Cell Cycle Regulation and p53 Activation by Protein Phosphatase 2Calpha *

Paula OfekDagger , Daniella Ben-MeirDagger , Zehavit Kariv-InbalDagger , Moshe Oren§, and Sara LaviDagger

From the Dagger  Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv 69978, Israel and § Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel

Received for publication, November 18, 2002, and in revised form, December 30, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein phosphatase 2C (PP2C) dephosphorylates a broad range of substrates, regulating stress response and growth-related pathways in both prokaryotes and eukaryotes. We now demonstrate that PP2Calpha , a major mammalian isoform, inhibits cell growth and activates the p53 pathway. In 293 cell clones, in which PP2Calpha expression is regulated by a tetracycline-inducible promoter, PP2Calpha overexpression led to G2/M cell cycle arrest and apoptosis. Furthermore, PP2Calpha induced the expression of endogenous p53 and the p53-responsive gene p21. Activation of the p53 pathway by PP2Calpha took place both in cells harboring endogenous p53, as well as in p53-null cells transfected with exogenous p53. Induction of PP2Calpha resulted in an increase in the overall levels of p53 protein as well as an augmentation of p53 transcription activity. The dephosphorylation activity of PP2Calpha is essential to the described phenomena, as none of these effects was detected when an enzymatically inactive PP2Calpha mutant was overexpressed. p53 plays an important role in PP2Calpha -directed cell cycle arrest and apoptosis because perturbation of p53 expression in human 293 cells by human papillomavirus E6 led to a significant increase in cell survival. The role of PP2Calpha in p53 activation is discussed.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein phosphorylation is accepted as a central event in both the maintenance of normal cell metabolism and the pathogenesis of disease and is an integral part of the response to extracellular signals, protein turnover, transcription regulation, and numerous other cellular processes. The highly conserved protein phosphatase 2C (PP2C)1 family is one of four major groups of serine/threonine phosphatases in eukaryotes (1, 2). This class consists of monomeric phosphatases and is distinguished from the other groups by its dependence on divalent ions such as Mg2+. Several independent reports suggest that different members of this family regulate transcription of genes controlling growth-related pathways in mammals (3-6). The roles played by PP2C in response to stress have been identified in Arabidopsis (7, 8) as well as in yeast and mammalian cells (5, 9, 10).

The human genome contains at least 6 PP2C paralogs (UniGene, National Institutes of Health), among which PP2Calpha (also referred to as PPM1A) is the most characterized member (11). A growing list of substrates has been suggested to be specifically dephosphorylated by PP2Calpha in eukaryotic cells (12-20). This broad substrate specificity suggests that PP2Calpha has diverse functions and that it may play a central role in the regulation of stress response, gene expression, and replication. Still, because of the absence of specific inhibitors and the presence of multiple paralogs, the precise role of PP2Calpha in mammalian cells is not known. It is, therefore, important to identify and better characterize its specific physiological function(s).

The p53 tumor suppressor exerts its antiproliferative effects, including growth arrest, apoptosis, and replicative cell senescence in response to various types of stress (21-26). The tumor-suppressor function of p53 involves its ability to act as a sequence-specific transcription factor (27). Numerous p53 target genes regulating cell cycle and apoptosis have been identified, including p21, which plays a critical role in the induction of cell cycle arrest (28, 29), as well as a large array of proapoptotic genes (26).

In normal cells, under nonstressed conditions, p53 is a short-lived protein whose activity is maintained at a low level through its interaction with MDM2, which targets it for proteasomal degradation (30). The precise molecular mechanisms involved in p53 activation are not completely understood. Posttranslational modifications such as phosphorylation, dephosphorylation (31, 32), and acetylation (33, 34) are all thought to be involved in this process. Some of these modifications may stabilize the protein by interfering with MDM2 binding, whereas others may transform it from a latent to an active form or alter its cellular localization (35, 36).

In the present study we analyze the role of PP2Calpha as a negative growth regulator. In cells containing endogenous wt-p53, PP2Calpha overexpression mediates cell cycle arrest in the G2/M phase followed by apoptosis. This PP2Calpha -directed growth arrest is imposed through the activation of p53. Although PP2Calpha induction considerably augments p53 transcriptional activation, cell cycle arrest, and apoptosis, attenuation of p53 rescues the growth-arrested phenotype and leads to increased survival. These findings implicate p53 as a downstream mediator of the antiproliferative effects of PP2Calpha .

