We previously showed that 2-(2-mercaptoethanol)-3-methyl-1,4-napthoquinone or Compound 5 (Cpd 5), a Cdc25A dual specificity protein phosphatase inhibitor, can induce prolonged, strong ERK phosphorylation, which was related to 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 Cpd 5-induced ERK phosphorylation triggered c-Myc phosphorylation, but suppressed c-Myc expression at the mRNA and protein levels. Furthermore, Cpd 5 inhibited c-Myc transcriptional activity and Myc/Max 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 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 (T202A/Y204F), which has lost its dual-phosphorylation sites, abolished 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 expression, a chromatin immunoprecipitation 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 binding ability to c-myc promoter, whereas MEK inhibitor U-0126 antagonized this inhibitory effect. These data suggest that c-Myc phosphorylation in response to persistent ERK phosphorylation by Cpd 5 negatively auto-regulates c-Myc expression, leading to the suppression of its target gene expression and cell cycle block.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]