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

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. an inhibitor of Cdc25 increased Erk which the hypothesis that Cdc25A regulates Erk phosphorylation status. Compound 5 has been described (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). interleukin-1α was from and the MEK inhibitor U-0126 from was from for and the of of a assay was from and was from and or in and in a of and NIH3T3 cells from from of human hepatoma cells have been Y. Wang Z. Kerns J. Wilcox Carr B.I. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). or NIH3T3 cells in the of a and to for the with Compound 5 or with in and with with against phospho-p38, or the of and with with fluorescent and cells by the using an at of and by analysis on the ArrayScan II using the described to J.A. Gough A. R.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to assess the of and nuclear using a modified as by et al. P. Res. PubMed Scopus Google Scholar). cells in to 5 for the of and by in of and on for by a and at for The was as in nuclear and on for and at to the nuclear proteins by and to probed with and using and an to the Erk activity in and nuclear was using a of nuclear or proteins with of and at with and with kinase 5 and in of kinase with and of protein and for at in and by using The antiproliferative activity of Compound 5 in with the MEK inhibitor U-0126 was by a described assay on of cellular using the J. PubMed Scopus Google Scholar). cells in and for with of Compound 5 in the or of the MEK inhibitor U-0126 by and cellular was as described J. PubMed Scopus Google Scholar). expression Cdc25A and Cdc25A in a by Roberts Cancer (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). by the the cells in the of a and with of in using the was with growth and the cells to for with Compound 5 for and protein and by and analysis for phospho-Erk and as described of protein expression on a and using the Compound 5 was to the phosphorylation of on a number of signaling proteins in the Erk including and (13Wang Z. Wang M. Carr B.I. J. Cell. Physiol. 2000; 183: 338-346Crossref PubMed Scopus (29) Google Scholar, S. Carr B.I. J. Cell. Physiol. 2000; 185: 386-393Crossref PubMed Scopus (30) Google Scholar). whether this in tyrosine phosphorylation was with a change in phospho-Erk nuclear cells with or Compound 5 for and with against a phosphorylated of Erk and by B that cells low of most of which was in the of cells with Compound 5 resulted in a in with nuclear accumulation and the of and nuclear phospho-Erk cells in two by the ArrayScan and the was by a described J.A. Gough A. R.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the nuclear area. within this nuclear is to as assess the of phospho-Erk in the a of by two was the nuclear within the is to as and to the or and are as by the from the an in the is of Erk activation or whether Compound 5 caused selective Erk nuclear accumulation by its effects with of signaling events that have also been reported to be in a tyrosine phosphorylation-dependent and are thought to for with Compound 5 or with or and for in A of cells in that Compound 5 to a in nuclear accumulation of phospho-Erk and phospho-p38, but a on and not the nuclear accumulation of In and but not the activity of Compound 5 was from that of the that Compound 5 is not a with the and phosphatase inhibitor demonstrated that and Erk are with kinetics in a of cell S. A. M. K. H. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). It was also reported that Erk activation is by of and the kinase inhibitor an involvement of in Erk the and kinetics of phospho-Erk and activation in cells. that of Erk and was at Compound 5. to Compound 5 caused a activation and nuclear accumulation with have also that the inhibitor not phospho-Erk nuclear accumulation not results that Compound 5 the tyrosine phosphatase inhibitor Compound 5 is a and its anti-phosphatase activity has been to of critical on dual-specificity and tyrosine phosphatases Y. Wang Z. Kerns J. Wilcox Carr B.I. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). whether its effects we cells with Compound 5 for 5 or by and compared the of phospho-Erk and accumulation with that a that of Compound 5 resulted in activation of Erk and consistent with a and inhibition of cellular phosphatases the results from the automated, analysis by biochemical with Compound 5 for the and nuclear and by using cells no nuclear consistent with the whole cell in Compound 5 caused a and in phospho-Erk nuclear In phospho-Erk in cells in the and increased a to Compound 5 5 The results from the analysis confirmed from the less solid-phase cytometry used the from Compound cells to whether the Erk phosphorylation resulted in an in Erk kinase activity. It is thought that upon phosphorylation by and in the a of Erk to the it and transcription as and C. J. Mol. Cell. Biol. PubMed Google Scholar). whether phosphorylated Erk was functional in Compound we its ability to phosphorylate the transcription factor Elk-1. was from Compound and and to an in vitro kinase assay using protein as a by and with B that nuclear phospho-Erk kinase activity and that its kinetics of activation well with its phosphorylation status. Compound nuclear phospho-Erk was functional and to phosphorylate its physiological substrate, Elk-1. possible of Erk or activation by Compound 5. whether inhibition of the direct upstream kinase for Erk, reduce phospho-Erk nuclear with the inhibitor U-0126 R.A. F. R.A. R.L. J.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) for with Compound 5 for an in the of the inhibitor, and on the ArrayScan II for nuclear accumulation of phospho-p38, and with results from Compound 5 caused a in nuclear phospho-Erk and phospho-p38, but a on of U-0126 caused inhibition of Compound Erk as or no on or data that MEK inhibition is to phospho-Erk nuclear accumulation by Compound 5. whether the activation of Erk or a role in the antiproliferative activity of Compound 5, cells with the of Compound 5 in the or of 5 for and with and cellular was by as described Y. Wang Z. Kerns J. Wilcox Carr B.I. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). that of the MEK inhibitor Compound cell growth suggests that activation of the Erk pathway is the major in the antiproliferative effects of Compound 5. In which has been in cell in many cell not to growth inhibition of cells by Compound 5 in the of cell growth of nuclear phospho-p38, but of phospho-Erk whether the accumulation of phospho-Erk was for we the ability of Compound 5 to phospho-Erk nuclear accumulation in a of mammalian cell using the ArrayScan that Compound phospho-Erk nuclear accumulation was not to but that the of response with cell on the of phospho-Erk and which showed to and increases over in nuclear phospho-Erk to Compound 5 not and cells less to Compound and cells not to Compound 5 with phospho-Erk nuclear accumulation at to not the induction of phospho-Erk nuclear accumulation by Compound 5 was not to but a more in vitro studies that Compound 5 is most 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 whether the effects of a treatment with Compound 5 on phospho-Erk nuclear accumulation could be to Cdc25A have that the tyrosine phosphorylation and activity of Raf-1, which is an upstream activator of Erk, are 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). we that expression of Cdc25A might reduce Erk phosphorylation and a novel assay to the actions of Compound 5 against cells as a in the of no phospho-Erk nuclear accumulation was with Compound 5 in these to low Cdc25A activity. that this have the and that with a small could be to an on the in Cdc25A expression Erk phosphorylation by in Erk phosphorylation the phosphatase activity of Cdc25A a Cdc25A not reduce Erk phosphorylation in these cells. whether Compound 5 was to Erk phosphorylation expression of with Cdc25A to for the of with or of Compound 5. that Compound 5 Erk phosphorylation to with the of cells to Compound 5 the of Erk phosphorylation in cells not upon Compound 5 treatment phospho-Erk in Compound cells in cells results support the hypothesis that Compound 5 with dephosphorylation of and dual-specificity phosphatases play a major role in signal transduction the kinase of growth factor are regulated by tyrosine at the cell are by a of kinases that phosphorylate to activation of of the Erk, and by phosphorylation on tyrosine and phosphatases and growth factor the signal at the of is by dephosphorylation of the on tyrosine and by the cell cycle phosphatase Cdc25A has been to regulate the tyrosine phosphorylation and activity of Raf-1, a key in the Erk signaling (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). In to their upstream kinases, to the phosphorylate and their protein which several transcription studies have spatial of phosphorylation-dependent signaling and to our have small molecules that might the subcellular localization of key signal in the of tyrosine phosphorylation. Here we have used a high-content, cell-based assay to the and spatial of key signaling molecules in response to Compound 5, a synthetic vitamin K with in activity (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) and antiproliferative activity (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) in a of cell Using this novel we that Compound 5 caused and nuclear accumulation of phospho-Erk and The activation of Erk by a to cause growth inhibition J. Naganathan S. Dowd P. Finn F.M. Carr B.I. Bioorg. Chem. 1995; 23: 101-108Crossref Scopus (43) Google Scholar, K. Southwick E.C. Kerns J. Rosi K. Carr B.I. Wilcox C. Lazo J.S. Cancer Res. 2000; 60: 1317-1325PubMed Google Scholar, Y. Wang Z. Kerns J. Wilcox Carr B.I. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) is activation of Erk is with and In and are thought to be of and Z. M. J. R.J. Science. 1995; 270: PubMed Scopus Google Scholar). the that activation might be a factor in the antiproliferative activity of Compound 5. In the of the inhibitor which Erk cells of In we that of cells with U-0126 not prevented Compound Erk but also protected cells from the growth effects of Compound 5. results support our that activation of the Erk pathway is in the growth effects of Compound 5 (13Wang Z. Wang M. Carr B.I. J. Cell. Physiol. 2000; 183: 338-346Crossref PubMed Scopus (29) Google Y. Wang Z. Kerns J. Wilcox Carr B.I. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). our is in with a of data an involvement of Erk in growth inhibition in cells M. Wang Y. S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), NIH3T3 cells S. A. 1997; PubMed Scopus Google Scholar), and MCF-7 cells (14Kar S. Carr B.I. J. Cell. Physiol. 2000; 185: 386-393Crossref PubMed Scopus (30) Google Scholar). In is evidence that not to but can be certain Z. 2000; 19: PubMed Scopus Google Scholar, C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, P. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The of Compound 5 to to a suggests specificity and that it is not a and are by a of extracellular as and In the protein-tyrosine phosphatase inhibitors and have been to R.A. A. C. D. P. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, F. M. Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), support for a and more with Compound 5. recently demonstrated that Compound 5 of the dual-specificity phosphatase family with a of and it is less against a prototype and 100-fold less against protein-tyrosine phosphatase (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). Compound 5 causes cell cycle arrest in G1 and which with phosphorylation of the Cdc25 substrates Cdk2, and Cdk4 (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). that the growth of Compound 5 might be to inhibition of the Cdc25 but in part to a lack of has been no direct evidence that Compound 5 Cdc25 phosphatases in the whether Cdc25A could Erk phosphorylation and be within cells by Compound 5, we a on that Cdc25A associates with the Raf-1 (15Galaktionov K. Jessus C. Beach D. Genes Dev. 1995; 9: 1046-1058Crossref PubMed Scopus (230) Google Scholar). evidence that Cdc25A regulates Raf-1 activity was 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 showed that of Raf-1 together with Cdc25A platelet-derived growth Raf-1 tyrosine phosphorylation in NIH3T3 cells. Raf-1 is one of the most upstream of the Erk J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). that Cdc25A in Erk phosphorylation and that an inhibitor of Cdc25A Erk phosphorylation to by the caused by Cdc25A the we which not to Compound 5 with increased phospho-Erk nuclear cells with of Compound 5 that not cause Erk hyperphosphorylation growth we to that Compound 5 the effects of the Cdc25A protein on Erk phosphorylation. we have for the evidence that Cdc25A regulates Erk phosphorylation in whole cells and that Compound 5 Cdc25A function in the Although the of Compound 5 for inhibition of the in vitro are an of for Cdc25A it is possible that inhibition of by Compound 5 also to Erk and A number of and nuclear which have substrate have been the Erk are by M. A. M. C. K. A. S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). and but also have activity Y. K. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). More recently, a has been reported T. T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The prototype dual-specificity phosphatase which to in the Erk J.M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), but its on kinases has not been The that Compound 5 suggests that it may have this we not have information whether Compound 5 has specificity for the but this information as we our to for inhibitors of In using the ArrayScan we to and selective activation of tyrosine phosphorylation-dependent signal transduction events by a small dual-specificity phosphatase inhibitor in intact cells. spatial analysis in a we demonstrated that this inhibitor dual-specificity cellular using and tools activation of the Erk pathway as the the antiproliferative activity of Compound 5 and direct evidence that it could with Cdc25A function in the that the of high-content, cell-based with a be a to inhibitors of a of cellular of and A. for and critical of this and Wang for