and two eluted with the salt gradient (peaks II and III). Peak I was concentrated to 35 ml by ultrafiltration in a 200-ml Amicon cell equipped with a PM-10 membrane. This sample was brought to 150 ml with buffer D (20 mM glycine, pH 9, 2 mM DTT, 1 µg/ml leupeptin and 1 µg/ml antipain) and applied to a Q-Sepharose column (20 cm 3 ) equilibrated with the same buffer. The column was washed with 50 ml of buffer D and developed with 200 ml of a 0-0.5 M NaCl gradient.

Fractions of 2.5 ml were collected. The most active ones (10 ml) were concentrated to 0.5 ml (with Vivaspin-2) and gel-filtered on a Superdex-200 10/300 GL column equilibrated with buffer E (25 mM Tris, pH 7.1, 1 mM DTT, 1 µg/ml leupeptin, 1 µg/ml antipain and 100 mM NaCl). Peaks II (18 ml) and III (21 ml) of the SP-Sepharose column were also concentrated and gel-filtered on Superdex-200 equilibrated with buffer E. Fractions of 0.5 ml were collected. The bands co-eluting with the RN5Pase activity in each Superdex-200 chromatography were cut out from a 14% (w/v) polyacrylamide-SDS gel and digested with trypsin or chymotrypsin. Peptides were analyzed by nanoelectrospray-ionisation tandem mass spectrometry as previously described [16].

Expression and purification of human low-molecular-weight protein-tyrosinephosphatases

The open reading frame of human low-molecular-weight protein-tyrosine-phosphatases (LMWPTPs) A and B (Accession Numbers AAP35800.1 and AAC52065.1), two isoforms generated by alternative splicing [17], were PCR-amplified using Pwo polymerase, human liver cDNA as template, a 5' primer containing the initiator codon (GCATATGGCGGAACAGGCTACCAAGT) in an NdeI site (in bold) and a 3' primer containing the putative stop codon (CGGATCCTCAGTGGGCCTTCTCCAAGAAC) flanked by a BamHI site (in bold). The 480-bp PCR-product was subcloned into pBlueScript.

Restrictions with BstUI, HaeIII and AvaII were performed to discriminate between the two isoforms, which were checked by sequencing. NdeI-BamHI fragments of each form were removed from pBlueScript and inserted into pET-3a. The resulting plasmids were used to transform E. coli BL21(DE3)pLys [START_REF] Studier | Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes[END_REF]. The expression and preparation of bacterial extracts were performed as described previously [START_REF] Fortpied | Plant ribulosamine/erythrulosamine 3-kinase, a putative protein-repair enzyme[END_REF]. The extract (12.5 ml) was diluted 3-fold with buffer F (25 mM Tris, pH 7.1, 5 mM DTT, 1 µg/ml leupeptin and 1 µg/ml antipain) and applied to a DEAE-Sepharose column (12 cm 3 ) equilibrated with the same buffer. This column was washed with 25 ml of buffer F and the flow-through and washing fractions of this column were pooled, concentrated and gel-filtered on Sephacryl S-200 (70 cm 3 ) equilibrated with buffer F. The fractions containing LMWPTP, as determined by SDS-PAGE analysis, were supplemented with 10% (w/v) glycerol and stored at -70°C. Protein concentration was estimated by measuring A 280 assuming extinction coefficients of 18 260 and 15 130 M -1 cm -1 for LMWPTP-A and LMWPTP-B, respectively. Starting from 250 ml of culture, we obtained for both isoforms ≈ 10 mg of pure protein.

Assay of phosphatase activities

FN6Pase and p-nitrophenyl-phosphatase activities were assayed as previously described [START_REF] Fortpied | Magnesium-dependent phosphatase-1 is a protein-fructosamine-6-phosphatase potentially involved in glycation repair[END_REF].

