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INTRODUCTION |
Approximately one-third of mammalian proteins are thought to be
post-translationally modified by phosphorylation (1). The human genome
contains hundreds of protein kinases (2), but the reversibility of the
phosphorylation process suggests that phosphatases also play a major
role in the regulation of protein phosphorylation. Although the roles
and cellular functions of kinases have been extensively studied,
protein phosphatases have received much less attention. A long held
view has been that phosphatases serve merely to reverse the actions of
protein kinases. More recently has it been recognized that phosphatases
may be as numerous and as tightly regulated as protein kinases, with as
widely varying substrate specificities and signaling functions (3). The
preference of certain phosphatases for one phosphorylated hydroxyamino
acid over others has resulted in the current classification of
phosphatases as protein serine/threonine-specific,
protein tyrosine-specific, and dual-specificity
phosphatases. Although several highly potent and selective inhibitors
of serine/threonine phosphatases have been isolated from natural
sources, selective protein-tyrosine phosphatase or dual-specificity
phosphatase inhibitors are still rare.
Protein kinases and phosphatases are part of a complex signaling
network of tightly regulated dynamic processes. The nature and details
of network organization are just beginning to be unveiled, but their
abundance, diversity, and substrate specificity alone cannot explain
how these molecules function to regulate complex biochemical pathways.
An emerging concept in signaling specificity is the subcellular
location at which signaling events occur (4). Most protein movements
within the cell are consistent with a random diffusion process.
However, it is now being recognized that spreading as well as
restriction of signaling events to certain regions of the cell is
driven by the availability of sites for protein-protein and
protein-second messenger interactions (5). Phosphorylation of many key
signaling molecules causes a subcellular redistribution that is
critical for biological activity (6-8).
Despite the potential importance that spatial regulation might have in
signal transduction and the considerable information that can be
derived from localization studies, a lack of readily available,
quantitative analytical tools to assess the subcellular localization of
multiple signal transduction molecules has impeded progress in this
area. Current fluorescent imaging techniques have low throughput and
are not well suited for the dissection of how complex signaling
networks are coordinated. In this report, we have used a novel,
automated, fluorescence-based, multiparametric, solid-phase cytometer,
the Cellomics ArrayScan II (9), to rapidly quantitate the effects of a
synthetic vitamin K analog, Compound 5, on the spatial regulation of a
subset of key signal transduction molecules.
Compound 5 was discovered to be a potent inhibitor of hepatoma cell
growth in a small targeted library of synthetic vitamin K analogs (10,
11). It has anti-phosphatase activity that is thought to contribute to
its antiproliferative activity. Most notably, it is one of the most
potent in vitro inhibitors of the Cdc25 phosphatase family
of dual-specificity phosphatases reported to date (12). In
vitro, Compound 5 is ~10- and 100-fold more potent against Cdc25
compared with the prototype dual-specificity phosphatase
VHR1 and protein-tyrosine
phosphatase 1B, respectively (12). Its ability to cause a dual-cell
cycle arrest in G1 and G2 phases as well as
increased phosphorylation of the Cdc25 substrates Cdc2 (Cdk1), Cdk2,
and Cdk4 is consistent with Cdc25 phosphatase inhibition by Compound 5 (12). Recent work from our laboratories has also demonstrated that
Compound 5 causes increased tyrosine phosphorylation on a number of
proteins, including the epidermal growth factor receptor and Erk in
hepatocytes (13) and MCF-7 cells (14), but it is unknown how Compound 5 enhances Erk phosphorylation or whether Compound 5 treatment changes
Erk subcellular localization. A possible link between the mitogenic
signal transduction and Cdc25A has been described by Galaktionov
et al. (15), who reported that Cdc25A associates with Raf-1,
a key upstream activator of Erk, in mammalian cells and frog oocytes.
More recently, evidence for a possible functional involvement of Cdc25A
in the Erk pathway was presented by Xia et al. (16), who
reported that coexpression of Cdc25A together with Raf-1 prevents Raf-1
activation in response to platelet-derived growth factor in NIH3T3
cells. Nonetheless, no direct evidence for Cdc25A involvement in Erk
phosphorylation or activity has been reported.
In this study, we demonstrated that Cdc25A expression could reduce Erk
phosphorylation and described a novel cell-based assay revealing that
Compound 5 directly interfered with Cdc25A function upon Erk
phosphorylation. Using quantitative, fluorescence-based, solid-phase
cytometry, we documented that Erk hyperphosphorylation by Compound 5 resulted in increased nuclear accumulation of kinase-active phospho-Erk. Thus, an inhibitor of Cdc25 increased Erk phosphorylation, which further supported the hypothesis that Cdc25A regulates Erk phosphorylation status.
