Phosphorylation regulates Myc expression via prolonged activation of the mitogen-activated protein kinase pathway.
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.
Mesh Terms:
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors, Blotting, Western, Butadienes, Carcinoma, Hepatocellular, Cell Cycle, Cell Cycle Proteins, Cell Line, Tumor, Electrophoretic Mobility Shift Assay, Enzyme Activation, Enzyme Inhibitors, Extracellular Signal-Regulated MAP Kinases, Gene Expression Regulation, Enzymologic, Gene Expression Regulation, Neoplastic, Humans, Immunoprecipitation, Liver Neoplasms, Nitriles, Oligonucleotide Array Sequence Analysis, Phosphorylation, Promoter Regions, Genetic, Protein Binding, Proto-Oncogene Proteins c-myc, RNA, Messenger, Signal Transduction, Transcription, Genetic, Transfection, Vitamin K, cdc25 Phosphatases
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors, Blotting, Western, Butadienes, Carcinoma, Hepatocellular, Cell Cycle, Cell Cycle Proteins, Cell Line, Tumor, Electrophoretic Mobility Shift Assay, Enzyme Activation, Enzyme Inhibitors, Extracellular Signal-Regulated MAP Kinases, Gene Expression Regulation, Enzymologic, Gene Expression Regulation, Neoplastic, Humans, Immunoprecipitation, Liver Neoplasms, Nitriles, Oligonucleotide Array Sequence Analysis, Phosphorylation, Promoter Regions, Genetic, Protein Binding, Proto-Oncogene Proteins c-myc, RNA, Messenger, Signal Transduction, Transcription, Genetic, Transfection, Vitamin K, cdc25 Phosphatases
J. Cell. Physiol.
Date: Jul. 01, 2006
PubMed ID: 16596619
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