All other enzymatic assays were performed at 30°C in a medium containing 25 mM Tris, 1 mM DTT and the indicated concentration of substrate. Ribuloselysine-5-phosphatase activity was measured through the formation of inorganic phosphate [19]. Activities on 32 Pcompounds were determined in 50 µl of a solution containing 1 µM of phospho-groups (i.e., ≈ 7000 cpm); this corresponded to a protein concentration of 0.5 mM (lysozyme), 15 µM (myelin basic protein) and 50 µM (haemoglobin). The reactions were stopped by the addition of 1 mg bovine serum albumin (as a carrier) and 100 µl of ice-cold 10% (w/v) trichloroacetic acid. The samples were centrifuged for 10 min at 10 000 g and the radioactivity in the supernatant was counted. Unless otherwise stated, RN5Pase activity was measured using lysozyme glycated with [ 32 P]ribose 5-phosphate as a substrate (tested at 1 µM of phosphogroups).

Phosphorylation by FN3K-RP of the dephosphorylation products of LMWPTP

Lysozyme glycated with ribose 5-phosphate (5 mg/ml) was incubated for 10 min at 37°C in 20 µl of a solution containing 25 mM Tris, pH 7.1, 1 mM DTT and 20 µg/ml LMWPTP-A.

The samples were then incubated in 50 µl of the same medium containing 1 mM MgCl 2 , 1 mM EGTA, 100 µM ATP•Mg, 10 6 cpm [γ-32 P]ATP and 60 µg/ml spinach leaf ribulosamine-3-kinase [START_REF] Fortpied | Plant ribulosamine/erythrulosamine 3-kinase, a putative protein-repair enzyme[END_REF] or 280 µg/ml human erythrocyte FN3K-RP [START_REF] Collard | Fructosamine 3-kinase-related protein and deglycation in human erythrocytes[END_REF]. After the indicated times, aliquots (12 µl) were spotted on P81 papers, washed and counted for radioactivity as described previously [START_REF] Delpierre | Identification, cloning, and heterologous expression of a mammalian fructosamine-3-kinase[END_REF].

RESULTS

Glycation of proteins with ribose 5-phosphate

To get a better appraisal of the relative reactivity of ribose 5-phosphate and other sugars in early glycation, we measured the incorporation of 14 C-labelled ribose 5-phosphate, ribose and glucose 6-phosphate into lysozyme and albumin. As shown in Figure 1, ribose 5-phosphate reacted with lysozyme and albumin at rates that were 10-and 6-fold higher than ribose, and 15-and 24-fold higher than glucose 6-phosphate, respectively. As glucose 6-phosphate is itself 3-to 8-fold [START_REF] Fortpied | Magnesium-dependent phosphatase-1 is a protein-fructosamine-6-phosphatase potentially involved in glycation repair[END_REF] more powerful than glucose as a glycating agent, we calculated that ribose 5-phosphate is about 80-fold more reactive than glucose towards proteins, being therefore a very significant glycating agent (see Discussion).

Tissue distribution and purification of ribulosamine-5-phosphatase

As RN5Ps are not substrates for FN3K-RP (see below), we searched for an enzyme able to dephosphorylate RN5Ps in tissues and erythrocyte extracts. For this purpose, we used a radiolabelled substrate obtained by incubating lysozyme with [ 32 P]ribose 5-phosphate. As shown in Figure 2, this substrate (tested at a concentration of phospho-group of 1 µM) was dephosphorylated in all rat tissue extracts that were tested. Interestingly, the highest activity was observed with extracts derived from erythrocytes, a cell type in which both FN3K-RP and FN3K are particularly active [11]. In all cases, the enzymatic activity was > 90% inhibited by 50 mM Hepes (a property of LMWPTP, see below).

