Downregulation of protein phosphatase 2A activity in HeLa cells at the G2-mitosis transition and unscheduled reactivation induced by 12-O-tetradecanoyl phorbol-13-acetate (TPA)

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European Journal of Cell Biology 84 (2005) 719–732 www.elsevier.de/ejcb

Downregulation of protein phosphatase 2A activity in HeLa cells at the G2-mitosis transition and unscheduled reactivation induced by 12-O-tetradecanoyl phorbol-13-acetate (TPA) Manuela Klingler-Hoffmanna, Holger Barthb, James Richardsa, Norbert Ko¨niga, Volker Kinzela, a

Former Department of Pathochemistry, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany b Department of Pharmacology and Toxicology, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany Received 26 January 2005; received in revised form 28 April 2005; accepted 29 April 2005

Abstract In the cell cycle the transition from G2 phase to cell division (M) is strictly controlled by protein phosphorylation–dephosphorylation reactions effected by several protein kinases and phosphatases. Although much indirect and direct evidence point to a key role of protein phosphatase 2A (PP2A) at the G2/M transition, the control of the enzyme activity prior to and after the transition are not fully clarified. Using synchronized HeLa cells we determined the PP2A activity (i.e. the increment sensitive to inhibition by 2 nM okadaic acid) in immunoprecipitates obtained with antibodies raised against a conserved peptide sequence (residues 169–182, Ab169/182) of the PP2A catalytic subunit (PP2A C). Two different substrates were offered: the phospho-peptide KR(p)TIRR and histone H1 phosphorylated by means of the cyclin-dependent protein kinase p34cdc2. The results indicate that in HeLa cells the specific activity of PP2A towards both substrates goes through a minimum in late G2 phase and stays low until metaphase. Treatment of G2 cells with TPA (10
7 M) caused a reactivation of the downregulated PP2A activity within 20 min, i.e. the same time frame within which TPA was shown earlier to block HeLa cells at the transition from G2 to mitosis [Kinzel et al., 1988. Cancer Res. 48, 1759–1762]. Activation of PP2A was also induced by TPA in mitotic cells. The low activity of PP2A in mitotic cells was accompanied by a strong reaction of mitotic PP2A C with anti-P-Tyr antibodies in Western blots, which was reversed by treatment of mitotic cells with TPA. The results suggest that the activity of cellular PP2A requires downregulation for the transition from G2 phase to mitosis. Unscheduled reactivation of PP2A induced by TPA in late G2 phase appears to inhibit the progress into mitosis. r 2005 Elsevier GmbH. All rights reserved. Keywords: PP2A; G2/mitosis; Downregulation; Unscheduled reactivation; Phorbol ester; TPA; G2 inhibition

Abbreviations: AT procedure, amethopterin/thymidine synchronization procedure; MPF, M-phase promoting factor; OA, okadaic acid; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; PP2A C, catalytic subunit of PP2A; TPA, 12-O-tetradecanoyl phorbol-13-acetate Corresponding author. Tel.: +49 6221 423 253; fax: +49 6221 423 249. E-mail address: [email protected] (V. Kinzel). 0171-9335/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2005.04.002

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Introduction The profound transformation of a cell that occurs as it traverses from interphase to mitosis is regulated at the molecular level to a great extent by a precisely timed cascade of protein phosphorylation/dephosphorylation reactions involving a number of conserved protein kinases and protein phosphatases. Well established is the cyclin-dependent protein kinase p34cdc2 alias cdc2 or cdk1 in complex with cyclin B named M-phase promoting factor (MPF) which becomes activated at the G2/M transition (for review, see Dunphy, 1994). Checkpoints in the cell cycle serve to control the completion of individual steps in the cascade prior to progression to the next and thus guarantee the cellular integrity, e.g. by allowing repair processes to take place. The delay of the transition from G2 phase to mitosis caused by a number of chemical and physical influences is a perfect example. Protein phosphatase 2A (PP2A) represents a highly regulated family of abundant phosphoserine/phosphothreonine phosphatases in eukaryotic cells (Cohen, 1989, 1991) which is indispensable in numerous cellular processes including the cell division cycle (for review see Mayer-Jaekel and Hemmings, 1994). The enzyme activity is regulated in several ways including different regulatory subunits, post-translational modifications, second messengers and inhibitory proteins and it may vary depending on individual substrates and intracellular location (for review, see Janssens and Goris, 2001). The catalytic subunit C (existing in two variants a and b), and a constant regulatory subunit A (or PR65; also existing in two isoforms a and b) represent core dimers to which one of several variable B subunits may be bound forming heterotrimers and giving rise to a number of PP2A isozymes. The expression of PP2A subunits has been found to be constant throughout the cell cycle (Ruediger et al., 1991). A role of PP2A and the regulation of its activity at the transition from G2-phase to cell division is indicated by the following observations. An extract prepared from frog oocytes which inhibited the entry into meiosis by preventing the activation of MPF (Cyert and Kirschner, 1988) was shown to contain enzymatically active PP2A as the inhibitory principle (Lee et al., 1991, 1994), thus suggesting that an active state of PP2A may counteract entry into cell division. In contrast, the specific inhibition of PP2A-type enzymes in various cells with low concentrations of okadaic acid (OA) has been shown to induce a premature entry into both meiosis and mitosis, suggesting that a less active or inactive state of PP2A may facilitate the transition from G2 phase to cell division (Goris et al., 1989; Felix et al., 1990; Yamashita et al., 1990). Ruediger et al. (1991) did not observe changes in the activity of PP2A during the cell cycle. Sontag et al.

