Hydrogen peroxide reversibly inhibits epidermal growth factor (EGF) receptor internalization and coincident ubiquitination of the EGF receptor and Eps15.
Recently, we demonstrated that hydrogen peroxide (H2O2) inhibits the internalization of the epidermal growth factor (EGF) receptor and the EGF-induced mono-ubiquitination of EGF receptor pathway substrate clone #15 (Eps15) in fibroblasts. In addition, it was suggested that EGF receptor internalization might be inhibited by H2O2 by inhibition of ubiquitination of proteins involved in endocytosis. Here, we show that H2O2 also inhibits the poly-ubiquitination of the EGF receptor in fibroblasts. Furthermore, recovery of the cells resulted in re-establishment of ubiquitination of both the EGF receptor and Eps15 and coincided with restoration of internalization of those receptors that had bound EGF in the presence of H2O2. In addition, EGF receptor internalization was inhibited by the sulphydryl reagent N-ethylmaleimide (NEM), indicating that intact SH groups might be required for receptor-mediated endocytosis. Furthermore, H2O2 rapidly induced an increase in the cellular ratio of GSSG:GSH (oxidized glutathione:reduced glutathione) and removal of H2O2 resulted in a fast restoration of the ratio of GSSG:GSH. Therefore, these results suggest a relation between the inhibition of internalization ubiquitination and an increase in GSSG:GSH ratio, which strengthens the hypothesis that H2O2 inhibits EGF receptor internalization by an inhibition of ubiquitination of proteins involved in EGF receptor-mediated endocytosis.
De Wit
R
R
Institute of Biomembranes, Department of Molecular Cell Biology, Utrecht University, Utrecht, The Netherlands. R.deWit@bio.uu.nl
Makkinje
M
M
Boonstra
J
J
Verkleij
A J
AJ
Post
J A
JA
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D005347:Fibroblasts / Q000502:physiology; D005978:Glutathione / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D008954:Models, Biological; D018384:Oxidative Stress / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D014452:Ubiquitins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; C486206:EPS15 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014452:Ubiquitins; D004815:Epidermal Growth Factor; D006861:Hydrogen Peroxide; D066246:ErbB Receptors; D005978:Glutathione; D019803:Glutathione Disulfide