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Transient and sustained ERK phosphorylation and nuclear translocation in growth control
Takahito Adachi1, Siddhartha Kar, Meifang Wang
1Department of Surgery, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pennsylvania 15213, USA.
Abstract:
Growth stimulation and inhibition are both associated with tyrosine phosphorylation. We examined the effects of epidermal growth factor (EGF), a growth stimulant, and compound 5 (Cpd 5), a protein-tyrosine phosphatase (PTPase) inhibitor, which inhibits the growth of the same Hep3B hepatoma cells. We found that both EGF and Cpd 5 induced tyrosine phosphorylation of EGF receptor (EGFR) and ERK. However, the phosphorylation caused by EGF was transient and that caused by Cpd 5 was prolonged. Furthermore, Cpd 5 action caused a strong nuclear phospho-ERK signal and induced phospho-Elk-1, a nuclear target of ERK activation, in contrast to the weak effects of EGF. An ERK kinase assay demonstrated that ERK activated by Cpd 5 could phosphorylate its physiological substrate, Elk-1. The MEK inhibitors PD098056 and U0126 abrogated both the induction by Cpd 5 of phospho-ERK, its nuclear translocation and phospho-Elk-1 and also antagonized its growth inhibitory effects. Furthermore, phospho-ERK phosphatase and phospho-Elk-1 activities were lost from nuclear extracts from Cpd 5 treated, but not EGF treated cells. In conclusion, the data show that Cpd 5 causes growth inhibition as a consequence of prolonged ERK and Elk-1 phosphorylation, likely a result of inhibition of multiple PTPases, including those acting on phospho-EGFR, on phospho-ERK, and on phospho-Elk-1, in contrast to the kinase driven transient activation resulting from EGF.
Insights
Compound 5 (Cpd 5), a protein-tyrosine phosphatase inhibitor, prolongs ERK and Elk-1 phosphorylation, inhibiting hepatoma cell growth. This contrasts with epidermal growth factor (EGF), which causes transient activation.
Area of Science:
- Cellular signaling and cancer biology.
- Investigating the role of tyrosine phosphorylation in cell growth regulation.
Background:
- Tyrosine phosphorylation is linked to both growth stimulation and inhibition.
- Epidermal growth factor (EGF) stimulates growth, while protein-tyrosine phosphatase inhibitors (PTPase inhibitors) like Compound 5 (Cpd 5) inhibit it in Hep3B hepatoma cells.
Purpose of the Study:
- To compare the effects of EGF and Cpd 5 on tyrosine phosphorylation pathways.
- To elucidate the mechanisms by which Cpd 5 inhibits hepatoma cell growth.
Main Methods:
- Treatment of Hep3B cells with EGF and Cpd 5.
- Analysis of tyrosine phosphorylation of EGF receptor (EGFR) and ERK.
- Assessment of nuclear phospho-ERK and phospho-Elk-1.
- ERK kinase assays and MEK inhibitor studies (PD098056, U0126).
- Enzyme activity assays on nuclear extracts.
Main Results:
- Both EGF and Cpd 5 induced tyrosine phosphorylation of EGFR and ERK, but Cpd 5's effect was prolonged.
- Cpd 5 caused strong nuclear phospho-ERK and induced phospho-Elk-1, unlike EGF.
- Cpd 5-activated ERK phosphorylated Elk-1.
- MEK inhibitors blocked Cpd 5's effects on phospho-ERK, Elk-1, and growth inhibition.
- Nuclear phospho-ERK and Elk-1 phosphatase activities were reduced by Cpd 5.
Conclusions:
- Cpd 5 inhibits hepatoma cell growth through prolonged ERK and Elk-1 phosphorylation.
- This prolonged phosphorylation is likely due to the inhibition of multiple PTPases.
- EGF induces transient activation, whereas Cpd 5 causes sustained inhibition via PTPase blockade.