The purification of ribulosamine-5-phosphatase (RN5Pase) was started by fractionation of a human erythrocyte lysate on DEAE-Sepharose at pH 8. RN5Pase was eluted from this column as a single peak, which was separated from the peak of FN6Pase/MDP-1 but coincided with the peak of p-nitrophenyl-phosphatase (pNPPase) activity (Figure 3 A). Unlike FN6Pase/MDP-1, RN5Pase was not dependent on the presence of Mg 2+ (not shown) but was inhibited by Hepes (IC 50 = 1.2 mM). Upon further purification on SP-Sepharose at pH 6 (Figure 3 B), about 60% of the RN5Pase activity was found in the flow-through and washing fractions ("peak I"), and the rest was eluted as two distinct peaks with ≈ 200 (peak II) and ≈ 300 mM NaCl (peak III). Peaks II and III were further purified by gel-filtration on Superdex-200. In both cases, the enzyme co-eluted with an 18-kDa polypeptide, as determined by SDS-PAGE analysis (Figure 4 B, C, E andF). This 18-kDa polypeptide was the only band present in some of the active fractions of the gel-filtration column on which peak III had been loaded.

The 18-kDa bands derived from peaks II and III were submitted to chymotrypsin digestion and analysed by electrospray ionisation-tandem mass spectrometry. This led in both cases to the identification of two peptides matching residues 40-55 and 117-132 of low-molecularweight protein-tyrosine-phosphatase-A (LMWPTP-A) (Figure 4 G). RN5Pase from peak I of the SP-Sepharose column was purified by chromatography on Q-Sepharose at pH 9 and Superdex-200. SDS-PAGE analysis of the fractions obtained at this stage revealed the presence of several bands co-eluting with the enzymatic activity (Figure 4 A and D), including an 18-kDa band that was also identified as LMWPTP-A (Figure 4 G). The other bands found in these fractions were digested with trypsin and mass spectrometry analysis led to their identification as (1) phosphohistidine phosphatase (NP_054891), (2) thioredoxin (NP_003320) and (3) SH3-domain binding glutamic acid-rich protein (NP_112576).

Expression and substrate specificity of LMWPTPs

Human LMWPTP-A and LMWPTP-B, which differs from LMWPTP-A by an internal sequence due to an alternative splicing event [START_REF] Dissing | Human red cell acid phosphatase (ACP1). The amino acid sequence of the two isozymes Bf and Bs encoded by the ACP1*B allele[END_REF]17], were overexpressed in E. coli and purified to near-homogeneity by chromatography on DEAE-Sepharose and gel-filtration on Sephacryl S-200 (not shown). The activities of both recombinant proteins and of FN6Pase/MDP1 were tested on 32 P-labelled RN5Ps bound to hen egg lysozyme or bovine haemoglobin, on fructosamine 6-phosphates (FN6Ps) bound to hen egg lysozyme and on phosphotyrosine bound to myelin basic protein. All substrates were used at the same, very low concentration of phospho-groups (1 µM). As illustrated in Table 1 and Figure 5, RN5Ps (whether bound to lysozyme or to haemoglobin) were the best substrates for both forms of LMWPTP, followed by protein-phosphotyrosine and FN6Ps, whereas a different substrate preference (FN6P >> RN5P > P-Tyr) was observed with FN6Pase/MDP1. Despite its high activity on protein-bound RN5Ps, LMWPTP-A was unable to dephosphorylate completely protein-RN5Ps : RN5Ps bound to lysozyme and haemoglobin were dephosphorylated to extents of 75% (Figure 5) and 55% (not shown), respectively. This suggests that some of the RN5P residues are inaccessible to the phosphatase. None of the two forms of LMWPTP enhanced the decomposition rate of ribulosamine 3-phosphates bound to lysozyme, indicating that they did not act as ribulosamine-3-phosphatases (not shown).

Interestingly, LMWPTP-A acted much better than LMWPTP-B on all four protein substrates (Table 1) as well as on free ribuloselysine 5-phosphate (activities at 1 mM : 1.6 ± 0.14 versus 0.08 ± 0.01 µmol/min/mg, n = 3), whereas a similar activity was found when pnitrophenylphosphate was used as a substrate (activities at 1 mM : 4.7 ± 0.35 versus 4.9 ± 0.49 µmol/min/mg, n = 3).

