title: Phosphorylation regulates Myc expression via prolonged activation of the mitogen-activated protein kinase pathway.
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.
authors.affiliation: Thomas E. Starzl Transplant Institute, Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Ziqiu
authors.initials: Z
authors.lastname: Wang
authors.forename: Lisheng
authors.initials: L
authors.lastname: Ge
authors.forename: Meifang
authors.initials: M
authors.lastname: Wang
authors.forename: Brian I
authors.initials: BI
authors.lastname: Carr
mesh_terms: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000235:genetics / Q000502:physiology; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D018797:Cell Cycle Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D008113:Liver Neoplasms / Q000473:pathology; D009570:Nitriles / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics* / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology; D014162:Transfection; D014812:Vitamin K / Q000031:analogs & derivatives / Q000494:pharmacology; D020687:cdc25 Phosphatases / Q000032:analysis / Q000235:genetics / Q000502:physiology
chemical_list: C446520:2-(2-hydroxyethylsulfanyl)-3-methyl-1,4-naphthoquinone; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002070:Butadienes; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; C499330:MAX protein, human; D009570:Nitriles; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; C113580:U 0126; D014812:Vitamin K; D048049:Extracellular Signal-Regulated MAP Kinases; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases