EGF receptor–ligand interaction generates extracellular hydrogen peroxide that inhibits EGFR-associated protein tyrosine phosphatases

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Abstract

Hydrogen peroxide (H2O2) has been shown to be an important modulator of intracellular phosphatase activity involved in cell signaling pathways, including signaling by members of the receptor tyrosine kinase family of receptors such as the epidermal growth factor receptor (EGFR). Intracellular H2O2 can be generated by mitochondria-dependent pathways, whereas we recently showed that H2O2 could be generated extracellularly by receptor–ligand interaction. Here, we show that H2O2 produced by EGF–EGFR interaction can modulate the activity of intracellular protein tyrosine phosphatases (PTPs). Using purified proteins, we found that EGFR–ligand interaction generates H2O2 that is capable of inhibiting the activity of PTP1B in vitro. Furthermore, the addition of catalase rescued phosphatase inhibition consequent to EGF–EGFR interaction. Using cells that overexpress EGFR, we found that the addition of extracellular catalase prevented EGF-induced inhibition of EGFR-associated phosphatase activity. Our findings suggest that extracellular H2O2 generated by EGFR–ligand interaction permeates the plasma membrane and inhibits EGFR-associated tyrosine phosphatase activity, thereby modulating downstream signal transduction pathways.

Section snippets

Materials and methods

Proteins. Recombinant human soluble EGFR (sEGFR; Research Diagnostics) was approximately 80% pure. Recombinant human EGF (R&D Systems) was >97% pure. Recombinant human granulocyte–macrophage-colony stimulating factor (GM-CSF) was a gift from Amgen, Inc. Bovine liver catalase was obtained from Sigma. Active recombinant human full-length PTP1B was obtained from Upstate Biotechnology.

H2O2quantitation. H2O2 generated by receptor–ligand interaction was quantitated using a PeroxiLuminol Kit (World

Results and discussion

On the basis of our previous finding that EGFR–ligand interaction could generate extracellular hydrogen peroxide (H2O2) [23], we sought to examine the role of H2O2 in modulating the activity of phosphatases involved with EGFR signaling. First, we utilized purified EGF ligand and the sEGFR that possesses the extracellular binding domain of the receptor. We quantitated H2O2 production by EGF–sEGFR interaction in vitro using a luminometry-based assay. The PeroxiLuminol assay system determines H2O2

Acknowledgments

The authors dedicate this paper to Dr. David W. Golde, our mentor and friend. This work was supported by grants from the Lebensfeld and Schultz Foundations.

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