Conversion of ribulosamine 5-phosphates to substrates for FN3K-RP

Protein-bound RN5Ps were neither phosphorylated by human FN3K-RP, nor by its spinach leaf homologue, as indicated by the lack of incorporation of radioactivity from [ 32 P]ATP into protein (Figure 6). As ribulosamine 3,5-bisphosphates are most likely more labile than ribulosamine 3-phosphates, we also measured the formation of [ 32 P] inorganic phosphate under the same conditions and found it to be undetectable. This confirmed the absence of phosphorylation of RN5Ps by both ketoamine-3-kinases. As expected, preincubation of lysozyme-bound RN5Ps with LMWPTP-A led to the formation of a substrate for both plant and human ribulosamine-3-kinases (Figure 6).

DISCUSSION

Previous work has shown that ribose 5-phosphate is a potent glycating agent [START_REF] Sandwick | Maillard reactions of ribose 5phosphate and amino acids[END_REF]. However, the technique used to measure the glycation rate-the formation of UV absorbing materialdoes not allow one to make a precise comparison of the reactivity of different sugars, because the formation of UV-absorbing material involves reactions downstream of the formation of Amadori products and therefore largely depends on the reactivity of the Amadori products themselves. The present work shows that ribose 5-phosphate reacts with proteins about 10fold more rapidly than ribose and 80-fold more rapidly than glucose. This higher reactivity was obtained with freshly prepared, purified radiolabelled compounds, and identical results were obtained with [ 14 C] and [ 32 P]ribose 5-phosphate (not shown). This indicates that there was no interference of contaminating, highly reactive radioactive impurities, which could potentially lead to an overestimation of the glycation rate [START_REF] Trueb | Nonenzymatic glycosylation of proteins. A warning[END_REF].

Since the intracellular ribose concentration appears to be < 10 µM (unpublished results) as compared to 6-45 µM for ribose 5-phosphate [START_REF] Casazza | The content of pentose-cycle intermediates in liver in starved, fed ad libitum and meal-fed rats[END_REF][START_REF] Vincent | Increase in phosphoribosyl pyrophosphate induced by ATP and Pi depletion in hepatocytes[END_REF], the much higher reactivity of ribose 5-phosphate compared to ribose indicates that ribulosamines most likely mainly arise from ribose 5-phosphate. At a (physiological) concentration of 25 µM, ribose 5-phosphate has a glycating power amounting to about 40% of that of 5 mM glucose, which indicates that it is indeed a significant glycating agent. It may therefore make sense to have a system allowing the deglycation of RN5Ps.

FN3K-RP and its plant homologue are devoid of RN5P-3-kinase activity. A phosphatase is therefore needed to convert RN5Ps to ribulosamines. We show in the present work that such a phosphatase is present in mammalian tissues and erythrocytes. The human erythrocyte enzyme was purified and identified as LMWPTP-A. This identification was based on mass spectrometry analysis of purified RN5Pase, on the finding that like LMWPTP it is not dependent on Mg 2+ but is inhibited by Hepes [START_REF] Zhang | Crystal structure of bovine low molecular weight phosphotyrosyl phosphatase complexed with the transition state analog vanadate[END_REF], and on the demonstration that recombinant LMWPTP-A acts as a RN5Pase. Erythrocyte LMWPTP-A separated into three peaks upon chromatography on SP-Sepharose. This is possibly the result of genetic heterogeneity (a common polymorphism replaces Gln105 by Arg, [26]), post-translational modification, oligomerization or association with other proteins.