(1995) measured the activity of microtubuli-associated PP2A in highly synchronized cells and found that the activity was diminished if not inhibited during G2 and in mitosis. Turowski et al. (1995) showed that the methylation state of PP2A C is altered during the cell cycle. The published evidence for the involvement of PP2A at a number of biochemical steps during the preparation of the cell for division has been comprehensively reviewed by Janssens and Goris (2001). PP2A is necessary to keep the cdk1/cyclin B complex in its inactive precursor form by regulating cdk-activating kinase (CAK) and Wee1 kinase activity. In addition, it is thought that PP2A operates upstream of cdc25-C (Clarke et al., 1993), the protein phosphatase which is involved in the final activation of cdk1 at the entry into cell division. A PP2A-type enzyme seems to be involved in keeping cdc25-C inactive prior to mitosis by dephosphorylation of activating sites (Clarke et al., 1993). In vertebrates cdc25-C is activated by phosphorylation at these serine and threonine residues by polo-like kinase (Karaiskou et al., 1998) and subsequently by cdk1 itself causing full activation of cdc25-C within a feedback loop responsible for maximal activation of cdk1 for mitosis (Hoffmann et al., 1993). Incubation of enzymatically active cdc25-C isolated from mitotic cells with PP2A in vitro has been shown to cause an inactivation of the former (Clarke et al., 1993) thus raising the possibility that PP2A operates upstream of cdc25-C. Moreover, PP2A represents a major enzyme that dephosphorylates other physiological products of cyclin-dependent protein kinases (Agostinis et al., 1992; Ferrigno et al., 1993; Mayer-Jaekel et al., 1994). Little is known about the actual regulation of PP2A activity prior to and during mitosis up to metaphase. The study of PP2A in mammalian cells has been hindered so far by the failure to stably overexpress functional PP2A by standard gene transfer techniques (Green et al., 1987; Wadzinski et al., 1992) due to an autoregulatory control exerted at the translational level (Baharians and Schonthal, 1998). To analyze PP2A activity at the G2/M transition and in mitosis we used highly synchronous HeLa cells and determined the phospho-peptide phosphatase activity ex vivo, i.e. in immunoprecipitates obtained with specific polyclonal anti-peptide antibodies raised against the PP2A catalytic subunit and determined the enzyme activity which was sensitive to a low concentration of OA. This fraction is considered to reflect PP2A. As substrates we used a phospho-peptide and phosphohistone H1 previously phosphorylated by means of cdk1 (alias p34cdc2). The elucidation of molecular events in the course of the cell cycle is facilitated by analysis of checkpoints which may also become obvious through the inhibition induced by various chemical and physical means. The well-known inhibitory influence of X-irradiation on the

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G2/M transition of mammalian cells has been shown to be mimicked by TPA (Kinzel et al., 1980). Similar to Xirradiation, treatment with TPA influences the G2mitosis transition in HeLa cells in two different ways. Xrays and TPA cause (i) a delayed passage through G2 of cells exposed in the first half of the previous S phase and (ii) an immediate and transient blockage of cells which are in G2 during the treatment (Kinzel et al., 1981; Xu et al., 2002). Important for this study is the second case. In this context it was shown that TPA rapidly prevents the activation of cdk1 (Barth and Kinzel, 1994). Subsequently, it was demonstrated that TPA causes a rapid decrease in the specific enzyme activity of already active cdc25-C in G2 cells (Barth et al., 1996), thus indicating that under these circumstances cdc25-C may not reach the threshold activity required to initiate activation of cdk1 for the transition to mitosis. Moreover, the specific activity of cdc25-C of mitotic cells treated with TPA was decreased as well. These results raised the possibility that treatment with TPA may effect the activity of PP2A of cells in G2 phase as well as in mitosis. The data presented indicate that the specific activity of PP2A goes through a minimum in the late G2 phase which lasts at least until metaphase and that the application of TPA to cells in G2 phase and in mitosis rapidly reactivates the enzyme. The untimely reactivation of downregulated PP2A in G2 cells by TPA prior to the respective G2-mitosis checkpoint may help to explain why the phorbol ester counteracts the G2/ mitosis transition in HeLa cells.

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upon reseeding in fresh culture medium in the absence of nocodazole. For the treatment of HeLa cells with 10
7 M TPA (Sigma) the phorbol ester was dissolved in acetone (0.2% final concentration in the medium).

Cell lysate For the preparation of cell lysates, cultures were washed twice with phosphate-buffered saline (PBS) at 4 1C either in dishes (adherent cells) or by sedimentation (non-adherent cells). Subsequently, the cells kept on ice were covered with or suspended in a small volume (about 0.2 ml per 107 cells) of lysis buffer (adapted from Turowski et al. (1995): 10 mM HEPES/NaOH, pH 7.5, 10 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 1.5 mM MgCl2, 25% glycerol, 0.1% Nonidet P 40, CompleteTM, a cocktail of protease inhibitors (Roche, 1 tablet per 10 ml) for about 15 min before they were scraped off and/or vortexed. Lysates not to be used for enzyme measurements contained, in addition, 1 mM vanadate and 50 mM sodium fluoride. The lysate was incubated on ice for another 15 min, centrifuged (13,000g) and the protein content of the supernatant was determined according to Bradford (1976) using bovine serum albumin as the standard.

Polyclonal peptide-specific antiserum against PP2A C, Ab169/182 The antiserum against the internal sequence GLSPSIDTLDHIRA182 of the PP2A C common to both isoforms a and b (Green et al., 1987; Favre et al., 1994; Stone et al., 1987; Arino et al., 1988) was obtained as follows: The peptide was prepared by an in-house peptide synthesis facility (Dr. H.R. Rackwitz; solid phase synthesis; standard Fmoc methodology). The peptide was purified by RP-HPLC, coupled to keyhole limpet hemocyanin (KLH) by the use of glutaraldehyde and sent for commercial immunization of rabbits according to standard procedures to Eurogentec (Belgium). The rabbits were finally bled after 80 days of immunization. Antisera were assayed for specificity by preincubation of diluted serum (1:1000) with the antigenic peptide (0.1 mM) for 30 min at room temperature. Preimmune sera served for further control.

