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

Vogt, Andreas; Adachi, Takahito; Ducruet, Alexander P.; Chesebrough, Jon; Nemoto, Kiyomitsu; Carr, Brian I.; Lazo, John S.

doi:10.1074/jbc.m100078200

Cited by 40 publications

(40 citation statements)

References 36 publications

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“…Whether there are other PTPs that are also inhibited in intact cells is considered likely, but has not been fully evaluated. In addition, we found that Cdc25A was a phosphatase both for phospho-ERK-2 and for phospho-EGFR (Vogt et al, 2001;. Several other p-ERK phosphatases have more recently been described and reviewed (Camps et al, 2000).…”

Section: Cellular Consequences Of K Vitamin Interaction With Ptpsmentioning

confidence: 64%

“…Cdc25B2 was by far the most sensitive substrate for Cpd 5 action, next was VHR, and PTP1B was two orders of magnitude less sensitive to the inhibitory actions of Cpd 5 (Tamura et al, 2000). We have found plentiful evidence that Ccd25A is a target of Cpd 5 growth in cell-free systems, in cells in culture and using recombinant phosphatase with authentic Cdk4p that was immunoprecipitated from our cells (Tamura et al, 2000;Wang et al, 2000;Vogt et al, 2001;Wang et al, 2001. Whether there are other PTPs that are also inhibited in intact cells is considered likely, but has not been fully evaluated.…”

Section: Cellular Consequences Of K Vitamin Interaction With Ptpsmentioning

confidence: 93%

“…On the other hand, in an EGFR mutated fibroblast NR-6 cell line, Cpd 5 could still activate ERK phosphorylation and inhibit cell growth , implying that Cpd 5 may have some effects on ERK phosphorylation by inhibiting its phosphatases. Recent study has shown that this ERK phosphatase is most likely Cdc25A, since over-expression of Cdc25A can abolish Cpd 5-induced ERK phosphorylation and nuclear translocation (Vogt et al, 2001). Growing evidence has shown that Cdc25A is a phosphatase for EGFR , Raf-1 (Galaktionov et al, 1995a;Xia et al, 1999), and ERK (Vogt et al, 2001), thus its inactivation by Cpd 5 must cause an imbalance between tyrosine phosphorylation and dephosphorylation, disturbing cell growth behavior.…”

Section: Prolonged Erk Phosphorylation Induced By Cpd 5 Is Related Tomentioning

confidence: 99%

“…The most prominent included the EGFR and ERK-2 (Nishikawa et al, 1995(Nishikawa et al, , 1999Ni et al, 1998;Kar and Carr, 2000;Wang et al, 2000;Vogt et al, 2001). We therefore focussed on proteins in the EGFR signaling cascade and likely PTP targets for K vitamin actions.…”

Section: Cellular Consequences Of K Vitamin Interaction With Ptpsmentioning

confidence: 99%

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K vitamins, PTP antagonism, and cell growth arrest

Carr

Wang

Kar

2002

Journal Cellular Physiology

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The main function of K vitamins is to act as co-factors for g-glutamyl carboxylase. However, they have also recently been shown to inhibit cell growth. We have chemically synthesized a series of K vitamin analogs with various side chains at the 2 or 3 position of the core naphthoquinone structure. The analogs with short thio-ethanol side chains are found to be more potent growth inhibitors in vitro of various tumor cell lines. Cpd 5 or [2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone] is one of the most potent. The anti-proliferation activity of these compounds is antagonized by exogenous thiols but not by non-thiol antioxidants. This suggests that the growth inhibition is mediated by sulfhydryl arylation of cellular glutathione and cysteine-containing proteins and not by oxidative stress. The protein tyrosine phosphatases (PTP) are an important group of proteins that contain cysteine at their catalytic site. PTPs regulate mitogenic signal transduction and cell cycle progression. PTP inhibition by Cpd 5 results in prolonged tyrosine phosphorylation and activation of several kinases and transcription factors including EGFR, ERK1/2, and Elk1. Cpd 5 could activate ERK1/2 either by signaling from an activated EGFR, which is upstream in the signaling cascade, or by direct inhibition of ERK1/2 phosphatase(s). Prolonged ERK1/2 phosphorylation strongly correlates with Cpd 5-mediated growth inhibition. Cpd 5 can also bind to and inhibit the Cdc25 family of dual specific phosphatases. As a result, several Cdc25 substrates (Cdk1, Cdk2, Cdk4) involved in cell cycle progression are tyrosine phosphorylated and thereby inhibited by its action. Cpd 5 could also inhibit both normal liver regeneration and hepatoma growth in vivo. DNA synthesis during rat liver regeneration following partial hepatectomy, transplantable rat hepatoma cell growth, and glutathione-S-transferase-pi expressing hepatocytes after administration of the chemical carcinogen diethylnitrosamine, are all inhibited by Cpd 5 administration. The growth inhibitory effect during liver regeneration and transplantable tumor growth is also correlated with ERK1/2 phosphorylation induced by Cpd 5. Thus, Cpd 5-mediated inhibition of PTPs, such as Cdc25 leads to cell growth arrest due to altered activity of key cellular kinases involved in signal transduction and cell cycle progression. This prototype K vitamin analog represents a novel class of growth inhibitor based upon its action as a selective PTP antagonist. It is clearly associated with prolonged ERK1/2 phosphorylation, which is in contrast with the transient ERK1/2 phosphorylation induced by growth stimulatory mitogens.