LMWPTP was initially identified as a phosphatase acting on low-molecular-weight substrates, most particularly FMN and p-nitrophenylphosphate, but very poorly on sugar phosphates [START_REF] Tsuboi | Acid phosphatase. VI. Kinetic properties of purified yeast and erythrocyte phosphomonoesterase[END_REF]. Later work showed it to act also as a protein-tyrosine-phosphatase, but not as a protein-serine/threonine-phosphatase. This, together with the presence of a sequence motif reminiscent of tyrosine phosphatases, led to this enzyme being renamed 'low-molecularweight protein-tyrosine-phosphatase'. We now show that this enzyme acts particularly well as a protein-RN5Pase. Interestingly, this substrate is structurally similar to one of the best lowmolecular-weight substrates, FMN, which contains a ribitol 5-phosphate portion. The crystal structure of LMWPTP shows it to have a deep and narrow catalytic pocket [START_REF] Su | The crystal structure of a low-molecular-weight phosphotyrosine protein phosphatase[END_REF]29,[START_REF] Zhang | Crystal structure of bovine low molecular weight phosphotyrosyl phosphatase complexed with the transition state analog vanadate[END_REF]. This is presumably why this enzyme is unable to remove phosphate groups bound to the first atom of an amino acid side-chain (serine and threonine phosphates), but that it is well active on tyrosine-phosphates and RN5Ps, where the phosphate is bound to the 5 th and 10 th atom of the amino acid side-chain, respectively. The low activity of LMWPTP on fructosamine 6phosphates is most likely due to the fact that the latter are essentially under their furanose forms [START_REF] Wiame | Identification of a pathway for the utilization of the Amadori product fructoselysine in Escherichia coli[END_REF], which are probably too bulky to enter the catalytic site. By contrast, RN5Ps are unable to cyclize. The lack of action of LMWPTP on ribulosamine 3-phosphates is presumably also related to the narrowness of the catalytic site, which is probably unable to accommodate a secondary phosphate group when the neighbouring carbons bear a hydroxyl group. The lack of ribulosamine-3-phosphatase activity is functionally important : its presence would prevent deglycation to take place since the latter involves phosphate elimination.

LMWPTP is unable to dephosphorylate all the RN5Ps that form on a protein that has reacted with ribose 5-phosphate. Similar observations have been made with other proteins that are bona fide protein-repair enzymes such as FN3K [START_REF] Delpierre | Identification of fructosamine residues deglycated by fructosamine-3-kinase in human hemoglobin[END_REF] and protein-isoaspartylmethyltransferase [START_REF] Johnson | Partial repair of deamidation-damaged calmodulin by protein carboxyl methyltransferase[END_REF]. This is most likely due to a problem of accessibility of some of the modified residues to these enzymes, as was previously discussed for fructosamine-6phosphatase/MDP-1 [START_REF] Fortpied | Magnesium-dependent phosphatase-1 is a protein-fructosamine-6-phosphatase potentially involved in glycation repair[END_REF].

In mammals and birds, two different forms of LMWPTP exist, which differ from each other by an internal sequence comprising residues 40-73 that border the catalytic cleft [START_REF] Dissing | Human red cell acid phosphatase (ACP1). The amino acid sequence of the two isozymes Bf and Bs encoded by the ACP1*B allele[END_REF]26].

We found that the A form is intrinsically much better than the B form in its action as a RN5Pase both on low-molecular-weight and protein-bound substrates, but that this is not the case for p-nitrophenylphosphate. This difference may possibly be due to the presence in LMWPTP-A of a glutamate (Glu50, replaced by Asn in the B-form), which could potentially form an ionic pair with the amino group of RN5Ps. FN3K-RP is widely distributed in mammalian tissues and is particularly active in erythrocytes, a cell type with no protein renewal and in which therefore protein-repair may be particularly critical. The tissue distribution of RN5Pase indicates that it is also widely distributed in tissues, consistently with other studies [START_REF] Wo | Sequencing, cloning, and expression of human red celltype acid phosphatase, a cytoplasmic phosphotyrosyl protein phosphatase[END_REF][START_REF] Magherini | Cloning of murine low molecular weight phosphotyrosine protein phosphatase cDNA: identification of a new isoform[END_REF], and particularly active in erythrocytes. Although we do not know at this stage if this high activity reflects or not a high relative content of LMWPTP-A, it is consistent with the idea that LMWPTP has a role in protein maintenance besides its role in cell signalling.

Further work is needed to appreciate the physiological importance of the dephosphorylation of RN5Ps as a protein repair mechanism. The identification of an enzyme that dephosphorylates RN5Ps is an important step in this endeavour.