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- For PP2Calpha -pcDNA3.1 (wt-PP2Calpha ), a rat cDNA library (37) was screened with probes derived from the rat PP2Calpha gene (38), yielding several PP2Calpha clones. A clone encoding the complete PP2Calpha cDNA was isolated and converted to a plasmid, enabling CMV promoter-driven transcription of the inserted cDNA. The complete coding sequence of PP2Calpha was amplified by PCR and cloned into expression vector pcDNA3.1 (Invitrogen), between the HindIII and ApaI sites. Sequence accuracy was verified by DNA sequencing after cloning.

For PP2Calpha D239A (mut-PP2Calpha ), PCR was performed to introduce the mutation D239A into PP2Calpha . Two PCR reactions were conducted in parallel, using PP2Calpha -pcDNA3.1 as the template. In the first, the upstream primer introducing the mutation in the sense direction was 5'-CTTGCATGTGCTGGCATCTGG-3'; the downstream primer was SP6- 5'-ATTTAGGTGACACTATAG-3'. In the second reaction, the upstream primer contained the internal PP2Calpha sequence 5'-ACACGGTGCAGATAGAAGTG-3' and the downstream primer contained the mutation in the antisense direction 5'-CCAGATGCCAGCACATGCAAG-3'. After purification, the resultant PCR products from the two parallel reactions were mixed and used as a template in a final PCR reaction mix containing the two external primers (the upstream primer with the internal PP2Calpha sequence and the downstream with the SP6 primer). The resultant PCR product was cloned into PP2Calpha -pcDNA3.1 via the EcoRI and ApaI sites. The sequence accuracy of the mut-PP2Calpha was verified by sequencing.

For inducible PP2Calpha (PP2Calpha -pcDNA4/TO), PP2Calpha (wt or mut) was isolated from the pcDNA3-based vectors described above, via the HindIII and ApaI sites and subcloned into the pcDNA4/TO vector (T-Rex system, Invitrogen).

A human wild-type p53 expression vector (pC53SN3) was kindly provided by B. Vogelstein. Reporter plasmids encoding firefly luciferase under the control of human mdm2 and cyclin G promoters were as described (39, 40). Firefly luciferase under the Rous sarcoma virus (RSV) promoter and Renilla luciferase under the cytomegalovirus (CMV) promoter were purchased from Promega.

Cells and Transfections-- Human embryonic 293 kidney cells (obtained from ATCC) were grown in Dulbecco's modified Eagle's medium and transfected by calcium phosphate/DNA precipitation (41). HCT116 human colorectal cancer cells, rendered p53-null by somatic gene knockout (42, 43) were grown in McCoy's 5A medium and transfected using the LipofectAMINETM reagent (Invitrogen). Culture medium was supplemented with 1% glutamine, 1% of a pen-strep-ampho solution (Biological Industries, Israel) and 10% fetal calf serum (Biological Industries, Israel).

Establishment of Inducible PP2Calpha Cells-- T-RexTM (Invitrogen) is a Tet-regulated mammalian expression system based on the binding of tetracycline (Tet) to a Tet repressor and derepression of the promoter controlling the expression of the gene of interest (44, 45). T-RexTM-293 cells stably expressing the regulatory plasmid pcDNA6/TR (Invitrogen) were transfected by calcium phosphate/DNA precipitation with PP2Calpha -pcDNA4/TO wt or mut, or an empty vector (pcDNA4/TO). At 48 h after transfection, cells were seeded into fresh medium containing blasticidin (5 µg/ml) and zeocin (200 µg/ml) (both purchased from Invitrogen). The selective medium was replaced every 3-4 days until foci became visible. Several clones were isolated and characterized. Clone 9, expressing high levels of wt-PP2Calpha upon Tet induction, was used in all of the experiments. Mut-PP2Calpha or TO (empty vector) stable transfected clones were pooled and used in the experiments described below. Stable cell lines were maintained in medium containing blasticidin and zeocin.

Retroviral Infection of T-RexTM-293 cells-- For constitutive expression of the E6 protein, T-RexTM-293 cells were infected with the recombinant retrovirus HPV16 E6, kindly provided by L. Sherman (46). The cells were grown to 75% confluence and infected at a multiplicity of infection of 2-5. Upon reaching confluence, cell cultures were split and subjected to G418 selection (1.2 mg/ml, Calbiochem). Pooled stable clones were used in all further experiments.

Protein Analysis-- Cells were harvested with phosphate-buffered saline containing 0.25 mM EDTA and lysed in 50 mM Hepes, 150 mM NaCl, 1% Triton X-100, pH 8.0, supplemented with protease inhibitors (CompleteTM solution, Boehringer-Mannheim) and 2 mM Na3VO4. The cell debris was pelleted, and the protein concentration was determined in the supernatant using the BCA reagent (Pierce). Proteins were separated by SDS-PAGE (41), transferred to a nitrocellulose membrane, and immunoblotted with the relevant primary antibodies, followed by peroxidase-conjugated IgG (Jackson) and West Pico Chemiluminescent Substrate (Pierce).

Monoclonal Anti-PP2Calpha Antibodies (9F4)-- Coding sequences of PP2Calpha were cloned into the pET-28b bacterial expression vector (Novagen), and the resulting plasmid was used to transform BL21 (DE3) Escherichia coli. Culture of the transformants grown overnight at 30 °C, following induction by 0.1 mM isopropyl-beta -D-thiogalactoside, led to overexpression of soluble and active PP2Calpha . The recombinant protein was purified on a nickel-agarose column (Qiagen) under nondenaturing conditions and used for the preparation of mouse monoclonal antibodies as described (38). Immunoglobulin heavy-chain isotyping was carried out with an IsoStrip Mouse Monoclonal Antibody Isotyping kit (Roche Molecular Biochemicals) according to the manufacturer's instructions.

Antibodies-- Polyclonal anti-p38 antibodies were obtained from Sigma. Monoclonal anti-human p53 (DO-1) antibodies were a generous gift from D. Lane. Polyclonal anti-p53 (CM1) antibodies were purchased from Novocastra, and anti-p21 (C-19) antibodies were purchased from Santa Cruz Biotechnology. Human specific anti-cleaved PARP antibodies (Asp214) were obtained from Cell Signaling Technology.

XTT Assay-- A colorimetric method based on the tetrazolium salt XTT (47) is based on the ability of metabolic active cells to reduce the tetrazolium salt XTT to orange-colored compounds of formazan. The test procedure includes cultivation of cells in a 96-well plate, addition of the XTT reagent, and incubation for 2-24 h, during which an orange color is formed. The greater the number of active cells in the well, the greater the activity of mitochondria enzymes, and the higher the concentration of the dye formed, which can then be measured and quantitated.

T-Rex-293 cells expressing wt-PP2Calpha , mut-PP2Calpha , or empty vector TO (2 × 104 cells/well in a 96-well plate) were incubated for 24, 48, and 72 h in the presence or absence of Tet, (1 µg/ml). 50 µl of XTT reaction solution (Biological Industries, Israel) was added to each well, and the plate was incubated at 37 °C for 2 h. The sample's absorbance was measured with an enzyme-linked immunosorbent assay reader at a wavelength of 450 nm. The reference absorbance (nonspecific readings) was measured at a wavelength of 630 nm.

Protein Phosphatase Activity: Malachite Green Assay-- This assay is specific for the PP2C family and distinguishes it from the other classes of protein phosphatases (PP1, PP2A, and PP2B). It is performed in the presence of okadaic acid that completely inhibits PP1 and PP2A, EGTA that neutralizes the Ca2+/calmodulin-dependent PP2B, and Mg2+ that activates PP2C. Thus, PP2C activity is measured as the Mg2+-dependent and okadaic acid-insensitive activity (1). The phosphopeptide substrate in our assay, FLRTpSCG is derived from AMP-activated protein kinase and was previously shown to be a good substrate for PP2Calpha (49).

Protein extracts were prepared from transfected cells, and free phosphate was removed with a VivaSpin concentrator (cutoff 10,000 Da). Phosphatase activity was then measured colorimetrically as described (48). Briefly, the assay was performed in 30 µl of assay buffer (50 mM Tris, pH 7.5, 0.1 mM EGTA) containing 5 µg of cell extract and 0.5 mM substrate FLRTpSCG (49), in the presence of 30 mM MgCl2, 5 µM okadaic acid, and 5 µg of bovine serum albumin. After an incubation of 30 min at 30 °C, the reaction was terminated by adding 70 µl of cold assay buffer, followed by 25 µl of malachite green/ammonium molybdate reagent. Measurements were taken at 630 nm in an enzyme-linked immunosorbent assay reader (Dynatech MR5000).

Plating Efficiency-- A quantity of 500 cells/well was seeded in a 24-well plate and grown for 10 days (unless otherwise specified). The resistant colonies were fixed with 4% formaldehyde in phosphate-buffered saline, stained with Giemsa stain (Sigma) and counted.

Flow Cytometry Analysis-- T-Rex-293 wt-PP2Calpha or mut-PP2Calpha cells were seeded (1.5 × 106 cells per 6 cm plate). After 24 h, Tet (1 µg/ml) was added. After 0, 24, 48, and 72 h, the cells were harvested, fixed with methanol, and resuspended in phosphate-buffered saline containing 0.1% NaN3. Propidium iodide (50 µg/ml) was added for nuclear staining, and the cells were analyzed in a fluorescence-activated cell sorter (FACS Caliber, Becton Dickinson).

Immunofluorescence Microscopy-- T-Rex-293 wt-PP2Calpha cells cultured on polylysin-coated cover slips were incubated for 0, 24, and 48 h with Tet (1 µg/ml), fixed, permeabilized and stained with anti-PP2Calpha (9F4) followed by fluorescein isothiocyanate-conjugated secondary antibody, as previously described (38). Cell nuclei were stained with propidium iodide. The slides were then observed under a confocal laser scanning microscope (Zeiss LSM510).

Luciferase Assay-- T-Rex-293 wt-PP2Calpha or mut-PP2Calpha cells (6 × 105 cells/well, in a 6-well plate) were transfected with a combination of plasmids (200 ng of each) encoding firefly luciferase under the control of the mdm2/cyclin G/or RSV promoters, and Renilla luciferase under the CMV promoter. The cells were incubated with or without Tet (1 µg/ml) for 48 h and then harvested.

p53-null HCT116 cells were plated at 6 × 105 cells/well, in a 6-well plate, and transfected with a combination of plasmids (500 ng of each) encoding firefly luciferase under the control of the mdm2 or RSV promoters, and Renilla luciferase under the CMV promoter, with and without wt-PP2Calpha (2 µg) and wt-p53 (0.5-3 µg). The cells were harvested 48 h later. Firefly and Renilla luciferase activities were determined with a commercial double luciferase kit (Promega) using a TD-20e luminometer (Turner Design). Values of luciferase activity driven by the different promoters tested were normalized for Renilla luciferase readings in the same extracts.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of Stable Cell Lines Expressing PP2Calpha -- To obtain stable cell lines expressing PP2Calpha , 293 cells were initially transfected with wild-type PP2Calpha (wt-PP2C) or mutant PP2Calpha D239A (mut-PP2C) expression vectors. However, overexpression of wt-PP2Calpha was found to be highly toxic to the cells and stable clones were not obtained. In contrast, cells expressing the mutated catalytically inactive PP2Calpha yielded high numbers of G418 stable clones, similar to cells expressing the empty vector.

Therefore, to obtain stable clones producing high levels of wt-PP2Calpha , we used the T-RexTM system in which the tetracycline-inducible promoter regulates PP2Calpha . T-RexTM-293 cells were transfected with pcDNA4-wt-PP2Calpha , pcDNA4-mut- PP2Calpha , or the empty vector pcDNA4-TO. Stable zeocin-resistant clones expressing high levels of wt- or mut-PP2Calpha upon the addition of Tet were isolated and characterized. As shown in Fig. 1A, both the wt and the mutated PP2Calpha -expressing clones displayed a pronounced induction of PP2Calpha upon Tet addition, whereas clones harboring the empty vector expressed only basal PP2Calpha levels. The phosphatase activity in the wt-PP2Calpha -expressing cells was significantly augmented after Tet induction (Fig. 1B). Interestingly, after induction of the mut-PP2Calpha , the phosphatase activity in the treated cell extracts became lower than in the untreated cell extracts. Hence, this mutant may act in a dominant-negative manner, reducing the activity of the endogenous PP2Calpha .


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Fig. 1.   Characterization of stable cell lines expressing PP2Calpha under the regulation of a Tet-regulated promoter. T-Rex-293 cells expressing wt-PP2Calpha , mut-PP2Calpha , or empty vector (TO), were incubated for 0, 24, and 48 h with 1 µg/ml Tet. A, protein extracts (10 µg) from each sample were electrophoresed and immunoblotted with anti-PP2Calpha antibodies. B, extracts from wt-PP2C or mut-PP2C cells incubated with Tet for 0, 24, and 48 h (white, gray, and black bars, respectively) were assayed for phosphatase activity by the malachite green assay. The graph shows the average of 4 samples ± S.D. from one of at least 3 independent experiments.

PP2Calpha Overexpression Inhibits Cell Proliferation and Colony Formation-- Cell proliferation was significantly inhibited by the induction of wt-PP2Calpha . As shown in Fig. 2, A and B, only 20% of the cells expressing wt-PP2Calpha remained viable 48 and 72 h after the addition of Tet, as measured by the XTT assay. In contrast, cells expressing mut-PP2Calpha displayed enhanced proliferation (up to 120%), supporting the notion that the mutant protein indeed acts in a dominant-negative manner, thereby reversing an inherent antiproliferative function of the endogenous PP2Calpha . Tet treatment itself did not affect cell growth.


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Fig. 2.   PP2Calpha overexpression inhibits cell proliferation and colony formation. A, T-Rex-293 cells expressing wt-PP2Calpha (black bars), mut-PP2Calpha (white bars), or an empty vector TO (gray bars) were incubated for 24, 48, and 72 h with Tet (1 µg/ml). In parallel, control cultures were incubated for the corresponding times without Tet. The cells were then assayed for viability using the XTT assay. The percent of viable Tet-treated cells relative to the control (nontreated cells) was calculated, and the average of 6 different wells from a representative experiment was plotted ± S.D. B, the same cells were lysed, electrophoresed, and immunoblotted with anti-PP2Calpha antibodies. C, T-Rex-293 wt or mut-PP2Calpha cells were seeded at different Tet concentrations (0, 0.5, 1.5, 6, 25, and 100 ng/ml) and grown for 10 days to form colonies, and then were fixed and stained with Giemsa dye. D, extracts were prepared from cells treated similarly, 24 h after Tet induction, and assayed for PP2Calpha expression level.

The impact of PP2Calpha overproduction on the ability of the cells to form colonies was examined by a colony formation assay. Incubation of T-Rex-293 wt-PP2Calpha cells with increasing levels of Tet led to enhanced PP2Calpha expression and to a reduced number of surviving colonies (Fig. 2, C and D, wt). After the addition of low levels of Tet (6 ng/ml), there was already complete inhibition of cell growth. High levels of the mutant protein had no effect on the clonogenic capacity of the cells (Fig. 2, C and D, mut), demonstrating that PP2Calpha phosphatase activity is indeed responsible for the growth- inhibited phenotype.

PP2Calpha Inhibits Cell Cycle Progression and Induces Apoptosis-- T-Rex-293 wt-PP2Calpha or T-Rex 293-TO cells were incubated with Tet (1 µg/ml) for 0, 24, 48, and 72 h. As shown in Fig. 3, PP2Calpha induction led initially to pronounced accumulation of the cells in the G2/M phase, followed 24 and 48 h later by the appearance of sub-G1 apoptotic cells. At 72 h, a considerable fraction (28%) of the cells underwent apoptosis. Cell cycle arrest and apoptosis apparently resulted from the augmented phosphatase activity, as the inactive mutated protein did not alter cell cycle distribution (data not shown). Correspondingly, the wt-PP2Calpha producers displayed typical apoptotic characteristics, including nuclear shrinkage and chromatin condensation (Fig. 3B, arrows). In addition, we observed cleavage of poly(ADP)-ribose polymerase (PARP), which is split by caspases during the execution phase of apoptosis (50). As shown in Fig. 4A, cells expressing wt-PP2Calpha displayed a significant increase in the level of cleaved PARP 48 h after Tet induction, whereas in the cells expressing the mutated phosphatase, cleaved PARP was not detected. Another typical parameter of apoptotic cells, DNA fragmentation, with the characteristic apoptotic DNA ladder was observed in the wt-PP2Calpha -expressing cells (Fig. 4B). Cumulatively, these findings demonstrate that PP2Calpha overproduction triggers an apoptotic response in 293 cells.


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Fig. 3.   PP2Calpha overexpression causes G2/M cell cycle arrest and apoptosis. A, T-Rex-293 PP2Calpha or TO (empty vector control) cells were incubated for 0, 24, 48, and 72 h with Tet (1 µg/ml) and analyzed by flow cytometry. Cell cycle distribution of the cells was monitored for each treatment and the data from one of several experiments is presented in the table. B, T-Rex-293 PP2Calpha cells were cultured on polylysin-coated coverslips. After 0, 24, and 48 h incubation in the presence of Tet (1 µg/ml), the cells were fixed, permeabilized, and stained with anti-PP2Calpha and fluorescein isothiocyanate-conjugated goat anti-mouse IgG (green). Cell nuclei were labeled with propidium iodide (red). Coverslips were mounted on microscope slides and observed under a confocal laser scanning microscope (Zeiss LSM510). The arrows point to cells that display apoptotic characteristics.


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Fig. 4.   PP2Calpha induces an apoptotic response. A, T-Rex-293 PP2Calpha cells (wt or mut) were incubated for 0, 24, and 48 h in Tet (1 µg/ml). The cells were lysed and the extracts were immunoblotted with anti-cleaved-PARP antibodies and anti-PP2Calpha antibodies. B, T-Rex-293 PP2Calpha cells were grown for 48 h with or without Tet (1 µg/ml). Low molecular weight DNA was extracted by the Hirt method (51), resuspended in TE buffer, separated on 1% agarose gel, and stained with ethidium bromide.

PP2Calpha Augments the Transcriptional Activity of p53-- p53 is a DNA sequence-specific transcription factor that exerts its primary effects by activating the transcription of specific target genes. As shown in Fig. 5, the levels of p53 and p21, a major transcriptional target of p53, were elevated in T-Rex-293 cells, concomitant with wt-PP2Calpha induction, 6 and 10 h after Tet addition. Under the same conditions, the expression of p38, used as a control, was unaffected (Fig. 5, wt). Interestingly, the induction of mut-PP2Calpha was accompanied by decreased expression of both p53 and p21. At 48 h after induction of mut-PP2Calpha , there remained only marginal expression of both proteins, implying a putative dominant-negative function of the mutant. As expected, the level of p38 was not influenced by the mutant phosphatase. To directly examine the effects of PP2Calpha on the activity of p53 as a transcription factor, we cotransfected cells with luciferase reporter constructs under promoters of known p53 transcriptional activation targets such as cyclin G and mdm2. As shown in Fig. 6, luciferase activity increased in response to the induction of wt-PP2Calpha , whereas expression of the mutated protein did not affect these promoters. To gauge the specificity of PP2Calpha in affecting promoter activity, we used a constitutively active RSV promoter linked to the luciferase gene as control. The luciferase activity of this construct was not altered by PP2Calpha induction (Fig. 6). These findings clearly demonstrate that the activation of the p53 pathway in 293 cells is a direct outcome of the phosphatase activity of PP2Calpha .


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Fig. 5.   p53 and p53-responsive genes are induced by PP2Calpha . T-Rex-293 PP2Calpha cells (wt or mut) were incubated with Tet (1 µg/ml) for 0, 3, 6, 10, 24, and 48 h. The cells were lysed and the extracts were immunoblotted with anti-PP2Calpha , anti-p53, anti-p21, and anti- p38 antibodies.


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Fig. 6.   PP2Calpha induces the transcriptional activity of p53 in 293 cells. T-Rex-293 PP2Calpha cells wild-type (black bars) or mutant (white bars) were transfected with firefly luciferase under the control of mdm2/cyclin G/or RSV promoters. A Renilla luciferase expressing vector directed by the CMV promoter was included as an internal control for transfection efficiency. At 48 h after PP2Calpha induction, the cells were lysed, and luciferase activity (relative to control samples without Tet) was determined. The plotted values are the average of four independent samples of a representative experiment ±S.D.

Because 293 cells express the adenoviral E1A and E1B proteins, which affect p53 expression and function, it was important to establish that PP2Calpha activates the p53 pathway in cells that do not express these viral proteins. To address this issue, we used HCT116 cells rendered p53-null by somatic gene knockout. The cells were transiently cotransfected with luciferase reporter constructs regulated by the mdm2 or RSV promoter and increasing amounts of p53 expression vectors (0 to 3 µg), with or without PP2Calpha . As shown in Fig. 7, PP2Calpha overproduction in the absence of p53 did not affect transcriptional activation of the mdm2 promoter. Increasing amounts of p53 enhanced mdm2 promoter activity up to 130-fold in cells lacking exogenous PP2Calpha . However, upon cotransfection of both PP2Calpha and p53, enhanced transcriptional activation was observed, reaching a maximal 470-fold level. Thus, PP2Calpha activates the p53 signaling pathway also in cells that do not express adenoviral proteins.


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Fig. 7.   PP2Calpha induces p53 transcriptional activity in a p53 dose-dependent manner. p53-null HCT116 cells were transfected with firefly luciferase under the control of mdm2 or RSV promoter, together with increasing amounts of a p53 expression vector, with and without wt-PP2Calpha (black and white bars, respectively). A CMV-directed Renilla luciferase expression vector was included as an internal control for transfection efficiency. At 48 h after transfection, the cells were lysed, and luciferase activity was determined relative to that of the control samples (not transfected by PP2Calpha and/or p53). All of the shown results were normalized for RSV-luciferase readings in the same extracts. The plotted values are the average of four independent samples of a representative experiment ±S.D.

The PP2Calpha Cytotoxic Effect Is Partially p53-dependent-- To examine the role of p53 in the PP2Calpha -mediated growth arrest response, we constructed T-Rex-293 PP2Calpha cells that stably express the human papilloma virus E6 protein, which targets p53 for ubiquitination and rapid degradation. In these cells, p53 expression was dramatically reduced (Fig. 8A). The effect of the induced PP2Calpha on cell survival and colony formation was compared between the E6-expressing cells and the parental cells, using the colony formation assay. As shown in Fig. 8B, in cells expressing E6, the antiproliferative effects of PP2Calpha were suppressed, and the number of colonies was dramatically increased. Although in the parental cells low levels of PP2Calpha induction (12 ng/ml Tet) led to a pronounced reduction in colony number and size, in the presence of E6 there was no detectable effect under these conditions. At higher Tet concentrations, the increased PP2Calpha expression completely abolished colony formation in the presence of p53, whereas similar PP2Calpha levels in the absence of p53 were barely toxic. These findings clearly indicate that p53 is directly involved in PP2Calpha -mediated growth inhibition and cytotoxicity.


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Fig. 8.   The PP2Calpha cytotoxic effect is partially p53-dependent. A, T-Rex-293 PP2Calpha cells (± E6) were incubated for 24 h with or without Tet (1 µg/ml), lysed, and analyzed by Western blot with anti-p53 and anti-PP2Calpha antibodies. B, parental T-Rex-293 PP2Calpha cells and T-Rex-293 PP2Calpha cells stably expressing human papilloma virus E6 cDNA (Control and +E6, respectively) were seeded (300 cells/well). Different Tet concentrations (0, 3, 12, 50, 200, and 1000 ng/ml) were added, and the cells were grown for 20 days. The resistant colonies were fixed and stained with Giemsa dye.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we show that overexpression of PP2Calpha in 293 cells can lead to cell cycle arrest in the G2/M phase and to apoptosis. We demonstrate that PP2Calpha specifically activates p53 and stimulates its transactivational function, and that p53 plays an important role in the antiproliferative effects of PP2Calpha .

PP2Calpha -mediated Activation of p53-- The assumption that PP2Calpha directly participates in p53 activation emerges from the following observations: increased levels of endogenous p53 and p21 were detected in the induced cells, concomitant with overexpression of wt-PP2Calpha (Fig. 5). Furthermore, expression of the mutant protein, PP2Calpha D239A, which displays a dominant-negative phenotype, dramatically reduced the levels of endogenous p53 and p21. In the T-Rex-293 cells expressing wt-PP2Calpha , we observed transcriptional activation of the p53-responsive mdm2 and cyclin G promoters (Fig. 6). Because 293 cells are transformed by the adenoviral E1A and E1B proteins, which deregulate the cell cycle (52, 53), we resorted to p53-null HCT116 human colon carcinoma cells. In these cells, the activity of the mdm2 promoter was increased upon ectopic p53 expression. Moreover, transfection with PP2Calpha significantly amplified this effect (Fig. 7). Due to the lack of PP2Calpha -specific inhibitors and the presence of multiple PP2C paralogs that might complement each other, we are unable at this stage to demonstrate whether physiological PP2Calpha expression is essential for p53 activation. Further studies in which the activity of the multiple PP2C isoforms will be inhibited should clarify this issue.

How Does PP2Calpha Modulate p53 Activity?-- The PP2Calpha directed activation of exogenous p53 controlled by the CMV promoter suggests that this process is not governed by enhanced p53 transcription and points to the involvement of posttranslational events. p53 and its regulatory proteins are subject to many post-translational modifications including phosphorylation (54, 23), and could therefore serve as substrates for PP2Calpha . Although phosphorylation has been most extensively linked to the activation of the p53 response, it may also play a role in the negative regulation of p53 stability. The COP9 signalosome complex was recently shown to target p53, phosphorylated on Thr155, for proteasome-dependent degradation (55). Similarly, phosphorylation by protein kinase C, of Ser376 and Ser378, at the C terminus of p53, enhances the ubiquitination and degradation of p53 in unstressed cells (56). Dephosphorylation of these sites in response to ionizing radiation, by an undefined phosphatase, may contribute to the DNA damage-induced stabilization of p53 (57).

Another possible target for PP2Calpha is MDM2, which binds directly to p53 and targets it for degradation through the ubiquitin-dependent proteolytic pathway (58, 59). This protein is responsible, at least in part, for maintaining low levels of p53 under conditions of normal cell growth. Recently, it was reported that MDM2 dephosphorylation between serine residues 244-260 augments p53 stability (60). Cdk2 phosphorylates Thr216 of MDM2 at the onset of the S phase. This residue is dephosphorylated by an unknown phosphatase, when the cell passes through the S phase (61). PP2Calpha is a good candidate for being this unidentified phosphatase. Interestingly, Cdk2 is also a known substrate of PP2Calpha (19, 62). Thus, PP2Calpha may act directly on MDM2 or indirectly through the dephosphorylation of Cdk2.

Another possible candidate for PP2Calpha dephosphorylation activity is p300, which acetylates p53 at C-terminal residues (63). Finally, PP2Calpha might indirectly lead to p53 phosphorylation by activating one or more of the kinases responsible for its activation. At this stage we cannot rule out the possibility that in 293 cells, PP2Calpha also contributes to p53 activation by affecting the adenoviral proteins.

PP2Calpha -mediated Growth Arrest and Apoptosis-- Shortly after PP2Calpha induction in T-Rex 293 cells, a substantial fraction of the cells undergoes cell cycle arrest in the G2/M phase, followed by apoptosis. Perturbation of the expression of the endogenous wild-type p53 by the human papilloma E6 gene led to suppression of the growth-arrested phenotype and enhanced survival (Fig. 8). Therefore, we conclude that PP2Calpha -mediated growth inhibition in 293 cells results, at least in part, from the activation of p53 checkpoints.

Multiple overlapping p53-dependent pathways control the G2/M transition. p53 is involved in the regulation of the cyclin-dependent kinase Cdc2, which is essential for entry into mitosis. Cdc2, as well as other CDKs, is active only when associated with cyclins. CDKs also interact with different proteins belonging to the family of CDK inhibitors. Expression of p21Waf/Cip/Sdi, the only CKI capable of interacting with essentially all the CDK complexes (64), regulates the transition between the different cell cycle phases and efficiently inhibits Cdc2, arresting the cells in the G2 phase (43). This effect appears to be mediated, at least in part, through the interference of the activating phosphorylation of Cdc2 at Thr161 (65). Thus, p21 inhibits Thr161 phosphorylation of Cdc2, enforcing the G2 DNA damage checkpoint. Interestingly, PP2C was recently shown to dephosphorylate Thr161 on Cdc2 in Xenopus oocytes (65). Hence, PP2Calpha -induced G2/M cell cycle arrest might occur through the regulation of Cdc2 activity via two independent pathways. The first, involves direct dephosphorylation of Cdc2 and removal of the activating phosphorylated Thr161. The second engages the induction of p21 expression through p53 activation and the inhibition of Cdc2 Thr161 phosphorylation.

Our data suggest that the PP2Calpha killing effect is at least partially p53-dependent (Fig. 8) and thus, additional pathways may therefore be involved in its apoptotic effect. For example, members of the PP2C family were shown to down-regulate the Wnt signaling pathway via modulation of GSK3beta phosphorylation, inhibiting transcription of LEF-1-modulated target genes (6). Furthermore, at least with regard to apoptosis in 293 cells, the possible interaction between PP2Calpha and adenoviral proteins E1A and E1B merits investigation.

    ACKNOWLEDGEMENTS

We are especially grateful to N. Kazhdan for very skillful assistance and to S. Karby and N. Smorodinsky for help in the preparation of the antibodies. We thank Z. Cohen for the DNA ladder picture, S. Wilder for generously providing constructs, reagents and cell lines, and L. Sherman for the E6 retroviral vector. We also thank O. Sagi-Assif for help with the fluorescence-activated cell sorter analysis and A. Barbul for assistance with the confocal microscopy.

    FOOTNOTES

* This work was supported by grants from Cap CURE, Israel and by a grant from the Ministry of Science Culture and Sports, Israel and the DKFZ (Deutsches Krebsforschungszentrum).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 972-3-6409832; Fax: 972-3-6422046; E-mail: lavisara@post.tau.ac.il.

Published, JBC Papers in Press, January 3, 2003, DOI 10.1074/jbc.M211699200

    ABBREVIATIONS

The abbreviations used are: PP2C, protein phosphatase 2C; CMV, cytomegalovirus; RSV, Rous sarcoma virus; CDK, cyclin-dependent kinase; Tet, tetracycline; wt, wild type; mut, mutant; PARP, poly(ADP)-ribose polymerase; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)- 2H-tetrazolium-5-carboxanilide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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