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EXPERIMENTAL PROCEDURES |
Reagents--
Compound 5 (2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone) has been described
previously (10). Human recombinant interleukin-1
(IL-1
) was from
R&D Systems (Minneapolis, MN). Mouse monoclonal anti-phospho-Erk
antibody (E10) and the MEK inhibitor U-0126 were from New England
Biolabs Inc. (Beverly, MA). Mouse monoclonal anti-Erk2 antibody was
from Upstate Biotechnology, Inc. (Lake Placid, NY). Primary antibodies
for phospho-p38 and the p65 subunit of NF-
B were components of a
commercially available assay kit (Cellomics, Pittsburgh, PA).
Anti-Oct-1 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA),
and anti-Hsp90 antibody was from BD Transduction Laboratories (San
Diego, CA). Secondary antibodies were AlexaFluor 488-conjugated goat
anti-mouse (phospho-Erk), goat anti-rabbit (phospho-p38 and
phospho-JNK), or donkey anti-goat (NF-
B) IgG (Molecular Probes,
Eugene, OR).
Cell Culture--
Cells were maintained in Dulbecco's minimum
essential medium containing 10% fetal bovine serum (Hyclone
Laboratories, Logan, UT) and 1% penicillin/streptomycin (Life
Technologies, Inc.) in a humidified atmosphere of 5% CO2
at 37 °C. HeLa, PC-3, DU-145, and NIH3T3 cells were from American
Type Culture Collection. Rat-1 fibroblasts were obtained from Dr.
Guillermo Romero (University of Pittsburgh). Hep3B human hepatoma cells
have been characterized previously (17).
Indirect Immunofluorescence--
Hep3B, HeLa, PC-3, DU-145,
Rat-1, or NIH3T3 cells (4000 cells/well) were plated in the wells of a
collagen-coated 96-well dark-well plate (Packard
ViewPlateTM) and allowed to attach overnight. Cells were
treated for the times indicated with Compound 5 or IL-1
, fixed with
3.7% formaldehyde in phosphate-buffered saline, and permeabilized with
phosphate-buffered saline/Triton X-100. Cells were stained with
antibodies against phospho-Erk, phospho-p38, phospho-JNK, or the 65-kDa
subunit of NF-
B and washed with phosphate-buffered saline/Tween 20. Nuclei were stained with Hoechst 33342 fluorescent dye, and
immunoreactive cells were visualized by the AlexaFluor 488-conjugated
secondary antibodies using an XF100 filter set at excitation/emission
wavelengths of 494/519 nm (AlexaFluor 488) and 350/461 nm (Hoechst).
Plates were analyzed by automated image analysis on the ArrayScan II system (Cellomics) using the previously described cytoplasm to nucleus
translocation algorithm (18). Control experiments omitting primary
antibodies were performed each time to assess the amount of nonspecific
background staining.
Cell Fractionation and Western Blotting--
Cytosolic and
nuclear fractions were prepared using a slightly modified procedure as
published by Schreiber et al. (19). Hep3B cells were plated
in 100-mm tissue culture dishes, exposed to 10 µM
Compound 5 for the indicated periods of time, and harvested by
centrifugation. Cell pellets were resuspended in 200 µl of hypotonic
buffer (10 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.2 mM
phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, and 0.5% Nonidet P-40), incubated on ice for 10 min, disrupted by repeated
aspiration through a 20-gauge needle, and centrifuged at 2500 × g for 15 min. The supernatant was collected as cytosolic extract. Nuclear pellets were resuspended in nuclear extraction buffer
(20 mM HEPES, pH 7.9, 10% glycerol, 1.5 mM
MgCl2, 400 mM KCl, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5 mM
dithiothreitol), incubated on ice for 1 h, and centrifuged at
13,000 × g to collect the nuclear fraction.
Solubilized proteins were resolved by 10% SDS-PAGE and transferred to
polyvinylidene difluoride membranes (PerkinElmer Life Sciences).
Membranes were probed with anti-phospho-Erk, anti-Oct-1, and anti-Hsp90
antibodies. Positive antibody reactions were visualized using
peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch
Laboratories, Inc., West Grove, PA) and an enhanced chemiluminescence
detection system (Renaissance, PerkinElmer Life Sciences) according to
the manufacturers' instructions.
Erk Activity Assay--
Erk activity in cytosolic and nuclear
fractions was determined using a nonradioactive immunoprecipitation kit
(Cell Signaling Technologies, Beverly, MA). Briefly, 200 µg of
nuclear or cytosolic proteins were incubated with 15 µl of
agarose-conjugated anti-phospho-Erk antibody and incubated overnight at
4 °C with gentle rocking. Immunoprecipitates were pelleted and
washed twice with kinase buffer (25 mM Tris, pH 7.5, 5 mM
-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, and 10 mM MgCl2). Pellets were resuspended in 50 µl
of kinase buffer supplemented with 200 µM ATP and 2 µg
of GST-Elk-1 fusion protein and incubated for 30 min at 30 °C.
