Phosphorylation regulates Myc expression via prolonged activation of the mitogen-activated protein kinase pathway

Ziqiu Wang, Lisheng Ge, Meifang Wang, Brian I. Carr

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)133-140
Number of pages8
JournalJournal of Cellular Physiology
Volume208
Issue number1
DOIs
Publication statusPublished - Jul 2006

Fingerprint

Phosphorylation
Mitogen-Activated Protein Kinases
Chemical activation
Gene expression
myc Genes
Dimerization
Cell growth
Gene Expression
cdc25 Phosphatases
Cells
Association reactions
Proto-Oncogene Proteins c-myc
MAP Kinase Kinase Kinases
MAP Kinase Signaling System
Chromatin Immunoprecipitation
Phosphoprotein Phosphatases
Mitogen-Activated Protein Kinase Kinases
Transcription
Growth
Genetic Promoter Regions

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology

Cite this

Phosphorylation regulates Myc expression via prolonged activation of the mitogen-activated protein kinase pathway. / Wang, Ziqiu; Ge, Lisheng; Wang, Meifang; Carr, Brian I.

In: Journal of Cellular Physiology, Vol. 208, No. 1, 07.2006, p. 133-140.

Research output: Contribution to journalArticle

Wang, Ziqiu ; Ge, Lisheng ; Wang, Meifang ; Carr, Brian I. / Phosphorylation regulates Myc expression via prolonged activation of the mitogen-activated protein kinase pathway. In: Journal of Cellular Physiology. 2006 ; Vol. 208, No. 1. pp. 133-140.
@article{58afdaa22a4842c4baedfc5c3f3428de,
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.",
author = "Ziqiu Wang and Lisheng Ge and Meifang Wang and Carr, {Brian I.}",
year = "2006",
month = "7",
doi = "10.1002/jcp.20649",
language = "English",
volume = "208",
pages = "133--140",
journal = "Journal of cellular and comparative physiology",
issn = "0021-9541",
publisher = "Wiley-Liss Inc.",
number = "1",

}

TY - JOUR

T1 - Phosphorylation regulates Myc expression via prolonged activation of the mitogen-activated protein kinase pathway

AU - Wang, Ziqiu

AU - Ge, Lisheng

AU - Wang, Meifang

AU - Carr, Brian I.

PY - 2006/7

Y1 - 2006/7

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=33744917003&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33744917003&partnerID=8YFLogxK

U2 - 10.1002/jcp.20649

DO - 10.1002/jcp.20649

M3 - Article

C2 - 16596619

AN - SCOPUS:33744917003

VL - 208

SP - 133

EP - 140

JO - Journal of cellular and comparative physiology

JF - Journal of cellular and comparative physiology

SN - 0021-9541

IS - 1

ER -