 |
INTRODUCTION |
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 PP2C
(also referred to as PPM1A)
is the most characterized member (11). A growing list of substrates has
been suggested to be specifically dephosphorylated by PP2C
in
eukaryotic cells (12-20). This broad substrate specificity suggests
that PP2C
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 PP2C
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 PP2C
as a negative
growth regulator. In cells containing endogenous wt-p53, PP2C
overexpression mediates cell cycle arrest in the G2/M phase
followed by apoptosis. This PP2C
-directed growth arrest is imposed
through the activation of p53. Although PP2C
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 PP2C
.
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EXPERIMENTAL PROCEDURES |
Plasmids--
For PP2C
-pcDNA3.1 (wt-PP2C
), a rat
cDNA library (37) was screened with probes derived from the rat
PP2C
gene (38), yielding several PP2C
clones. A clone encoding
the complete PP2C
cDNA was isolated and converted to a plasmid,
enabling CMV promoter-driven transcription of the inserted cDNA.
The complete coding sequence of PP2C
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 PP2C
D239A (mut-PP2C
), PCR was performed to
introduce the mutation D239A into PP2C
. Two PCR reactions were
conducted in parallel, using PP2C
-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 PP2C
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 PP2C
sequence and the downstream with the SP6 primer). The resultant PCR
product was cloned into PP2C
-pcDNA3.1 via the EcoRI
and ApaI sites. The sequence accuracy of the mut-PP2C
was verified by sequencing.
For inducible PP2C
(PP2C
-pcDNA4/TO), PP2C
(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 PP2C
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 PP2C
-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-PP2C
upon Tet induction, was used in all of the experiments. Mut-PP2C
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-PP2C
Antibodies (9F4)--
Coding
sequences of PP2C
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-
-D-thiogalactoside, led to overexpression of soluble and active PP2C
. 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-PP2C
, mut-PP2C
, 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 PP2C
(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-PP2C
or mut-PP2C
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-PP2C
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-PP2C
(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-PP2C
or mut-PP2C
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-PP2C
(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 |
Generation of Stable Cell Lines Expressing PP2C
--
To obtain
stable cell lines expressing PP2C
, 293 cells were initially
transfected with wild-type PP2C
(wt-PP2C) or mutant PP2C
D239A (mut-PP2C) expression vectors. However,
overexpression of wt-PP2C
was found to be highly toxic to the cells
and stable clones were not obtained. In contrast, cells expressing the
mutated catalytically inactive PP2C
yielded high numbers of G418
stable clones, similar to cells expressing the empty vector.
Therefore, to obtain stable clones producing high levels of wt-PP2C
,
we used the T-RexTM system in which the
tetracycline-inducible promoter regulates PP2C
.
T-RexTM-293 cells were transfected with
pcDNA4-wt-PP2C
, pcDNA4-mut- PP2C
, or the empty vector
pcDNA4-TO. Stable zeocin-resistant clones expressing high levels of
wt- or mut-PP2C
upon the addition of Tet were isolated and
characterized. As shown in Fig.
1A, both the wt and the
mutated PP2C
-expressing clones displayed a pronounced induction of
PP2C
upon Tet addition, whereas clones harboring the empty vector
expressed only basal PP2C
levels. The phosphatase activity in the
wt-PP2C
-expressing cells was significantly augmented after Tet
induction (Fig. 1B). Interestingly, after induction of the
mut-PP2C
, 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 PP2C
.

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Fig. 1.
Characterization of stable cell lines
expressing PP2C under the regulation of a
Tet-regulated promoter. T-Rex-293 cells expressing wt-PP2C ,
mut-PP2C , 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-PP2C 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.
|
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PP2C
Overexpression Inhibits Cell Proliferation and
Colony Formation--
Cell proliferation was significantly inhibited
by the induction of wt-PP2C
. As shown in Fig.
2, A and B, only
20% of the cells expressing wt-PP2C
remained viable 48 and 72 h after the addition of Tet, as measured by the XTT assay. In contrast,
cells expressing mut-PP2C
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 PP2C
. Tet treatment
itself did not affect cell growth.

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Fig. 2.
PP2C overexpression
inhibits cell proliferation and colony formation. A,
T-Rex-293 cells expressing wt-PP2C (black
bars), mut-PP2C (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-PP2C antibodies. C, T-Rex-293 wt or mut-PP2C
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 PP2C expression level.
|
|
The impact of PP2C
overproduction on the ability of the cells to
form colonies was examined by a colony formation assay. Incubation of
T-Rex-293 wt-PP2C
cells with increasing levels of Tet led to
enhanced PP2C
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 PP2C
phosphatase activity is indeed responsible for the growth- inhibited phenotype.
PP2C
Inhibits Cell Cycle Progression and Induces
Apoptosis--
T-Rex-293 wt-PP2C
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, PP2C
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-PP2C
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-PP2C
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-PP2C
-expressing cells (Fig. 4B). Cumulatively, these
findings demonstrate that PP2C
overproduction triggers an apoptotic
response in 293 cells.