169

Materials and methods Cell culture and synchronization HeLa cells were cultured as monolayers and synchronized as described (Kinzel et al., 1981, 1988; Mueller and Kajiwara, 1969). Briefly, for the study of the G2 phasemitosis transition cells were synchronized at the G1–S border (and in S) by treatment with amethopterin (10
6 M, Calbiochem) for 16 h and released by thymidine (10 mg per 106 cells, Sigma) – the so called AT procedure. The adherent cell fraction was obtained after removal of the loosely attached mitotic cells by shake off. The cell cycle progress of the adherent fraction was followed by FACS analysis of the DNA content after DAPI staining (Kinzel et al., 1981). The mitotic activity was determined microscopically. The non-adherent fraction collected by shake off consisted mainly of mitotic cells at different states. More highly enriched mitotic cells were obtained by synchronization with nocodazole (2.7  10
7 M, Sigma) for 21 h. Here the harvest obtained by shake off consisted of about 98% cells in metaphase. More than 90% of those divided

Further antibodies and antisera used Anti-PP2A C peptide295/309 antibody (monoclonal, mouse) and anti-P-Tyr 4G10 (monoclonal, mouse) (UBI, USA); anti-peptide antiserum against the PP2A A subunit PR 65 (rabbit, polyclonal) raised with PR 65 peptide177/196 (present in both, PR 65a and b; synthesized in-house and KHL-coupled as above)

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(Eurogentec); anti-peptide antiserum against the PP2A B subunit PR 55a (rabbit, polyclonal) raised with PR 55a peptide14/27 (Calbiochem); anti-peptide antiserum against PPX (alias PP4) (rabbit, polyclonal) raised with PPX peptide294/307 (Calbiochem); antiserum against p34cdc2 C-terminus (rabbit, polyclonal) (Gibco). All secondary antibodies were conjugated with peroxidase: goat anti-rabbit and sheep anti-mouse (Dianova); pig anti-rabbit (Genosys Biotech., England).

Immunoprecipitation For immunoprecipitation, aliquots of cell lysates (0.1–2 mg protein) were precleared with 50 ml of a 1:2 dilution of protein-A-agarose beads (Roche) in lysis buffer (1 ml) for 2 h at 4 1C and sedimented by centrifugation. Immunoprecipitation of PP2A was carried out by an incubation with Ab169/182 (4 ml) overnight at 4 1C on a slow rotator. Subsequently, the sample was further rotated with 20 ml of a 1:2 dilution of protein-A-agarose for 2 h at 4 1C, the immunoprecipitates were collected by centrifugation and washed 3 times with 50 mM Tris/HCl, pH 7.5. Analysis of the supernatant as well as of the protein A-bound proteins by SDS-PAGE and immunoblotting using Ab169/182 exhibited PP2A C only in the protein A-bound fraction indicating that the enzyme was removed by immunoprecipitation.

Determination of PP2A activity For this purpose the dephosphorylation usually of two different substrates – a phospho-peptide and p34cdc2-phosphorylated histone H1 – was measured in parallel. Both assays were done in the absence and in the presence of OA (2 nM) to which PP2A is especially sensitive (IC50 ¼ 0:1 nM with a maximum inhibition at 1 nM (Haystead et al., 1989; Gauss et al., 1997)); the plus OA samples were preincubated with OA for 10 min. Hence PP2A activity is defined as the phospho-peptide phosphatase activity inhibited by 2 nM OA (Cohen et al., 1989; Sola et al., 1991). The phospho-peptide phosphatase activity of immunoprecipitated PP2A was assayed using a microtiter plate assay (UBI, Lake Placid). By the use of the phospho-peptide at 0.5 mM concentration it emerged that immunoprecipitates from 0.1 mg cell lysate protein allowed the measurement under linear conditions for at least 30 min. The immuno-precipitates were incubated in 50 ml with the synthetic phospho-peptide KR(p)TIRR (0.5 mM; a modified sequence of the EGF receptor which becomes phosphorylated by PKC (Harder et al., 1994)) for 30 min at room temperature. The immunoprecipitates were removed by centrifugation. The released phosphate was determined by the addition of

100 ml malachite green solution (one volume 4.2% (w/v) ammonium molybdate in 4 M HCl was added to 3 volumes of 0.045% malachite green, 0.01% (v/v) Tween 20 was added before use) to 20 ml of the supernatant. The absorbance at 595 nm was determined after 15 min using an ELISA reader. The calibration curve was linear up to at least 1 nmol Pi. The analysis of the phospho-histone H1 phosphatase activity was carried out with PP2A immunoprecipitated from cell lysate as described above. For phosphorylation of histone H1 the protein kinase p34cdc2 was immunoprecipitated from mitotic HeLa cells synchronized with nocodazole (Mayer-Jaekel et al., 1994). Mitotic cell lysate equivalent to 1 mg protein was incubated with a polyclonal antiserum (4 ml) against p34cdc2 (rabbit; from Gibco, Germany) and the immunoprecipitate was collected with protein A sepharose. Washed kinase beads were incubated with histone H1 and [g-32P]ATP for 30 min at 37 1C. The specific radioactivity of histone H1 was in the range of 2  105–106 cpm/nmol. The amount of 32P-histone H1 dephosphorylated within 30 min at 30 1C by immunoprecipitates obtained with Ab169/182 from 0.1 mg lysate protein of asynchronous HeLa cells was 30%, a prerequisite to stay within linear conditions (Cohen et al., 1988). Under the conditions used, linearity of dephosphorylation was observed until at least 50 min. For routine assays, immunoprecipitates were resuspended in 20 ml lysis buffer. The phosphatase reaction was initiated by the addition of 10 ml 32P-histone H1 (final concentration 30 mM) and was carried out for 30 min at 30 1C. The reaction was stopped by the addition of SDS sample buffer and the entire supernatant was subjected to SDSPAGE (10% gels). The gels were stained with Coomassie blue, dried, and autoradiographed. Moreover, the histone bands were excised, incubated in 2 ml 30% H2O2 overnight at room temperature, mixed with 2 ml scintillation cocktail (Aquasafe 500, Zinsser Analytik, Frankfurt, Germany) and analyzed by liquid scintillation counting. Alternatively, the radioactivity was determined by phospho-imaging. Each determination with or without OA was performed in triplicate. The difference of the mean values represents the OA-sensitive protein phosphatase activity, i.e. the PP2A activity. In the case of (almost) missing PP2A activity sometimes a computed negative value may result simply because of variations of the measurements within the OA minus and the OA plus groups. Experiments were carried out at least 2 times; usually 3 times.

