Abstract
Extracellular signal‐regulated kinase (ERK) plays a central role in regulating cell growth, differentiation, and apoptosis. We previously found that 2‐(2‐mercaptoethanol)‐3‐methyl‐1,4‐napthoquinone or Compound 5 (Cpd 5), is a Cdc25A protein phosphatase inhibitor and causes prolonged, strong ERK phosphorylation which is triggered by epidermal growth factor receptor (EGFR) activation. We now report that Cpd 5 can directly cause ERK phosphorylation by inhibiting Cdc25A activity independently of the EGFR pathway. We found that Cdc25A physically interacted with and de‐phosphorylated phospho‐ERK both in vitro and in cell culture. Inhibition of Cdc25A activity by Cpd 5 resulted in ERK hyper‐phosphorylation. Transfection of Hep3B human hepatoma cells with inactive Cdc25A mutant enhanced Cpd 5 action on ERK phosphorylation, whereas over‐expression of Cdc25A attenuated this Cpd 5 action. Furthermore, endogenous Cdc25A knock‐down by Cdc25A siRNA resulted in a constitutive‐like ERK phosphorylation and Cpd 5 treatment further enhanced it. In EGFR‐devoid NR6 fibroblasts and MEK (ERK kinase) mutated MCF7 cells, Cpd 5 treatment also resulted in ERK phosphorylation, providing support for the idea that Cpd 5 can directly act on ERK phosphorylation by inhibiting Cdc25A activity. These data suggest that phospho‐ERK is likely another Cdc25A substrate, and Cpd 5‐caused ERK phosphorylation is probably regulated by both EGFR‐dependent and EGFR‐independent pathways. © 2005 Wiley‐Liss, Inc.