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• W2102586514 abstract "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 κB. Immunoblot analysis confirmed Erk phosphorylation and nuclear accumulation, andin 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. 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 κB. Immunoblot analysis confirmed Erk phosphorylation and nuclear accumulation, andin 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. VH-1-related phosphatase extracellular signal-regulated kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase interleukin-1α nuclear factor κ B 90-kDa heat shock protein c-Jun N-terminal kinase polyacrylamide gel electrophoresis glutathioneS-transferase mitogen-activated protein kinase stress-activated protein kinase mitogen-activated protein kinase phosphatase Approximately one-third of mammalian proteins are thought to be post-translationally modified by phosphorylation (1Zolnierowicz S. Bollen M. EMBO J. 2000; 19: 483-488Crossref PubMed Scopus (155) Google Scholar). The human genome contains hundreds of protein kinases (2Venter J.C. Adams M.D. Myers E.W. Li P.W. Mural R.J. Sutton G.G. Smith H.O. Yandell M. Evans C.A. Holt R.A. Gocayne J.D. Amanatides P. Ballew R.M. Huson D.H. Wortman J.R. et al.Science. 2001; 291: 1304-1351Crossref PubMed Scopus (10633) Google Scholar), 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 (3Tonks N.K. Neel B.G. Cell. 1996; 87: 365-368Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar). 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 (4Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1900) Google Scholar). 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 (5Teruel M.N. Meyer T. Cell. 2000; 103: 181-184Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Phosphorylation of many key signaling molecules causes a subcellular redistribution that is critical for biological activity (6Brunet A. Roux D. Lenormand P. Dowd S. Keyse S. Pouyssegur J. EMBO J. 1999; 18: 664-674Crossref PubMed Scopus (517) Google Scholar, 7Dalal S.N. Schweitzer C.M. Gan J. DeCaprio J.A. Mol. Cell. Biol. 1999; 19: 4465-4479Crossref PubMed Scopus (240) Google Scholar, 8Liu F. Rothblum-Oviatt C. Ryan C.E. Piwnica-Worms H. Mol. Cell. Biol. 1999; 19: 5113-5123Crossref PubMed Scopus (98) Google Scholar). 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 (9Giuliano K.A. DeBiasio R.L. Dunlay T. Gough A. Volosky J.M. Zock J. Pavlakis G.N. Taylor D.L. J. Biomol. Screening. 1997; 2: 249-259Crossref Scopus (187) Google Scholar), 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 (10Nishikawa Y. Carr B.I. Wang M. Kar S. Finn F. Dowd P. Zheng Z.B. Kerns J. Naganathan S. J. Biol. Chem. 1995; 270: 28304-28310Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar,11Kerns J. Naganathan S. Dowd P. Finn F.M. Carr B.I. Bioorg. Chem. 1995; 23: 101-108Crossref Scopus (43) Google Scholar). 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 (12Tamura K. Southwick E.C. Kerns J. Rosi K. Carr B.I. Wilcox C. Lazo J.S. Cancer Res. 2000; 60: 1317-1325PubMed Google Scholar). 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 (12Tamura K. Southwick E.C. Kerns J. Rosi K. Carr B.I. Wilcox C. Lazo J.S. Cancer Res. 2000; 60: 1317-1325PubMed Google Scholar). 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 (12Tamura K. Southwick E.C. Kerns J. Rosi K. Carr B.I. Wilcox C. Lazo J.S. Cancer Res. 2000; 60: 1317-1325PubMed Google Scholar). 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 (13Wang Z. Wang M. Carr B.I. J. Cell. Physiol. 2000; 183: 338-346Crossref PubMed Scopus (29) Google Scholar) and MCF-7 cells (14Kar S. Carr B.I. J. Cell. Physiol. 2000; 185: 386-393Crossref PubMed Scopus (30) Google Scholar), 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 Galaktionovet al. (15Galaktionov K. Jessus C. Beach D. Genes Dev. 1995; 9: 1046-1058Crossref PubMed Scopus (230) Google Scholar), 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. (16Xia K. Lee R.S. Narsimhan R.P. Mukhopadhyay N.K. Neel B.G. Roberts T.M. Mol. Cell. Biol. 1999; 19: 4819-4824Crossref PubMed Scopus (42) Google Scholar), 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. Compound 5 (2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone) has been described previously (10Nishikawa Y. Carr B.I. Wang M. Kar S. Finn F. Dowd P. Zheng Z.B. Kerns J. Naganathan S. J. Biol. Chem. 1995; 270: 28304-28310Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Human recombinant interleukin-1α (IL-1α) 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-κB 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-κB) IgG (Molecular Probes, Eugene, OR). 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% CO2at 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 (17Nishikawa Y. Wang Z. Kerns J. Wilcox C.S. Carr B.I. J. Biol. Chem. 1999; 274: 34803-34810Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). 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-1α, 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-κB 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 (18Ding G.J. Fischer P.A. Boltz R.C. Schmidt J.A. Colaianne J.J. Gough A. Rubin R.A. Miller D.K. J. Biol. Chem. 1998; 273: 28897-28905Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Control experiments omitting primary antibodies were performed each time to assess the amount of nonspecific background staining. Cytosolic and nuclear fractions were prepared using a slightly modified procedure as published by Schreiber et al. (19Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3917) Google Scholar). Hep3B cells were plated in 100-mm tissue culture dishes, exposed to 10 μmCompound 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 mmMgCl2, 10 mm KCl, 0.2 mmphenylmethylsulfonyl 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 mmMgCl2, 400 mm KCl, 0.2 mm EDTA, 0.2 mm phenylmethylsulfonyl fluoride, and 0.5 mmdithiothreitol), 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 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 β-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. 