SDS gel electrophoresis, isoelectric focusing, immunoblotting SDS-PAGE was carried out according to Laemmli (1970). Isoelectric focusing (IEF) for two dimensional

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separations was done in the Immobiline Dry Strip system supplied by Pharmacia Biotech (Freiburg, Germany) according to the manufacturer’s instructions. Proteins were electroblotted onto Immobilone-P membranes, incubated either for 1 h at room temperature or overnight at 4 1C in blocking buffer (PBS containing 2% (w/v) bovine serum albumin and 0.05% Tween-20) and further in PBS with 0.05% Tween-20 containing the antibodies in appropriate dilution for 1 h at room temperature (polyclonal) or overnight at 4 1C (monoclonal). After incubation with horseradish peroxidasecoupled anti-rabbit IgG appropriately diluted in blocking buffer enhanced chemiluminescene was detected with the Amersham ECL system. For measurements by MALDI-mass spectrometry (MS), immunoprecipitated proteins were separated by SDS-PAGE. Coomassie blue-stained protein bands were excised, cut into small pieces and – after incubation with buffer and acetonitrile – dried, soaked in a small volume of buffer with trypsin (50 mM NH4HCO3, 5 mM CaCl2, 10% acetonitrile, 12.5 ng/ml trypsin (modified, Promega, Madison, USA)) and incubated at 37 1C overnight (kindly performed in house by Dr. Tore Kempf). MALDI-MS analysis of the supernatants was kindly performed in house by Dr. Martina Schno¨lzer (Central Protein Analysis Facility).

Results Characterization of peptide antisera against PP2A subunits from HeLa cell An important tool for this work was the anti-peptide antibody Ab169/182 against PP2A C which was raised and named according to Favre et al. (1994). This antigenic site of the catalytic subunit was chosen since it was likely to be recognized by such an antibody independent of modification(s) at the C-terminus known to be involved in the regulation of the enzyme (Favre et al., 1994). The antibody detected a major protein at about 36 kDa by Western blot (WB) analysis in cell lysates, a response which was lost after preincubation with the antigenic peptide (Fig. 1, left panel). Preimmune serum was ineffective (not shown). A cross reaction with the related protein phosphatase PPX (alias PPP4; 65% sequence homology (Cohen, 1991)) was not detectable as the PPX-specific antiserum detected in HeLa cells a single band clearly below PP2A C at about 34 kDa, a response which could be erased by preincubation with the respective PPX peptide294/307 (Fig. 1, right panel). A cross reaction of the antibody with PP1 catalytic subunit was excluded using phosphatase isolates obtained from HeLa cells by microcystin affinity chromatography (to

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Fig. 1. Specificity of Ab169/182 analyzed by Western blotting. Lysate of asynchronous HeLa cells (0.1 mg protein per lane) was separated by SDS-PAGE, blotted onto PVDF membranes and assayed with Ab169/182 and anti-PPX antiserum without (
) or with (+) preincubation with PP2A C peptide169/182 or PPX peptide294/307, respectively, as indicated (100 mM, 30 min, room temperature).

be published). WB analysis of HeLa cell lysates with anti-peptide antisera against other PP2A subunits detected PR 65 and PR 55a at the appropriate apparent molecular weights in gels (data not shown). Immunoprecipitation of HeLa cell lysates either with affinity-purified Ab169/182 or with a monoclonal antipeptide Ab against PP2A C295/309 and subsequent Western blotting with Ab169/182 resulted in single bands each at about 36 kDa (not shown). After immunoprecipitation with Ab169/182 for enzyme activity measurements, the remaining cell lysate was devoid of PP2A C as revealed by Western blotting with the same antibody. Protein staining of immunoprecipitates separated by SDS-PAGE resulted in a number of bands. Analysis of a tryptic digest from a band at about 36 kDa by MALDIMS exhibited a number of mass values characteristic of tryptic PP2A C peptides (Table 1).

OA-sensitive protein phosphatase activity in immunoprecipitates from HeLa cell lysates PP2A is characterized by its inhibition by very low concentrations of OA. To analyze if OA-sensitive phosphatase activity can be immunoprecipitated with Ab169/182, the antibody was incubated with cell lysate from asynchronous HeLa cells. Immunoprecipitates were assayed in presence or absence of OA (2 nM) with the synthetic phospho-peptide KR(p)TIRR and with histone H1 phosphorylated by p34cdc2. The latter has been considered a specific substrate of the PP2A holoenzyme containing in addition to PR 65 the variable PR55a subunit (Sola et al., 1991). The data (Fig. 2A, B demonstrate that in the presence of OA less phosphate is

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Table 1.

M. Klingler-Hoffmann et al. / European Journal of Cell Biology 84 (2005) 719–732

Mass values of tryptic peptides from a 36-kDa protein immunoprecipitated by Ab169/182

Mass value [MH]+

Theoretical mass value (monoisotopic)

Sequence of tryptic PP2A C peptides

795.33 951.84 1340.84 1793.25 2402.96

794.40 950.46 1339.66 1791.78 2402.10

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Fig. 2. Comparison of protein phosphatase activity in immunoprecipitates of asynchronous HeLa cells in the absence and presence of okadaic acid (OA). Immunoprecipitates (obtained with Ab169/182 from 0.1 mg protein aliquots of cell lysate) were assayed at room temperature with phosphopeptide KR(p)TIRR (0.5 mM) (A) as the substrate in the absence (black columns) or presence (gray columns) of OA (2 nM); the released phosphate was determined by the malachite green assay at 595 nm. (B) In addition, immunoprecipitates were assayed for phosphatase activity at 30 1C with histone H1 (30 mM) (radioactively phosphorylated by means of p34cdc2; for experimental details, see Materials and methods). Here the remaining histone-bound phosphate was determined. Samples were preincubated with OA for 10 min; the assays were performed for 30 min. Shown are the results of triplicate determinations.

released than in its absence, i.e. OA-sensitive phosphatase activity can be effectively immunoprecipitated. The OA-sensitive phosphatase activity, i.e. the difference between the results measured without and with 2 nM OA, is subsequently referred to as PP2A activity.