LEGENDS TO THE FIGURES Figure 1 Glycation of proteins with ribose 5-phosphate, ribose or glucose 6-phosphate

Hen egg lysozyme (A) or bovine serum albumin (B) were incubated with [ 14 C]ribose 5phosphate (R5P; triangles and squares), ribose (circles and diamonds) or glucose 6-phosphate (G6P; cross) and the indicated concentrations of unlabelled compound. After different times, the radioactivity bound to proteins was measured, as described in [START_REF] Fortpied | Magnesium-dependent phosphatase-1 is a protein-fructosamine-6-phosphatase potentially involved in glycation repair[END_REF]. Means ± S.E.M. (n = 3).

Figure 2 Tissue distribution of ribulosamine-5-phosphatase

The enzymatic activity was determined on crude extracts (1-3 mg protein/ml in the assay), using lysozyme glycated with [ 32 P]ribose 5-phosphate as a substrate (tested at 1 µM of phospho-group). Conditions used for these assays were such that the activity was proportional with time and with extract concentration and that less than 20% of the protein-RN5Ps were dephosphorylated. The results are the means ± S.E.M. for independent determinations performed on samples from 3 different animals. They are expressed as the activity per mg of protein or haemoglobin (in the case of erythrocytes).

Figure 3 Purification of ribulosamine-5-phosphatase from human erythrocytes

(A) A human erythrocyte extract was applied to a DEAE-Sepharose column and a linear NaCl gradient was applied. A 415 (circles), RN5Pase (squares), pNPPase (diamonds) and FN6Pase (triangles) were assayed. The flow-through and washing fractions (1000 ml; not shown) contained 17 mg/ml hemoglobin and less than 10% of the total RN5Pase activity. (B)

RN5Pase was further purified on SP-Sepharose at pH 6. Three peaks of phosphatase were found, one in the flow-through and washing fractions (I) and two eluted with the NaCl gradient (II and III).

Figure 4

 4 Figure 4 Further purification of the three peaks of RN5Pase on Superdex-200 and copurification with an 18-kDa polypeptide

Figure 5

 5 Figure 5 Dephosphorylation of protein substrates by human LMWPTP-A

Figure 6

 6 Figure 6 Formation of a substrate for FN3K-RP upon incubation of lysozyme with ribose 5-phosphate and dephosphorylation by LMWPTP-A

  Fig.1

Fig. 2 E r y t h r o c y t e

 2 Fig.2

Table 1 Substrate specificity of human LMWPTPs and MDP-1

 1 The activites were determined with pure recombinant enzymes. Means ± S.E.M. (n = 3) or

	individual values.
		Specific activity a (nmol/min/mg)
	Substrate	LMWPTP-A LMWPTP-B MDP-1/FN6Pase
	Lysozyme glycated with [ 32 P]ribose-5P	5.95 ± 0.005 0.28 ± 0.007	1.46 ± 0.156
	Lysozyme glycated with [ 32 P]glucose-6P	0.26 ± 0.121 0.04 ± 0.001	10.8 ± 0.298
	Myelin basic protein-[ 32 P]P-Tyr	0.93 ± 0.038 0.14 ± 0.014	0.31 ± 0.049
	Hemoglobin glycated with [ 32 P]ribose-5P	2.63 ; 3.05	0.66 ; 0.86	1.16 ; 1.29

a At 1 µM of phospho-groups.

Biochemical Journal

Immediate Publication. Published on 2 May 2007 as manuscript BJ20061485 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 2 May 2007 as manuscript BJ20061485 © 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

The authors thank Dr. Maria Veiga-da-Cunha and Catherine Peel for their help in the determination of the tissue distribution. This work was supported by grants from the European Foundation for the Study of Diabetes, the Juvenile Diabetes Foundation, the Interuniversity Attraction Pole Programme -Belgian Science Policy (Network P6/05) and the Actions de Recherches Concertées of the French Community of Belgium. R.G. is fellow of the Fonds pour l'Encouragement à la Recherche dans l'Industrie et dans l'Agriculture. D.V. was supported by the Interuniversity Attraction Pole Program -Belgian Science Policy (P6/28).