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 (20Rago R. Mitchen J. Wilding G. Anal. Biochem. 1990; 191: 31-34Crossref PubMed Scopus (387) Google Scholar). 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 (20Rago R. Mitchen J. Wilding G. Anal. Biochem. 1990; 191: 31-34Crossref PubMed Scopus (387) Google Scholar). 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) (16Xia K. Lee R.S. Narsimhan R.P. Mukhopadhyay N.K. Neel B.G. Roberts T.M. Mol. Cell. Biol. 1999; 19: 4819-4824Crossref PubMed Scopus (42) Google Scholar). 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). 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 (13Wang Z. Wang M. Carr B.I. J. Cell. Physiol. 2000; 183: 338-346Crossref PubMed Scopus (29) Google Scholar, 14Kar S. Carr B.I. J. Cell. Physiol. 2000; 185: 386-393Crossref PubMed Scopus (30) Google Scholar). 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, Aand D). Fig. 1 B 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. 1 E). 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 (18Ding G.J. Fischer P.A. Boltz R.C. Schmidt J.A. Colaianne J.J. Gough A. Rubin R.A. Miller D.K. J. Biol. Chem. 1998; 273: 28897-28905Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Hoechst 33342 staining (Fig. 1, Aand 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. 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-1α; immunostained with anti-phospho-Erk, anti-phospho-p38, anti-phospho-JNK, or anti-NF-κB 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-κB. In contrast, IL-1α activated all three stress-response mediators (p38, JNK, and NF-κB), but not Erk. Thus, the activity profile of Compound 5 was distinct from that of the cytokine IL-1α, suggesting that Compound 5 is not a general stress-inducing agent. 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 (21Ludwig S. Hoffmeyer A. Goebeler M. Kilian K. Hafner H. Neufeld B. Han J. Rapp U.R. J. Biol. Chem. 1998; 273: 1917-1922Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). 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. 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 (17Nishikawa Y. Wang Z. Kerns J. Wilcox C.S. Carr B.I. J. Biol. Chem. 1999; 274: 34803-34810Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). 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. 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.5 A). Untreated cells had almost no nuclear phospho-Erk, consistent with the whole cell images in Fig. 1 B. 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. 5 A). The results from the immunoblot analysis thus confirmed those from the less arduous solid-phase cytometry studies. 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 (22Chen R.H. Sarnecki C. Blenis J. Mol. Cell. Biol. 1992; 12: 915-927Crossref PubMed Google Scholar). 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.5 B 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. 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 (23Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2751) Google Scholar) 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. 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 μmU-0126 for 72 h. Cells were harvested and stained with Hoechst 33258, and cellular DNA was quantified by fluorometry as previously described (17Nishikawa Y. Wang Z. Kerns J. Wilcox C.S. Carr B.I. J. Biol. Chem. 1999; 274: 34803-34810Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). 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). 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. Because in vitro studies had shown that Compound 5 is most effective against the Cdc25 family of dual-specificity phosphatases (12Tamura K. Southwick E.C. Kerns J. Rosi K. Carr B.I. Wilcox C. Lazo J.S. Cancer Res. 2000; 60: 1317-1325PubMed Google Scholar), 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 (16Xia K. Lee R.S. Narsimhan R.P. Mukhopadhyay N.K. Neel B.G. Roberts T.M. Mol. Cell. Biol. 1999; 19: 4819-4824Crossref PubMed Scopus (42) Google Scholar). Thus, we hypothesized that ectopic expression of Cdc2" @default.
• W2102586514 created "2016-06-24" @default.
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• W2102586514 creator A5032116757 @default.
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• W2102586514 date "2001-01-01" @default.
• W2102586514 modified "2023-09-28" @default.
• W2102586514 title "Spatial Analysis of Key Signaling Proteins by High-content Solid-phase Cytometry in Hep3B Cells Treated with an Inhibitor of Cdc25 Dual-specificity Phosphatases" @default.
• W2102586514 cites W1781374595 @default.
• W2102586514 cites W1873548662 @default.
• W2102586514 cites W1914194999 @default.
• W2102586514 cites W1966778168 @default.
• W2102586514 cites W1969294711 @default.
• W2102586514 cites W1972172786 @default.
• W2102586514 cites W1973769612 @default.
• W2102586514 cites W1976381867 @default.
• W2102586514 cites W1976385326 @default.
• W2102586514 cites W1977158130 @default.
• W2102586514 cites W1980534531 @default.
• W2102586514 cites W1993454824 @default.
• W2102586514 cites W2003362851 @default.
• W2102586514 cites W2003940624 @default.
• W2102586514 cites W2016730049 @default.
• W2102586514 cites W2022723787 @default.
• W2102586514 cites W2030592815 @default.
• W2102586514 cites W2038908150 @default.
• W2102586514 cites W2039568202 @default.
• W2102586514 cites W2043373422 @default.
• W2102586514 cites W2043593059 @default.
• W2102586514 cites W2047929370 @default.
• W2102586514 cites W2049769334 @default.
• W2102586514 cites W2056823054 @default.
• W2102586514 cites W2059844384 @default.
• W2102586514 cites W2065032678 @default.
• W2102586514 cites W2065568194 @default.
• W2102586514 cites W2068502127 @default.
• W2102586514 cites W2080352303 @default.
• W2102586514 cites W2083750996 @default.
• W2102586514 cites W2084986398 @default.
• W2102586514 cites W2086262311 @default.
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