PP2A in asynchronous and mitotic cells In order to determine if the low PP2A activity reported by Sontag et al. (1995) for the microtubuleassociated enzyme from mitotic cells might be paralleled by that measurable in immunoprecipitates obtained with Ab169/182 from lysed mitotic HeLa cells, we compared synchronized mitotic with asynchronous HeLa cells. The results show that the PP2A activity of HeLa cells in mitosis is lower than that in asynchronous cells (Fig. 3).

GEPHVTR302 YGNANVWK144 284 YSFLQFDPAPR294 75 SPDTNYLFMGDYVDR89 50 CPVTVCGDVHGQFHDLMELFR70 137

Fig. 3. Comparison of PP2A activity of asynchronous HeLa cells with that of mitotic cells. Immunoprecipitates of asynchronous cells (black columns) and mitotic cells (gray columns) were assayed (A) with the phospho-peptide or (B) with phospho-histone H1. Mitotic cells were obtained by use of nocodazole. Assay conditions were as given in Fig. 2. Shown are the differences obtained in the absence and presence of OA from triplicate determinations.

The PP2A activities measured with the phospho-peptide are mirrored by that obtained with phospho-histone H1. A low PP2A activity has been observed in mitotic HeLa cells obtained by nocodazole treatment (Fig. 3) or by shake off from monolayer cultures after presynchronization with the AT procedure (data not shown). The low enzyme activity therefore appears to be characteristic of mitotic cells independent of the synchronization procedure. Data from WB analysis indicate that it was the specific enzyme activity which is decreased in mitotic cells. Lysates from asynchronous and mitotic cells exhibited no obvious differences in the detection of PP2A C with Ab169/182 (see below) and none in the case of the regulatory subunits PR 65 and of PR 55a with respective antibodies (data not shown). Similarly, immunoprecipitates obtained with Ab169/182 from asynchronous and mitotic cells did not exhibit any obvious differences in the detection of PP2A C by WB analysis with Ab169/182 (see below). These results show (i) that the lysates of asynchronous and mitotic cells contain equal amounts of PP2A C and (ii) that comparable amounts were immunoprecipitated. This interpretation is in agreement with the notion that the intracellular concentration of PP2A C remains constant during the cell

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cycle (Virshup et al., 1989). These data as well as the further results indicate that Ab169/182 recognizes PP2A C independent of changes in the specific activity of the enzyme described in this paper. The inhibitory principle did apparently not interfere with the immunoprecipitation of the enzyme by Ab169/182.

PP2A in G2 cells In order to determine the beginning of the decay of PP2A activity towards mitosis in the HeLa cell cycle the cells were synchronized in G2 by the AT procedure. The maximum of G2 cells in the adherent cell fraction (X60%) was obtained at about 971 h after release from synchronizing blockage with amethopterin by addition of thymidine (Fig. 4A). At this time about 30% were in G1 and a small percentage in prophase. Prior to the enzyme analysis of the adherent cell fraction the nonadherent (i.e. largely mitotic) cells were shaken off and discarded; the adherent cells were lysed and immunoprecipitated with Ab169/182. The PP2A activity of the immunoprecipitates was measured with the phospho-peptide (Fig. 4C) and independently with phospho-histone H1 as substrates (Fig. 4D). It is evident that in both cases the PP2A activity passed through a minimum. Three independent experiments were carried out separately with each substrate. Dependent on the synchrony essentially comparable results were obtained. In the individual experiments the minima were at 8, 9, respectively, 10 h with phospho-peptide and at 8, respectively, twice at 9 h with phospho-histone H1, i.e. when the majority of cells approached G2 and entered mitosis. Beyond 9–10 h the PP2A activity of the attached cells started to rise, apparently because divided cells, i.e. early G1 cells had reattached to the vessel. The data show that the PP2A activity decreases already in G2 while the cell is still attached to the culture vessel. The detached state of mitotic cells as such does not seem to be responsible for the low PP2A activity. The low PP2A activity in G2 cells appears to result from a decrease in specific activity as 7–11 h after release from synchronizing blockage lysates of the adherent cell fraction did neither exhibit differences in the amount of PP2A C detected by immunoblotting (Fig. 4B) nor in the amount of PR65 subunit (not shown), and immunoprecipitates obtained with Ab169/182 from such lysates also did not exhibit any differences in the immunoblotting analyses for PP2A C or co-precipitated PR 65 (not shown).

Fig. 4. PP2A activity of HeLa cells synchronized by the AT procedure at the G2/mitosis transition. (A) Typical cell cycle distributions of an AT synchronization experiment 7–11 h after release from blockage evaluated by FACS analysis of the adherent cell fraction: 2n cells (K); 4n cells (J), and by counting mitotic figures per field (&) (mf/field) independently. The maximal percentage of G2 cells (4n in the adherent fraction) is at about 9 h after release (465%) followed 1 h later by the maximal mitotic activity (470%). (B) Western blot analysis of cell lysates (0.1 mg each) for PP2A C with Ab169/182. (C, D) PP2A activity (difference of measurements without and with 2 nM OA) of immunoprecipitates determined as in Fig. 2 with phospho-peptide as the substrate (C) and with phospho-histone H1 (D). Shown are results from triplicate determinations.

Reaction of PP2A C with anti-P-Tyr antibodies A decrease of the specific enzyme activity of PP2A has been reported after in vitro phosphorylation of PP2A C

by src-tyrosine kinase (Chen et al., 1992) at tyrosine. Therefore, we compared the reactivity of PP2A C from mitotic and non-mitotic HeLa cells with anti-P-Tyr