Immunoprecipitates were boiled in SDS-PAGE sample buffer and analyzed
by Western blotting using anti-phospho-Elk-1 antibody.
Growth Inhibition Assay--
The antiproliferative activity of
Compound 5 in combination with the MEK inhibitor U-0126 was measured by
a previously described assay based on fluorometric quantitation of
total cellular DNA content using the fluorochrome Hoechst 33258 (20).
Briefly, cells were grown in 96-well microplates and treated every day for 3 days with various concentrations of Compound 5 in the presence or
absence of the MEK inhibitor U-0126 (5 µM). Cells were
lysed by repeated freeze-thawing, and cellular DNA was quantitated as described (20).
Cell Transfections--
Mammalian expression plasmids encoding
full-length wild-type Cdc25A and catalytically inactive C430S mutant
Cdc25A in a pcDNA3 vector were generously provided by Dr. Thomas
Roberts (Dana Farber Cancer Institute) (16). Transfections were carried
out by the LipofectAMINE method following the manufacturer's
instructions (Life Technologies, Inc.). Briefly, HeLa cells
(100,000/well) were plated in the wells of a 6-well plate and
transfected with 0.5 µg of cDNA in Opti-MEM transfection medium
using LipofectAMINE PlusTM reagent (Life Technologies,
Inc.). Three hours after transfection, the medium was replaced with
complete growth medium, and the cells were allowed to recover for
48 h. Cells were treated with 0-20 µM Compound 5 for 30 min, and protein lysates were prepared and analyzed by SDS-PAGE
and Western blot analysis for phospho-Erk and Erk2 levels as described
above. For quantitation of protein expression levels, x-ray films were
scanned on a Molecular Dynamics personal SI densitometer and analyzed
using the ImageQuant software package (Version 4.1, Molecular Dynamics,
Inc., Sunnyvale, CA).
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RESULTS |
A High-content, Fluorescence-based Assay for Phospho-Erk Nuclear
Accumulation--
Compound 5 was previously found to induce the
prolonged phosphorylation of tyrosines on a number of signaling
proteins in the Erk cascade, including Erk1 and Erk2 (13, 14). We first asked whether this increase in tyrosine phosphorylation was associated with a change in phospho-Erk nuclear accumulation. Hep3B cells were
incubated either with vehicle (Me2SO) (Fig.
1, A-C) or Compound 5 (D-F) for 30 min and immunostained with antibodies against
a dually phosphorylated (Thr202/Tyr204) form of
Erk (B, C, E, and F).
Nuclei were visualized by Hoechst 33342 staining (Fig. 1, A
and D). Fig. 1B shows that vehicle-treated cells
had very low levels of phospho-Erk, most of which was diffusely distributed in the cytoplasm. Treatment of cells with Compound 5 resulted in a substantial increase in total phospho-Erk, with prominent
nuclear accumulation (Fig. 1E). Overlay images (Fig. 1,
C and F) illustrate the quantitation of
cytoplasmic and nuclear phospho-Erk levels. Fluorescently labeled cells
were analyzed in two separate channels by the ArrayScan II, and the
cytoplasmic-to-nuclear distribution was determined by a previously
described algorithm (18). Hoechst 33342 staining (Fig. 1, A
and D) defined the nuclear area. Phospho-Erk fluorescence
intensity within this nuclear area is referred to as "cytonuclear
intensity." To assess the amount of fluorescently labeled phospho-Erk
in the cytoplasm, a set of concentric rings spaced by two pixels was
placed around the nuclear boundary. Phospho-Erk fluorescence intensity
within the ring area is referred to as "cytoring intensity."
Both cytonuclear and cytoring intensities were normalized to the total
cytonuclear or cytoring area and are expressed as average intensity per
pixel. All cytoplasmic-to-nuclear difference values were calculated by
subtracting the average cytoring intensity per pixel from the average
cytonuclear intensity per pixel. Thus, an increase in the cytonuclear
difference value is indicative of Erk activation through
phosphorylation, translocation, or both.

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Fig. 1.
Quantitation of phospho-Erk nuclear
accumulation in Compound 5-treated Hep3B cells by the cytoplasm to
nucleus translocation algorithm.
Untreated (A-C) and Compound 5-treated
(D-F) Hep3B cells were stained with Hoechst 33342 fluorescent dye (A and D) or with
anti-phospho-Erk antibody followed by a fluorescently tagged secondary
antibody (B, C, E, and F).
Images were acquired in two separate channels on an ArrayScan II system
and analyzed for both nuclear and cytoplasmic phospho-Erk expression.
Nuclear masks were generated from Hoechst 33342-stained nuclei, and
analysis parameters were adjusted to exclude irregularly shaped or
sized nuclei as well as aggregate cells. For determination of
cytoplasmic intensity, the nuclear boundary was eroded by two pixels
and fitted with two concentric circles placed around the nuclear mask
(C and F). Cytonuclear differences were
calculated by subtracting the average cytosolic fluorescence pixel
intensity from the average nuclear fluorescence pixel intensity.
Bar = 55 µm.
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Induction of Phospho-Erk and Phospho-p38, but Not NF-
B, by
Compound 5--
We next examined whether Compound 5 caused selective
Erk nuclear accumulation by comparing its effects with those of other signaling events that have also been reported to be activated in a
tyrosine phosphorylation-dependent manner and are thought to mediate stress responses. Cells were treated for 30 min with either
10 µM Compound 5 or 25 ng/ml IL-1
; immunostained with anti-phospho-Erk, anti-phospho-p38, anti-phospho-JNK, or anti-NF-
B p65 antibodies; and analyzed for differences in cytoplasmic-to-nuclear fluorescence intensity. A total of 100 cells were imaged in each well.
Fig. 2 shows that Compound 5 led to a
dramatic increase in nuclear accumulation of phospho-Erk and
phospho-p38, but had only a moderate effect on phospho-JNK and did not
affect the nuclear accumulation of NF-
B. In contrast, IL-1
activated all three stress-response mediators (p38, JNK, and NF-
B),
but not Erk. Thus, the activity profile of Compound 5 was distinct from
that of the cytokine IL-1
, suggesting that Compound 5 is not a
general stress-inducing agent.

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Fig. 2.
Selective activation of Erk and p38 (but not
NF- B) by Compound 5. Hep3B
cells (4000 cells/well) were plated in each of the 96 wells of a
dark-well plate; treated with Compound 5 (cpd 5) or vehicle;
and stained with anti-phospho-Erk, anti-phospho-p38, anti-phospho-JNK,
and anti-NF- B p65 antibodies. A minimum of 100 cells/well were
analyzed with the previously described cytoplasm to nucleus
translocation algorithm (18) on the ArrayScan II.
Cytoplasmic-to-nuclear difference values were calculated as described
in the legend to Fig. 1 and normalized to the maximum signal obtained
(10 µM Compound 5 for Erk and p38 and 25 ng/ml IL-1
for JNK and NF- B). The data shown are the means ± S.D. from
quadruplicate wells and are from a single experiment that was repeated
at least two times with identical results.
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Kinetics of Erk and p38 Activation by Compound 5--
Experiments
with the stress inducer and phosphatase inhibitor sodium arsenite
previously demonstrated that p38 and Erk are activated with different
kinetics in a variety of cell lines (21). It was also reported that Erk
activation is abrogated by dominant-negative forms of p38 and the
p38-specific kinase inhibitor SB-203580, suggesting an involvement of
p38 in Erk activation. We thus examined the concentration dependence
and kinetics of phospho-Erk and phospho-p38 activation in Hep3B cells.
Fig. 3 shows that maximum stimulation of
both Erk and p38 was obtained at 10 µM Compound 5. Moreover, continuous exposure to 10 µM Compound 5 caused
a progressively greater activation and nuclear accumulation with
similar temporal characteristics (Fig. 3, upper panel). We
have also found that the p38 inhibitor SB-203580 did not inhibit
phospho-Erk nuclear accumulation (data not shown). These results
suggest that Compound 5 acts differently than the nonspecific tyrosine
phosphatase inhibitor sodium arsenite.

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Fig. 3.
Kinetics and concentration dependence of
Compound 5-induced phospho-Erk and phospho-p38 nuclear
accumulation. Hep3B cells were incubated for 30 min with
increasing concentrations of Compound 5 (upper panel) or for
the indicated amounts of time with 10 µM Compound 5 (lower panel). Average cytonuclear differences were obtained
by quantitation of phospho-Erk or phospho-p38 staining. Data are the
means ± S.E. from quadruplicate wells, with ~100 cells being
scored in each well.
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Irreversibility of Compound 5 Action--
Compound 5 is a
sulfhydryl-arylating agent, and its sustained anti-phosphatase activity
has been ascribed to covalent modification of critical cysteine
residues on dual-specificity and tyrosine phosphatases (17). To
test whether its effects were irreversible, we treated cells with
Compound 5 for 5 or 10 min, followed by washout, and compared the
magnitude of phospho-Erk and phospho-p38 accumulation with that
obtained after a 30-min continuous exposure. Fig.
4 shows that short pulses of Compound 5 resulted in substantial activation of both Erk and p38, consistent with
a rapid and persistent inhibition of cellular phosphatases after
compound removal.

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Fig. 4.
