Spatial Analysis of Key Signaling Proteins by High-content Solid-phase Cytometry in Hep3B Cells Treated with an Inhibitor of Cdc25 Dual-specificity Phosphatases*

Andreas VogtDagger , Takahito Adachi§, Alexander P. DucruetDagger , Jon ChesebroughDagger , Kaoru NemotoDagger , Brian I. Carr§, and John S. LazoDagger

From the Departments of Dagger  Pharmacology and § Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Received for publication, January 4, 2001, and in revised form, March 14, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein phosphorylation frequently results in the subcellular redistribution of key signaling molecules, and this spatial change is critical for their activity. Here we have probed the effects of a Cdc25 inhibitor, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, or Compound 5, on the spatial regulation and activation kinetics of tyrosine phosphorylation-dependent signaling events using two methods: (i) high-content, automated, fluorescence-based, solid-phase cytometry and (ii) a novel cellular assay for Cdc25A activity in intact cells. Immunofluorescence studies demonstrated that Compound 5 produced a concentration-dependent nuclear accumulation of phospho-Erk and phospho-p38, but not nuclear factor kappa B. Immunoblot analysis confirmed Erk phosphorylation and nuclear accumulation, and in vitro kinase assays showed that Compound 5-activated Erk was competent to phosphorylate its physiological substrate, the transcription factor Elk-1. Pretreatment of cells with the MEK inhibitor U-0126 prevented the induction by Compound 5 of phospho-Erk (but not phospho-p38) nuclear accumulation and protected cells from the antiproliferative effects of Compound 5. Overexpression of Cdc25A in whole cells caused dephosphorylation of Erk that was reversed by Compound 5. The data show that an inhibitor of Cdc25 increases Erk phosphorylation and nuclear accumulation and support the hypothesis that Cdc25A regulates Erk phosphorylation status.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- Compound 5 (2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone) has been described previously (10). Human recombinant interleukin-1alpha (IL-1alpha ) 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-kappa 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-kappa 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-1alpha , 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-kappa 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 beta -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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

Induction of Phospho-Erk and Phospho-p38, but Not NF-kappa 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-1alpha ; immunostained with anti-phospho-Erk, anti-phospho-p38, anti-phospho-JNK, or anti-NF-kappa 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-kappa B. In contrast, IL-1alpha activated all three stress-response mediators (p38, JNK, and NF-kappa B), but not Erk. Thus, the activity profile of Compound 5 was distinct from that of the cytokine IL-1alpha , 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-kappa 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-kappa 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-1alpha for JNK and NF-kappa 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.

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.

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.

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.

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-1alpha for JNK and NF-kappa B. Results are from a single representative experiment that was repeated at least two times.

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 (black-square) 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).

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-kappa B and, to a lesser extent, JNK suggests specificity and that it is not a general stress-inducing stimulus. Both NF-kappa 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-kappa 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.

    ACKNOWLEDGEMENTS

We thank Donald B. DeFranco (University of Pittsburgh) and Kenneth A. Giuliano (Cellomics) for helpful discussions and critical review of this manuscript and Meifung Wang for excellent technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA 78039, CA 52995, and CA 82723; the Fiske Drug Discovery Fund; and a seed grant from the Pittsburgh Tissue Engineering Initiative.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: Dept. of Pharmacology, Biomedical Science Tower E-1340, University of Pittsburgh, Pittsburgh, PA 15261. Tel.: 412-648-9319; Fax: 412-648-2229; E-mail: lazo@pitt.edu.

Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M100078200

    ABBREVIATIONS

The abbreviations used are: VHR, VH-1-related phosphatase; Erk, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; IL-1alpha , interleukin-1alpha ; NF-kappa B, nuclear factor kappa  B; Hsp90, 90-kDa heat shock protein; JNK, c-Jun N-terminal kinase; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase; SAPK, stress-activated protein kinase; MKP, mitogen-activated protein kinase phosphatase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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