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antibodies in immunoblots. For this purpose, mitotic cells were collected by different methods and the enzyme was immunoprecipitated from lysates with Ab169/182 and compared with PP2A C precipitated from asynchronous HeLa cells (Fig. 5A). The PP2A C from mitotic cells exhibits in all cases a much stronger staining with antiP-Tyr antibodies than that from asynchronous cells (Fig. 5A, right). The reactivity with Ab169/182 , however, did not differ (Fig. 5A, left). The reactivity with Ab169/ 182 and with anti-P-Tyr antibodies also co-localized in immunoblots from two-dimensional gels (IEF in the first and SDS-PAGE in the second dimension) of lysate from mitotic cells. PP2A C was usually represented by two proteins focusing between pH 5 and 5.5 (see also Chen et al., 1994) (Fig. 5B). The strong reaction of mitotic PP2A C with anti-P-Tyr antibodies appears to be correlated with a low PP2A activity (see Fig. 3). In the case of G2 cells, however, this correlation was not detectable. WB analysis of immunoprecipitated PP2A (Ab169/182) from G2-enriched cell fractions assayed 7–11 h after release from synchronizing blockage did not exhibit a pronounced staining with anti-PTyr antibodies at 8 or 9 h (not shown), i.e. the time of the lowest enzyme activity (Fig. 4). Despite considerable effort it has been so far impossible to detect phospho-Tyr residues in mitotic PP2A C directly. Attempts to label mitotic PP2A C

metabolically with 32Pi failed: (i) HeLa cells preincubated during an amethopterin/thymidine synchronization with 32Pi did not proceed into mitosis. (ii) Incubation of mitotic cells (obtained with nocodazole) with 32Pi did not lead to a PP2A C-related radioactive signal after separation by SDS-PAGE. It has not been possible to detect P-Tyr residues by electrospray tandem MS in tryptic peptides of PP2A C purified by microcystin affinity chromatography from mitotic cells (Lehmann et al., data to be published). The sequence covered by the tryptic fragments, even though not complete, included the C-terminus of the enzyme containing Tyr 307, the residue reported by Chen et al. (1992) to be phosphorylated in vitro. It should be noted, however, that it has been possible in those experiments to confirm Thr 320 phosphorylation of copurified mitotic PP1 catalytic subunit (Kwon et al., 1997; Puntoni and Villa-Moruzzi, 1997). Element mass spectrometry (LC-ICP-MS) of tryptic peptides from mitotic PP2A C also failed to detect phosphorus although this procedure was sensitive enough to find phosphorus in an equivalent amount of catalytic subunit of mitotic PP1 (to be published). It cannot be excluded at present that during the purification of mitotic PP2A C or the tryptic digestion required for MS–MS analysis the enzyme may become dephosphorylated at Tyr residue(s) during some step, e.g. due to the proneness to autodephosphorylation of such residues (Chen et al., 1992, 1994).

Effect of TPA treatment on the PP2A activity of cells in G2 phase

Fig. 5. Reactivity of PP2A C from HeLa cells with anti-P-Tyr antibodies. (A) PP2A C was immunoprecipitated by Ab169/182 from lysates (2 mg protein each) of asynchronous and mitotic HeLa cells. Immunoprecipitates were separated by SDSPAGE, blotted onto PVDF membranes and detected (WB) with Ab169/182 (left) or with anti-P-Tyr antibodies (right). Lanes 1, 3: asynchronous cells, adherent fraction; lanes 2, 4: mitotic cells. (B) Mitotic HeLa cells obtained by nocodazole treatment were lysed and protein (1 mg) was precipitated by 4 volumes of acetone (at
20 1C). The rehydrated sample was separated by isoelectric focusing (anode (+) left; cathode (
) right) and SDS-PAGE, blotted and detected (WB) with anti-PTyr antibodies (right) and after stripping re-probed with Ab169/182 (left).

Our earlier work suggested the possibility that TPA may inhibit the transition from G2 phase to mitosis in HeLa cells by influencing the activity of PP2A (Barth et al., 1996). To test this we treated HeLa cells in G2 phase with TPA and measured their PP2A activity in immunoprecipitates. For this purpose cells were synchronized with the AT procedure and treated at the time when the population contained the maximal number of G2 cells and the lowest PP2A activity (Fig. 4; 9 h values). The cells were treated for 20 min with TPA or with solvent control. As a control for the minimal PP2A activity in G2 cells the analysis of untreated cells at 9 and 10 h was included. TPA caused in G2 cells a substantial reactivation of PP2A towards both substrates as shown in Fig. 6. For the explanation of apparently ‘‘negative’’ data, see the Materials and methods section. A 10-min treatment with TPA was not sufficient to effect reactivation (not shown). Treatment with TPA did not change the immunoprecipitability of PP2A C with Ab169/182 as detected by WB analysis (data not shown). Direct addition of TPA to the phosphatase assays did not

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Fig. 6. Influence of TPA on PP2A activity of HeLa cells in G2 phase. HeLa cells synchronized by the AT procedure were treated 9 h after release from blockage, i.e. at the expected G2 maximum (see Fig. 4A) for 20 min with TPA (10
7 M) (TPA) or with acetone (0.2%) (Ac) for solvent control. Cells were lysed, 0.1 mg protein aliquots were immunoprecipitated with Ab169/182 and assayed for PP2A activity (difference of measurements without and with 2 nM OA ) as described in Fig. 2 either with phospho-peptide (A) or with phosphohistone H1 (B). For control of the expected low PP2A activity in G2 phase (see Fig. 4C, D) in the individual experiments, in addition, untreated cells were assayed 9 and 10 h after release from synchronizing blockage as indicated. Shown are the results obtained from triplicate determinations. For the explanation of apparently ‘‘negative’’ data see the Materials and methods section.

influence the enzyme activity (not shown). An increase in the PP2A activity was also observed in lysates of TPA-treated G2 cells (not shown). The results indicate that TPA induces a reactivation of downregulated PP2A in G2 cells.