Pulse treatment with Compound 5 and partial
activation of Erk and p38. Cells were treated for various lengths
of time with Compound 5, followed by compound removal and incubation in
fresh medium. After a total of 30 min, cells were washed, fixed, and
stained with anti-phospho-Erk (open bars) or
anti-phospho-p38 (closed bars) antibodies. Numeric values
for phospho-Erk and phospho-p38 nuclear accumulation were obtained as
described in the legend to Fig. 1, and data were normalized to the
maximum signal obtained (30 min of continuous exposure to Compound 5).
Data are the means ± S.E. from quadruplicate wells. Similar
results were obtained in a second independent experiment.
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Biochemical Analysis Confirms Phospho-Erk Nuclear
Accumulation--
We next validated the results from the automated,
fluorescence-based analysis by conventional biochemical methods. Cells
were treated with 10 µM Compound 5 for the indicated
times, lysed, separated into cytosolic and nuclear fractions, and
analyzed by Western blotting using anti-phospho-Erk antibody (Fig.
5A). Untreated cells had
almost no nuclear phospho-Erk, consistent with the whole cell images in
Fig. 1B. Within minutes, Compound 5 caused a
time-dependent and sustained increase in phospho-Erk
nuclear accumulation. In contrast, cytosolic phospho-Erk levels in
control cells were higher than those in the nucleus and increased only
after a longer exposure to Compound 5 (30 min) (Fig. 5A).
The results from the immunoblot analysis thus confirmed those from the
less arduous solid-phase cytometry studies.

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Fig. 5.
Confirmation of Compound 5-mediated
phospho-Erk activation by immunoblot analysis and in vitro
kinase assay. Hep3B cells were grown to subconfluency in
100-mm dishes, treated for the indicated lengths of time with Compound
5 (10 µM), and harvested. A, nuclear and
cytoplasmic fractions of treated and untreated Hep3B cells were
separated by SDS-PAGE and immunoblotted with anti-phospho-Erk antibody
(p-Erk). Equal protein loading and the quality of the
cellular separation procedure were demonstrated by reprobing the
identical blots with anti-Oct-1 (nuclear marker) or anti-Hsp90
(cytosolic marker) antibodies. B, proteins from nuclear and
cytosolic fractions were immunoprecipitated with anti-phospho-Erk
antibody-agarose conjugate, and the immunoprecipitates were subjected
to an in vitro kinase assay using recombinant GST-Elk-1
fusion protein. Reaction mixtures were separated by SDS-PAGE and
immunoblotted with anti-phospho-Elk-1 antibody. The data shown are
representative of three experiments with similar results.
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Phosphorylated Erk from Compound 5-treated Cells Is Activated and
Phosphorylates Elk-1--
We then used the identical lysates from
Compound 5-treated cells to investigate whether the observed Erk
phosphorylation resulted in an increase in Erk kinase activity. It is
thought that upon phosphorylation by MEK1 and MEK2 in the cytosol, a
fraction of Erk translocates to the nucleus, where it phosphorylates
and activates transcription factors such as c-Fos, c-Jun, and Elk-1
(22). To investigate whether phosphorylated Erk was functional in
Compound 5-treated cells, we examined its ability to phosphorylate the transcription factor Elk-1. Phospho-Erk was immunoprecipitated from
Compound 5-treated and untreated cells, and immunoprecipitates were
subjected to an in vitro kinase assay using recombinant
GST-Elk-1 fusion protein as a substrate. Assay mixtures were separated
by SDS-PAGE and immunoblotted with anti-phospho-Elk-1 antibody. Fig. 5B shows that nuclear phospho-Erk had kinase activity and
that its kinetics of activation correlated well with its
phosphorylation status. Compound 5-induced nuclear phospho-Erk was thus
functional and able to phosphorylate its physiological substrate,
Elk-1.
MEK Inhibition and Nuclear Translocation of Phospho-Erk,
Phospho-p38, or Phospho-JNK--
We next investigated possible
consequences of Erk or p38 activation by Compound 5. We first examined
whether inhibition of MEK, the direct upstream activating kinase for
Erk, would reduce phospho-Erk nuclear accumulation. Cells were
pretreated with the MEK1/MEK2 inhibitor U-0126 (23) for 45 min,
stimulated with Compound 5 for an additional 30 min in the presence of
the inhibitor, and analyzed on the ArrayScan II for nuclear
accumulation of phospho-Erk, phospho-p38, and phospho-JNK. Consistent
with results from Fig. 2, Compound 5 caused a robust increase in
nuclear phospho-Erk and phospho-p38, but had only a partial effect on
phospho-JNK (Fig. 6). Inclusion of 10 µM U-0126 caused almost complete inhibition of Compound
5-induced Erk activation, but, as expected, had little or no effect on
p38 or JNK activation. These data suggest that MEK inhibition is
sufficient to inhibit phospho-Erk nuclear accumulation by Compound
5.

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Fig. 6.