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Fig. 3.
PP2C overexpression
causes G2/M cell cycle arrest and
apoptosis. A, T-Rex-293 PP2C 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 PP2C
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-PP2C 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.
PP2C induces an
apoptotic response. A, T-Rex-293 PP2C 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-PP2C antibodies. B,
T-Rex-293 PP2C 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.
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PP2C
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-PP2C
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-PP2C
was accompanied by decreased expression of
both p53 and p21. At 48 h after induction of mut-PP2C
, 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 PP2C
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-PP2C
, whereas
expression of the mutated protein did not affect these promoters. To
gauge the specificity of PP2C
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 PP2C
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 PP2C
.

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Fig. 5.
p53 and p53-responsive genes are induced by
PP2C . T-Rex-293 PP2C 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-PP2C , anti-p53, anti-p21, and anti- p38 antibodies.
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Fig. 6.
PP2C induces the
transcriptional activity of p53 in 293 cells. T-Rex-293 PP2C
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 PP2C 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.
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|
Because 293 cells express the adenoviral E1A and E1B proteins, which
affect p53 expression and function, it was important to establish that
PP2C
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 PP2C
. As shown in Fig.
7, PP2C
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 PP2C
. However, upon
cotransfection of both PP2C
and p53, enhanced transcriptional
activation was observed, reaching a maximal 470-fold level. Thus,
PP2C
activates the p53 signaling pathway also in cells that do not
express adenoviral proteins.

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Fig. 7.
PP2C 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-PP2C (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 PP2C 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.
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The PP2C
Cytotoxic Effect Is Partially
p53-dependent--
To examine the role of p53 in the
PP2C
-mediated growth arrest response, we constructed T-Rex-293
PP2C
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
PP2C
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 PP2C
were suppressed, and the number of
colonies was dramatically increased. Although in the parental cells low
levels of PP2C
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 PP2C
expression completely abolished colony formation
in the presence of p53, whereas similar PP2C
levels in the absence
of p53 were barely toxic. These findings clearly indicate that p53 is
directly involved in PP2C
-mediated growth inhibition and
cytotoxicity.

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Fig. 8.
The PP2C cytotoxic
effect is partially p53-dependent. A,
T-Rex-293 PP2C 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-PP2C antibodies. B, parental T-Rex-293
PP2C cells and T-Rex-293 PP2C 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.
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|
 |
DISCUSSION |
In the present study, we show that overexpression of PP2C
in
293 cells can lead to cell cycle arrest in the G2/M phase
and to apoptosis. We demonstrate that PP2C
specifically activates p53 and stimulates its transactivational function, and that p53 plays
an important role in the antiproliferative effects of PP2C
.
PP2C
-mediated Activation of p53--
The assumption that
PP2C
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-PP2C
(Fig. 5). Furthermore, expression of the mutant protein,
PP2C
D239A, which displays a dominant-negative phenotype,
dramatically reduced the levels of endogenous p53 and p21. In the
T-Rex-293 cells expressing wt-PP2C
, 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 PP2C
significantly amplified
this effect (Fig. 7). Due to the lack of PP2C
-specific inhibitors
and the presence of multiple PP2C paralogs that might complement each
other, we are unable at this stage to demonstrate whether physiological
PP2C
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 PP2C
Modulate p53 Activity?--
The PP2C
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 PP2C
. 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 PP2C
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). PP2C
is
a good candidate for being this unidentified phosphatase.
Interestingly, Cdk2 is also a known substrate of PP2C
(19, 62).
Thus, PP2C
may act directly on MDM2 or indirectly through the
dephosphorylation of Cdk2.
Another possible candidate for PP2C
dephosphorylation activity is
p300, which acetylates p53 at C-terminal residues (63). Finally,
PP2C
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, PP2C
also
contributes to p53 activation by affecting the adenoviral proteins.
PP2C
-mediated Growth Arrest and Apoptosis--
Shortly after
PP2C
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 PP2C
-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,
PP2C
-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 PP2C
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 GSK3
phosphorylation, inhibiting transcription of
LEF-1-modulated target genes (6). Furthermore, at least with regard to
apoptosis in 293 cells, the possible interaction between PP2C
and
adenoviral proteins E1A and E1B merits investigation.