Effect of TPA treatment on the PP2A activity of cells in mitosis The results obtained with G2 cells raised the possibility that the low PP2A activity of mitotic cells was also sensitive to incubation with the phorbol ester, especially since TPA was shown earlier to elicit specific responses in mitotic cells (Klein et al., 1997). Treatment with TPA for 20 min of mitotic cells obtained by different methods caused in all instances an activation of the PP2A activity. In Fig. 7 the results obtained with immunoprecipitates from mitotic cells (nocodazole treatment or AT synchronization) are shown. The fact that TPA induced reactivation of PP2A activity in adherent G2 cells as well as in detached mitotic cells indicates that this reactivation occurs independent of the physical state of the cell. WB analysis of lysates of TPA-treated G2 cells and mitotic cells did not point to changes in the protein level

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Fig. 7. Influence of TPA on PP2A activity of HeLa cells in mitosis. HeLa cells were synchronized in mitosis by nocodazole treatment (A) or by the AT procedure (B) and treated for 20 min with acetone (0.2%, solvent control) (black columns) or with TPA (10
7 M) (gray columns). Immunoprecipitates (from 0.1 mg protein aliquots) obtained with Ab169/182 were assayed for PP2A activity (difference of measurements without and with 2 nM OA) using phospho-histone H1 as the substrate. Shown are the results obtained from triplicate determinations.

of the enzyme when compared with solvent-treated control cells (not shown). Moreover, the treatment with TPA did not alter the precipitability of PP2A C with Ab169/182 as evident from WB analysis of cell lysate (see below) and of immunoprecipitates (not shown). Therefore, the results indicate that TPA had induced an increase of the specific enzyme activity of previously less active PP2A in G2 as well as in mitotic cells. In asynchronous HeLa cells, which in contrast to mitotic cells exhibit a pronounced PP2A activity, the enzyme was not further stimulated upon treatment with TPA (data not shown), thus indicating that it is the downregulated form of PP2A which is subject to activation induced by TPA. The observations that neither the decrease of PP2A activity in G2 phase and mitosis nor the stimulation by TPA of cellular PP2A in these cells appeared to be accompanied by a change in the amount of enzyme protein suggested again that a reversible protein modification may contribute to the regulation of the specific activity of the enzyme.

Reactivity of PP2A C from TPA-treated mitotic cells with anti-P-Tyr antibodies The pronounced reaction of mitotic PP2A C with anti-P-Tyr antibodies was accompanied by a low enzyme activity. Based on our previous results we hypothesized that a TPA-effected reactivation of PP2A in mitotic cells should go along with a decrease of the staining of PP2A C with an anti-P-Tyr antibody. This

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Fig. 8. Influence of TPA on the reactivity of PP2A C from mitotic HeLa cells with anti-P-Tyr antibodies. HeLa cells were synchronized in mitosis by nocodazole treatment and incubated for 20 min with acetone (0.2%, solvent control) (lanes 1, 3) or with TPA (10
7 M) (lanes 2, 4). Cell lysates (2 mg protein aliquots) were immunoprecipitated by the use of Ab169/182. The precipitates were separated by SDS-PAGE, blotted onto PVDF membranes and assayed (WB) with Ab169/182 for control (left panel) or anti-P-Tyr antibodies (right panel) as indicated.

was shown to be the case (Fig. 8). The TPA-induced reduction in the reactivity of blotted PP2A C with the anti-P-Tyr antibodies was observed independent of the method by which the mitotic cells had been obtained. These data indicate that the reactivity with this antibody reflects a property of mitotic PP2A C which is related to a low specific enzyme activity and that it can be reversed by treatment of the cells with TPA leading to a more active state.

Discussion Indirect evidence had indicated that the enzyme activity of PP2A might be strictly regulated at the G2–M phase transition (Lee et al., 1991, 1994; Felix et al., 1990; Yamashita et al., 1990; Kinoshita et al., 1993). In this paper we approached the determination of the PP2A activity of HeLa cells at the transition from G2 phase to mitosis and in metaphase. In addition, we asked the question if TPA, a compound known from our studies to rapidly induce an inhibition of the G2–mitosis transition (Kinzel et al., 1980, 1988), may affect the cellular PP2A activity as earlier work had suggested (Barth et al., 1996). The results show that the specific enzyme activity of PP2A goes through a minimum in G2 phase and remains low until metaphase. Treatment of cells in G2 phase and in mitosis with TPA induces a reactivation of the PP2A activity within 20 min. The unscheduled reactivation of PP2A prior to the G2–M checkpoint is likely to be involved in TPAeffected G2 inhibition. Immunoprecipitates from cell lysates were used for the determination of the PP2A activity. The work with immunoprecipitates suffers from the immobilized state of the enzyme as such as well as from the fact that the enzyme activity may be influenced by the bound antibody. The advantage, however, of using immuno-

precipitates for the phosphatase analysis consists of the relatively native conditions without any major modification/demodification and/or selective enrichment by any isolation procedure. The measurements were done under linear conditions (see Materials and methods section), thus allowing a quantitative comparison within the same experimental group. The molecular cause for the differences in the specific enzyme activities apparently did not interfere with the immunoprecipitability by Ab169/182. The substrates chosen for the determination of the enzyme activity, the phospho-peptide as well as the histone H1 phosphorylated by p34cdc2 have been frequently used for the determination of PP2A activity. The latter has even been considered specific for trimeric PP2A containing the variable subunit PR55a (Sola et al., 1991). The pattern of the phosphatase activity obtained from cells in different states was measured without exception with both substrates, indicating that the same enzyme entity was probably responsible for the results. The measurements by Sontag et al. (1995) of microtubule-associated PP2A using phosphorylated myosin light chains as the substrate also showed a low activity of the enzyme obtained from cells in G2 and mitosis. Our results, although obtained with immunoprecipitates from total cell lysate and different substrates, run in parallel with their observations. The decrease in late G2 could result from a number of reasons considering the variability in the subunit composition of the PP2A enzymes and the known possibilities for covalent regulation as well as for noncovalent control of the enzyme activity by activators and inhibitors including shifts in the substrate specificity (for review see (Janssens and Goris, 2001)). However, the model system did not allow evaluation of these possibilities in more detail. Src kinase has been shown to catalyze the phosphorylation of PP2A C at Tyr in vitro and to attenuate the phosphatase activity at the same time (Chen et al., 1992). In subsequent studies, the reactivity of PP2A C with anti-P-Tyr antibody was taken as a measure for the modification (Chen et al., 1994). In our experiments the reactivity of PP2A C with anti-P-Tyr antibodies is also inversely correlated with the activity when the enzyme of mitotic cells is compared with that of asynchronous cells. The easily detectable immunoreactivity in the blot is contrasted by a low enzyme activity towards both substrates. Moreover, the TPA-induced stimulation of the mitotic enzyme activity is accompanied by a decrease of the reaction with anti-P-Tyr antibodies in the blot. These observations seem to indicate that the reactivity of PP2A C with anti-P-Tyr antibodies reflects circumstances which are involved in the control of the enzyme. In our hands physical demonstration of P-Tyr in mitotic PP2A C has failed so far (to be published). If we