Inhibition of phospho-Erk (but not
phospho-p38 or phospho-JNK) nuclear translocation by a MEK
inhibitor. Hep3B cells were pretreated with 10 µM
U-0126 for 45 min and subsequently with vehicle (open bars),
10 µM Compound 5 (cpd 5; closed
bars), or a mixture of 10 µM Compound 5 and 10 µM U-0126 (hatched bars). After 30 min, cells
were fixed and stained with anti-phospho-Erk, anti-phospho-p38, or
anti-phospho-JNK antibodies. The data shown are the means ± S.D.
(normalized to the maximum signal obtained) from quadruplicate wells.
The conditions for maximum stimulation were as follows: 10 µM Compound 5 for Erk and p38 and 25 ng/ml IL-1 for
JNK and NF- B. Results are from a single representative experiment
that was repeated at least two times.
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The MEK Inhibitor U-0126 Protects Cells from the Antiproliferative
Effects of Compound 5--
To determine whether the activation of Erk
or p38 played a role in mediating the antiproliferative activity of
Compound 5, cells were incubated with the indicated concentrations of
Compound 5 in either the presence or absence of 5 µM
U-0126 for 72 h. Cells were harvested and stained with Hoechst
33258, and cellular DNA was quantified by fluorometry as previously
described (17). Fig. 7 shows that
inclusion of the MEK inhibitor significantly reduced Compound
5-mediated cell growth inhibition. This strongly suggests that
activation of the Erk pathway is the major determinant in the
antiproliferative effects of Compound 5. In contrast, p38 activation,
which has been implicated in cell death in many cell types, did not
appear to mediate growth inhibition of Hep3B cells by Compound 5 since,
in the presence of U-0126, cell growth continued despite high levels of
nuclear phospho-p38, but depressed levels of phospho-Erk (see Fig.
6).

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Fig. 7.
Protection of cells by the MEK inhibitor
U-0126 from Compound 5-mediated growth inhibition. Hep3B cells
were grown in 12-well tissue culture plates and treated every 24 h
with the indicated concentrations of Compound 5 in the presence ( )
or absence ( ) of 5 µM U-0126. After 3 days, the medium
was removed, and cell number was estimated by fluorometric quantitation
of cellular DNA as described under "Experimental Procedures." Data
are the means ± S.D. from seven independent experiments performed
in duplicate. **, p < 0.005; ***, p < 0.001 (as determined by two-tailed Student's t test,
assuming unequal variances).
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Effects of Compound 5 on Phospho-Erk Nuclear Accumulation Are Cell
Type-dependent--
To determine whether the observed
accumulation of phospho-Erk was specific for Hep3B cells, we examined
the ability of Compound 5 to induce phospho-Erk nuclear accumulation in
a variety of mammalian cell lines using the ArrayScan II. We found that
Compound 5-induced phospho-Erk nuclear accumulation was not unique to
Hep3B cells, but that the magnitude of response varied with cell type.
Cell lines fell into three categories based on the magnitude of
phospho-Erk induction. Strong responders were NIH3T3, Rat-1, and Hep3B
cells, which showed up to 24-, 75-, and 57-fold increases over control cells, respectively, in nuclear phospho-Erk levels 30 min after exposure to 10 µM Compound 5 (data not shown). DU-145 and
PC-3 prostate cancer cells were less responsive (2-3-fold increase after 30-min exposure to 10 µM Compound 5), and HeLa
cells did not respond to Compound 5 with enhanced phospho-Erk nuclear
accumulation at concentrations up to 30 µM (data not
shown). Thus, the induction of phospho-Erk nuclear accumulation by
Compound 5 was not limited to Hep3B cells, but instead constituted a
more generalized phenomenon.
Compound 5 Restores Phospho-Erk Levels after Cdc25A
Overexpression--
Because in vitro studies had shown that
Compound 5 is most effective against the Cdc25 family of
dual-specificity phosphatases (12), we investigated whether the effects
of a brief treatment with Compound 5 on phospho-Erk nuclear
accumulation could be attributed to Cdc25A inhibition. Previous reports
have revealed that the tyrosine phosphorylation status and activity of
Raf-1, which is an upstream activator of Erk, are controlled by Cdc25A
(16). Thus, we hypothesized that ectopic expression of Cdc25A might reduce Erk phosphorylation and provide a novel assay system to examine
the acute actions of Compound 5 against intracellular Cdc25A. We
selected HeLa cells as a model because in the absence of ectopic
Cdc25A, no phospho-Erk nuclear accumulation was seen with Compound 5 in
these cells, possibly due to low endogenous Cdc25A activity. We
predicted that this model would therefore have the lowest background
and that any effect seen with a small molecule could be assigned to an
action on the ectopically expressed Cdc25A. As illustrated in Fig.