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Section: Cellular Consequences Of K Vitamin Interaction With Ptpsmentioning

confidence: 64%

Section: Cellular Consequences Of K Vitamin Interaction With Ptpsmentioning

confidence: 93%

Section: Prolonged Erk Phosphorylation Induced By Cpd 5 Is Related Tomentioning

confidence: 99%

Section: Cellular Consequences Of K Vitamin Interaction With Ptpsmentioning

confidence: 99%

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K vitamins, PTP antagonism, and cell growth arrest

Carr

Wang

Kar

2002

Journal Cellular Physiology

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“…However, we were unable to detect the prototype EGFR PTPase SH-PTP1 in Hep3B cells and found no inhibition of another prototype EGFR PTPase, SH-PTP2, by Cpd 5 (data not shown). Because we previously observed that Cdc25A activity was inhibited by vitamin K analogs in cell-free conditions and in cell cultures (31,34,35), we explored the possibility that EGFR hyperphosphorylation might be related to inhibition of Cdc25A activity. We first examined the physical association of EGFR with Cdc25A.…”

Section: Cpd 5 Produces Persistent Egfr But Not Insulin Receptormentioning

confidence: 99%

Identification of Epidermal Growth Factor Receptor as a Target of Cdc25A Protein Phosphatase

Wang¹,

Wang²,

Lazo³

et al. 2002

Journal of Biological Chemistry

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Cellular Assays and Their Application in Drug Discovery

Albrecht

Brodbeck‐Hummel

Hoever

et al. 2008

Protein Science Encyclopedia

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Originally published in: Molecular Biology in Medicinal Chemistry. Edited by Theodor Dingermann, Dieter Steinhilber and Gerd Folkers. Copyright © 2004 Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3‐527‐30431‐8 The sections in this article are Introduction Positioning Cellular Assays Impact on Drug Discovery Classification of Cellular Assays Progress in Tools and Technologies for Cellular Compound Profiling Membrane Proteins and Fast Cellular Responses Receptors FLIPR Technology for Detection of Intracellular Calcium Release Competitive Immunoassay for Detection of Intracellular cAMP Enzyme Fragment Complementation (EFC) Technology Membrane Transport Proteins Ion Channels MDR Proteins Gene and Protein Expression Profiling in High‐throughput Formats Reporter Gene Assays in Lead Finding Reporter Gene Assays in Lead Optimization Spatio‐temporal Assays and Subpopulation Analysis Phosphorylation Stage‐specific Antibodies Target‐protein‐specific Antibodies Protein–GFP Fusions Fluorescence Resonance Energy Transfer (FRET) GPCR Activation using Bioluminescence Resonance Energy Transfer (BRET) Protein Fragment Complementation Assays (PCA) Phenotypic Assays Proliferation/Respiration/Toxicity Apoptosis Differentiation Monitoring Cell Metabolism Other Phenotypic Assays Acknowledgments

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

Cited by 40 publications

References 36 publications

K vitamins, PTP antagonism, and cell growth arrest

K vitamins, PTP antagonism, and cell growth arrest

Identification of Epidermal Growth Factor Receptor as a Target of Cdc25A Protein Phosphatase

Cellular Assays and Their Application in Drug Discovery

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