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postulate an in vivo Tyr phosphorylation in mitotic cells such a result may, however, not be surprising in view of published observations that argue for a rapid autodephosphorylation in vitro unless the enzyme is not continuously inhibited with OA and which may even take place under exceptional conditions such as blotting (Chen et al., 1992, 1994). In the course of any purification and digestion procedure of PP2A C the Tyr-phosphorylated enzyme may take the slightest chance to autodephosphorylate with the result that P-Tyr residues escape the physical detection. Therefore, firm evidence for a Tyr phosphorylation is yet to be shown. The tendency for autodephosphorylation may explain the observation that the attenuated PP2A activity in G2 cells was not accompanied by an increased reactivity of PP2A C with anti-P-Tyr antibodies in WB. In this case about p40% of the cells were not in G2, i.e. carried active PP2A C which could possibly be responsible for such dephosphorylation of the remainder during the subsequent analysis. Alternatively, Tyr phosphorylation of PP2A may not occur in G2 but only in mitosis. In this case, however, an additional mechanism for the decrease of PP2A activity in late G2 has to be assumed. The phosphorylation of proteins at the entry into cell division represents a widely used cellular mechanism to regulate their biological activity in a compatible manner (Shalloway and Shenoy, 1991; Roche et al., 1995). In the case of PP2A the downregulation of the catalytic function by modifying the catalytic subunit would guarantee the attenuation at the central part of the otherwise heteromeric enzyme. The catalytic subunit of another abundant protein phosphatase, PP1, has indeed been shown to be downregulated in mitosis by phosphorylation (in this case at a threonine residue, Thr 320; (Kwon et al., 1997; Puntoni and Villa-Moruzzi, 1997)). Several protein kinases, among them the Src kinase, were indeed shown to be upregulated towards mitosis (Shalloway and Shenoy, 1991; Roche et al., 1995). Since we failed to detect phosphorylation of Tyr residues of mitotic PP2A C physically so far in these experiments, it is possible that the anti-P-Tyr antibodies detect another important epitope of the protein. On the basis of our data we are convinced that the reactivity of mitotic PP2A C with anti-P-Tyr antibodies reflects an essential modification of the enzyme influencing its activity. The biochemical nature of this modification is yet to be elucidated. As a possibility for the untimely reactivation of mitotic PP2A by treatment with TPA the reversion of an inhibitory post-translational modification might be considered. Provided Tyr phosphorylation of PP2A C is responsible for or at least partly involved in the attenuation of the enzyme activity in mitotic cells, an activation induced by TPA could be triggered by an

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induced dephosphorylation. We have shown earlier that TPA-induced signaling works in mitotic cells as well as in G2 cells (Klein et al., 1997). Treatment with TPA could activate either the autodephosphorylating capacity of PP2A itself (Chen et al., 1992, 1994) or a P-Tyr phosphatase (Heimerl et al., 1995; den Hertog et al., 1995). Therefore it is in principle possible that the TPA-induced reversal of the pronounced reactivity of mitotic PP2A C with the anti-P-Tyr antibody might be effected in this way. Experiments with OA had already indicated that PP2A is involved in the G2/M transition: injection of OA into oocytes causes the appearance of active MPF (Goris et al., 1989; Picard et al., 1991); moreover, OA induces the activation of cdk 1 in extracts obtained from cells in interphase (Felix et al., 1990) and it elicits prematurely mitosis-specific events in BHK cells synchronized in the S phase (Yamashita et al., 1990). Collectively, these observations suggest that the downregulation of the PP2A activity is essential to allow the necessary activation of MPF for the initiation of mitosis-specific events. The premature activation of the otherwise attenuated PP2A activity in late G2 phase by treatment of HeLa cells with TPA and the simultaneous inhibition in G2 support a negative role of active PP2A on the G2/M transition. In view of the rapidness of the events elicited by TPA in G2 cells the phorbol ester causes an unscheduled activation of the already downregulated enzyme within the cell which is practically ready to enter mitosis, i.e. very late in G2. We have shown earlier by time-lapse analysis that HeLa cells continue to enter prophase for about 20 min after starting the treatment with TPA (10
7 M), then no further cells followed, they stopped in G2 (Kinzel et al., 1988), i.e. exactly after the time required to reactivate PP2A. It is unknown if the radiomimetic activity of TPA in the G2/mitosis transition leading to the immediate G2 inhibition utilizes pathways which are also activated by X-rays. This requires a detailed separate study. The immediate G2 delay elicited by X-rays has been shown to depend on the ataxia telangiectasia mutated (ATM) protein kinase (Xu et al., 2002). ATM-dependent cellular responses induced by ionizing radiation extend to the control of certain protein kinases and protein phosphatases as well as to the phosphorylation status of a number of proteins (Guo et al., 1999; Chen et al., 2001; Sapkota et al., 2002; Saito et al., 2002; Gatei et al., 2003) including influences in the nuclear PP1 as well as PP2A (Guo et al., 2002a, b). Active PP2A seems to interfere with the activation of MPF firstly through prevention of activating (via CAK) and/or support of inhibiting (probably via Wee1) phosphorylations (Lee et al., 1994; Kinoshita et al., 1993); see also (Janssens and Goris, 2001) and secondly by prohibiting the phosphatase cdc25-C to become fully activated by multiple phosphorylations rendering it

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incapable of removing inhibitory phosphorylations from MPF. In G2 cells TPA was shown earlier to rapidly prevent the activation of cdc25-C as well as that of p34cdc2, the catalytic subunit of MPF (Barth and Kinzel, 1994; Barth et al., 1996). The unscheduled reactivation of PP2A in G2 cells may not allow cdc25-C and subsequently MPF to reach the threshold phosphorylation or dephosphorylation state required for their full activation to pass from G2 phase to mitosis.

Acknowledgments We thank Drs. Ingrid Hoffmann and Jennifer Reed for critically reading the manuscript and Angelika Lampe-Gegenheimer for expert secretarial assistance. Part of the work was supported by the Deutsche Forschungsgemeinschaft.

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