8 (upper panels), ectopic
Cdc25A expression reduced Erk phosphorylation by 50%
(p < 0.05) (Fig. 8, lower panel). This
reduction in Erk phosphorylation absolutely required the intrinsic
phosphatase activity of Cdc25A because a catalytically inactive Cdc25A
mutant (C430S) did not reduce Erk phosphorylation in these
cells. We then asked whether Compound 5 was able to restore Erk
phosphorylation after ectopic expression of wild-type Cdc25A. Cells
transiently transfected with wild-type Cdc25A were allowed to recover
for 48 h and, during the last 30 min of recovery, treated with
vehicle or increasing concentrations of Compound 5. Fig. 8 (lower
panel) shows that Compound 5 gradually restored Erk
phosphorylation to mock/control levels. Consistent with the inherent
unresponsiveness of HeLa cells to Compound 5 (see above), the levels of
Erk phosphorylation in untransfected cells were not markedly changed
upon Compound 5 treatment (Fig. 8, upper panels).
Furthermore, phospho-Erk levels in Cdc25A-expressing, Compound
5-treated cells never exceeded those in untransfected cells (Fig. 8,
lower panel). These results support the hypothesis that
Compound 5 interferes with Cdc25A-mediated dephosphorylation of
Erk.

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Fig. 8.
Restoration of Erk phosphorylation in cells
overexpressing Cdc25A by Compound 5. Upper panels, HeLa
cells were transfected with plasmids encoding wild-type or C430S mutant
Cdc25A and allowed to recover for 48 h. Where indicated, cells
were treated with various concentrations of Compound 5 during the last
30 min of recovery, lysed, and analyzed for phospho-Erk levels by
Western blot analysis using anti-phospho-Erk
(phospho-p44/p42) antibody. Protein bands were quantified by
densitometry. p42 indicates the total Erk2 loading control.
Lower panel, Erk phosphorylation values are shown as a
percent of the mock-transfected control, averaged (means ± S.E.)
from the number of experiments indicated in the bars. *,
p < 0.05; **, p < 0.005 (as
determined by two-tailed Student's t test, assuming unequal
variances) compared with the mock-transfected control. pErk,
phospho-Erk.
|
|
 |
DISCUSSION |
Protein-tyrosine and dual-specificity phosphatases play a major
role in receptor-mediated signal transduction events. For example, the
kinase activities of growth factor receptors are regulated by tyrosine
autophosphorylation. Signals initiated at the cell surface are
transmitted by a series of cytoplasmic kinases that sequentially
phosphorylate each other, eventually leading to activation of members
of the MAPK superfamily, viz. Erk, p38, and SAPK/JNK, by
dual phosphorylation on tyrosine and threonine residues.
Protein-tyrosine phosphatases dephosphorylate tyrosines and thus
inactivate growth factor receptors, whereas the signal at the level of
MAPK is attenuated by dephosphorylation of the MAPKs on both tyrosine
and threonine by specific MKPs. Recently, the cell cycle phosphatase
Cdc25A has been proposed to regulate the tyrosine phosphorylation and
activity of Raf-1, a key element in the Erk signaling cascade (16).
In contrast to their upstream activating kinases,
tyrosine/threonine-phosphorylated MAPKs translocate to the nucleus,
where they phosphorylate and activate their respective protein targets, which include several transcription factors. Very few studies have
addressed spatial aspects of phosphorylation-dependent
signaling events; and to our knowledge, none have investigated small
molecules that might perturb the subcellular localization of key signal transducers in the context of tyrosine phosphorylation. Here we have
used a high-content, cell-based assay to evaluate the temporal and
spatial dynamics of three key parallel signaling molecules in response
to Compound 5, a synthetic vitamin K analog with in vitro
anti-phosphatase activity (12) and antiproliferative activity (10) in a
variety of cell lines. Using this novel methodology, we found that
Compound 5 caused rapid and irreversible nuclear accumulation of
phospho-Erk and phospho-p38. The observed activation of Erk by a
compound known to cause growth inhibition (11-12, 17) is somewhat
surprising since brief activation of Erk is often associated with
mitogenesis and survival. In contrast, JNK and p38 are thought to be
mediators of stress responses and apoptosis (24). We considered the
possibility that p38 activation might be a factor in the
antiproliferative activity of Compound 5. In the presence of the MEK1
inhibitor U-0126, however, which inhibits Erk activation, Hep3B cells
grew despite having high levels of phospho-p38. In contrast, we found
that pretreatment of cells with U-0126 not only prevented Compound
5-induced Erk phosphorylation, but also protected cells from the growth
inhibitory effects of Compound 5. These results strongly support our
previous suggestion that prolonged activation of the Erk pathway is
causally involved in the growth inhibitory effects of Compound 5 (13,
17). Moreover, our conclusion is in agreement with a growing body of
data documenting an involvement of Erk in growth inhibition in neuronal
cells (25), NIH3T3 cells (26), and MCF-7 cells (14). In addition, there is increasing evidence that p38 does not appear to exclusively mediate
cytotoxicity, but can be cytoprotective under certain conditions
(27-29).
The inability of Compound 5 to induce NF-
B and, to a lesser extent,
JNK suggests specificity and that it is not a general stress-inducing
stimulus. Both NF-
B and JNK are activated by a variety of
extracellular stimuli such as oxidative stress and inflammatory
cytokines. In addition, the broad protein-tyrosine phosphatase
inhibitors vanadate and pervanadate have been found to induce NF-
B
(30, 31), providing further support for a unique and more specific
action associated with Compound 5. We recently demonstrated that
Compound 5 selectively inhibits members of the dual-specificity
phosphatase family with a median inhibitory value of 4 µM
for Cdc25B2 and Cdc25A, whereas it is 10-fold less active
against VHR, a prototype MKP, and 100-fold less active against
protein-tyrosine phosphatase 1B (12). Furthermore, Compound 5 causes
cell cycle arrest in both G1 and G2, which
correlates with enhanced phosphorylation of the Cdc25 substrates Cdk1,
Cdk2, and Cdk4 (12). We suggested that the growth inhibitory properties of Compound 5 might be due to inhibition of the Cdc25 family; but in
large part due to a lack of appropriate assays, there has been no
direct evidence that Compound 5 inhibits Cdc25 phosphatases in the
cell. To investigate whether Cdc25A could affect Erk phosphorylation status and be inhibited within cells by Compound 5, we devised a
chemical complementation strategy based on earlier observations that
Cdc25A associates with the Raf-1 oncoprotein (15). Functional evidence
that Cdc25A regulates Raf-1 activity was obtained by Xia et
al. (16), who showed that overexpression of Raf-1 together with
wild-type Cdc25A reduces platelet-derived growth factor-mediated Raf-1
tyrosine phosphorylation in NIH3T3 cells. Raf-1 is one of the most
important upstream activators of the Erk cascade (32). We thus
hypothesized that Cdc25A overexpression would result in decreased Erk
phosphorylation and that an inhibitor of Cdc25A would restore Erk
phosphorylation to normal levels by chemically complementing the
loss-of-function phenotype caused by Cdc25A overexpression. To simplify
the analysis, we chose HeLa cells, which did not respond to Compound 5 with increased phospho-Erk nuclear accumulation. By treating
Cdc25A-overexpressing cells with concentrations of Compound 5 that did
not cause Erk hyperphosphorylation under normal growth conditions, we
were able to demonstrate that Compound 5 specifically inhibited the
effects of the overexpressed Cdc25A protein on Erk phosphorylation.
Thus, we have obtained, for the first time, evidence that Cdc25A
regulates endogenous Erk phosphorylation status in whole cells and that
Compound 5 affects Cdc25A function in the cell.
Although the concentrations of Compound 5 required for inhibition of
the MKP VHR in vitro are an order of magnitude higher than
those required for Cdc25A inhibition, it is possible that inhibition of
MKPs by Compound 5 also contributes to Erk and p38 activation. A number
of cytosolic and nuclear MKPs, which have overlapping substrate
specificities, have been described. For example, the Erk isoforms are
selectively inhibited by MKP-3, whereas M3/6 selectively
dephosphorylates JNK (33). MKP-1 and MKP-2 preferentially
dephosphorylate JNK, but also have some activity toward p38 (34, 35).
More recently, a p38-specific phosphatase, MKP-5, has been reported
(36). The prototype dual-specificity phosphatase VHR, which seems to
reside in the nucleus, dephosphorylates Erk (37), but its effect on
other kinases has not been examined. The fact that Compound 5 only
partially activated JNK suggests that it may have some selectivity. At
this time, we do not have any information about whether Compound 5 has
any specificity for the different MKPs, but this information should
become available as we expand our chemical complementation strategy to
probe for cell-active inhibitors of MKPs.
In summary, using the ArrayScan II, we were able to quickly and
quantitatively probe selective activation of tyrosine
phosphorylation-dependent signal transduction events by a
small molecule dual-specificity phosphatase inhibitor in intact cells.
By performing fluorescence-based spatial analysis in a high-throughput
compatible format, we demonstrated that this inhibitor selectively
activated dual-specificity phosphatase-dependent cellular
events. Subsequent analyses using both genetic and pharmacological tools identified activation of the Erk pathway as the dominant component mediating the antiproliferative activity of Compound 5 and
provided direct evidence that it could interfere with Cdc25A function
in the cell. We propose that the combination of high-content, cell-based analyses with a chemical complementation approach will be a
powerful technique to identify cell-active inhibitors of a variety of
cellular targets.