Abstract
We previously showed that prolonged and strong ERK phosphorylation induced by Compound 5 (Cpd 5), a Cdc25A protein phosphatase inhibitor, was involved in its mechanism of cell growth inhibition. To study the relationship between ERK phosphorylation and cell growth inhibition, we used Cpd 5 as a tool to investigate ERK-regulated c-Myc expression in Hep3B hepatoma cells. We found that ERK phosphorylation caused by Cpd 5 induced c-Myc phosphorylation, but suppressed c-Myc expression at the mRNA and protein levels. Furthermore, Cpd 5 inhibited c-Myc transcriptional activity and DNA binding ability, and this inhibition was antagonized by ERK kinase (MEK) inhibitor U-0126, implying that the ERK pathway was involved in regulating c-Myc expression. Since the participation of c-Myc protein in transcription requires its dimerization with Max protein, we examined the Myc-Max association in Cpd 5-treated cells and found that Cpd 5 suppressed Myc-Max dimerization. Transfection of Hep3B cells with mutated ERK (T188A/Y190F), which has lost its dual-phosphorylation sites, attenuated the actions of Cpd 5 on Myc-Max association. To further demonstrate whether Myc phosphorylation by Cpd 5-induced ERK activation was able to directly regulate c-myc gene expression, a chromatin immunoprecipitation (ChIP) assay was used to examine the binding of phospho-Myc to the c-myc promoter region. We found that phospho-Myc induced by Cpd 5 had lost its ability to bind to the c-myc promoter, whereas MEK inhibitor U-0126 antagonized this inhibitory effect. These data suggest that an increase in c-Myc phosphorylation in response to prolonged ERK phosphorylation negatively auto-regulates c-Myc gene expression, leading to the suppression of its target gene expression and cell cycle block.
Copyright 2006 Wiley-Liss, Inc.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / genetics
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Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / physiology
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Blotting, Western
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Butadienes / pharmacology
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Carcinoma, Hepatocellular / pathology
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Cell Cycle / genetics
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Cell Cycle / physiology
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Cell Cycle Proteins / analysis
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / physiology
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Cell Line, Tumor
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Electrophoretic Mobility Shift Assay
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Enzyme Activation / drug effects
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Enzyme Activation / physiology
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Enzyme Inhibitors / pharmacology
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Extracellular Signal-Regulated MAP Kinases / drug effects
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Extracellular Signal-Regulated MAP Kinases / genetics
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Extracellular Signal-Regulated MAP Kinases / physiology*
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Gene Expression Regulation, Enzymologic
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Gene Expression Regulation, Neoplastic / drug effects
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Gene Expression Regulation, Neoplastic / physiology*
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Humans
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Immunoprecipitation
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Liver Neoplasms / pathology
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Nitriles / pharmacology
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Oligonucleotide Array Sequence Analysis
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Phosphorylation / drug effects
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Promoter Regions, Genetic / genetics
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Promoter Regions, Genetic / physiology
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Protein Binding / drug effects
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Protein Binding / physiology
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Proto-Oncogene Proteins c-myc / genetics*
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Proto-Oncogene Proteins c-myc / physiology*
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RNA, Messenger / analysis
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RNA, Messenger / genetics
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Signal Transduction / drug effects
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Signal Transduction / genetics
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Signal Transduction / physiology*
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Transcription, Genetic / drug effects
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Transcription, Genetic / physiology
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Transfection
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Vitamin K / analogs & derivatives
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Vitamin K / pharmacology
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cdc25 Phosphatases / analysis
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cdc25 Phosphatases / genetics
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cdc25 Phosphatases / physiology
Substances
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2-(2-hydroxyethylsulfanyl)-3-methyl-1,4-naphthoquinone
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Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
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Butadienes
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Cell Cycle Proteins
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Enzyme Inhibitors
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MAX protein, human
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Nitriles
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Proto-Oncogene Proteins c-myc
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RNA, Messenger
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U 0126
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Vitamin K
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Extracellular Signal-Regulated MAP Kinases
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CDC25C protein, human
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cdc25 Phosphatases