The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling

Abstract

The epidermal growth factor receptor (EGFR) plays a critical role in regulating airway epithelial homeostasis and responses to injury. Activation of EGFR is regulated by redox-dependent processes involving reversible cysteine oxidation by reactive oxygen species (ROS) and involves both ligand-dependent and -independent mechanisms, but the precise source(s) of ROS and the molecular mechanisms that control tyrosine kinase activity are incompletely understood. Here, we demonstrate that stimulation of EGFR activation by ATP in airway epithelial cells is closely associated with dynamic reversible oxidation of cysteine residues via sequential sulfenylation and S-glutathionylation within EGFR and the non-receptor-tyrosine kinase Src. Moreover, the intrinsic kinase activity of recombinant Src or EGFR was in both cases enhanced by H₂O₂ but not by GSSG, indicating that the intermediate sulfenylation is the activating modification. H₂O₂-induced increase in EGFR tyrosine kinase activity was not observed with the C797S variant, confirming Cys-797 as the redox-sensitive cysteine residue that regulates kinase activity. Redox-dependent regulation of EGFR activation in airway epithelial cells was found to strongly depend on activation of either the NADPH oxidase DUOX1 or the homolog NOX2, depending on the activation mechanism. Whereas DUOX1 and Src play a primary role in EGFR transactivation by wound-derived signals such as ATP, direct ligand-dependent EGFR activation primarily involves NOX2 with a secondary role for DUOX1 and Src. Collectively, our findings establish that redox-dependent EGFR kinase activation involves a dynamic and reversible cysteine oxidation mechanism and that this activation mechanism variably involves DUOX1 and NOX2.

Keywords: DUOX1; NOX2; Src; cysteine; epidermal growth factor receptor (EGFR); epithelium; hydrogen peroxide; redox signaling; sulfenic acid.

FIGURE 1.

ATP-dependent activation of EGFR and Src is temporally coupled to cysteine sulfenylation and S-glutathionylation. H292 cells were stimulated with 100 μm ATP, and time-dependent changes in of EGFR phosphorylation at Tyr(P)-845 and Tyr(P)-1068 as well as EGFR sulfenylation (-SOH) and S-glutathionylation (-SSG) were analyzed by Western blot as detailed under “Experimental Procedures” (A and D), and similar phosphorylation (Tyr(P)-416), sulfenylation, and S-glutathionylation was evaluated in Src (B and E) as well as sulfenylation and S-glutathionylation PTP1B (C and F). D–F, densitometry analysis of two-three independent experiments. Data represent the mean ± S.D. (maximal band density ratios scaled to 1.0) showing the time-dependent association of phosphorylation and cysteine oxidation of Src and EGFR.

FIGURE 2.

DUOX1 activation induces sequential sulfenylation and S-glutathionylation of EGFR, Src, and PTP1B. A, scheme illustration of experimental approach to assess sequential cysteine sulfenylation and S-glutathionylation. B and C, MTE cells from WT and Duox1^−/− mice were preloaded with BioGEE and stimulated with ATP (100 μm; 10 min) in the absence or presence of dimedone (dim). B, Western blotting analysis of S-glutathionylation of EGFR, Src and PTP1B. C, Western blot of immunoprecipitated EGFR for streptavidin (reflecting incorporation of biotin-GSH), α-dimedone, or α-EGFR. Western blots are representative of two-three independent experiments.

FIGURE 3.

H₂O₂ enhances EGFR and Src tyrosine kinase activity via cysteine sulfenylation. A, effect of H₂O₂ or GSSG on intrinsic kinase activity of recombinant EGFR. B, Western blotting analysis of sulfenylation and S-glutathionylation of recombinant EGFR by H₂O₂ or GSSG (100 μm) using dimedone (DCP-Bio1) and α-GSH antibody, respectively. C, effects of GSH on H₂O₂-dependent increase in recombinant EGFR-tyrosine kinase activity. Note that listed GSH concentrations do not include GSH (333 μm) that was originally included in the EGFR storage buffer. D, comparison of H₂O₂-enhanced tyrosine kinase activity WT recombinant EGFR and C797S variant. E, effect of H₂O₂ and GSSG on intrinsic tyrosine kinase activity of recombinant Src. Data represent the mean ± S.D. of at least n = 4 replicates; Student's t test; **, p < 0.01; ****, p < 0.001. Western blots are representative of two-three independent experiments.

FIGURE 4.

Distinct roles for DUOX1 and NOX2 on ATP-dependent EGFR transactivation and direct ligand-dependent EGFR activation. A, H292 cells were transfected with siRNA against DUOX1, Src, or NOX2 and stimulated with ATP (100 μm) or EGF (100 ng/ml), and tyrosine phosphorylation and cysteine sulfenylation of EGFR and Src was determined by Western blot. B, Western blotting analysis of ATP- or EGF-stimulated MTE cells from WT and Duox1^−/− mice with or without siRNA silencing of NOX2 for Src and EGFR phosphorylation and sulfenylation. C–E, analysis of extracellular H₂O₂ production by H292 cells after siRNA-mediated silencing of DUOX1 and NOX2 (C), siRNA silencing (D), or pharmacological inhibition (E) of Src with PP2 (1 μm). Western blots are representative of two independent experiments. Data represent the mean ± S.D. from four-six replicates of three independent experiments. Two-way ANOVA; n = 4–6; *, p < 0.05; **, p < 0.01; ***, p < 0.005, ****, p < 0.001. NS, not significant.

FIGURE 5.

DUOX1 and NOX2 have stimulus-dependent impact on EGFR downstream signaling. A, representative Western blotting analysis of 100 μm ATP- and 100 ng/ml EGF-stimulated H292 cells of phosphorylation of STAT3 and ERK1/2 as a function of siRNA-mediated silencing of DUOX1 and NOX2. Quantification represents the mean ± S.D. of phosphorylation changes of STAT3 (B) and ERK1/2 (C) normalized ratio of phospho/total of NS siRNA EGF treatment of 1.0. **, p < 0.01; ***, p < 0.005, ****, p < 0.001. NS, not significant.

FIGURE 6.

Direct ligand-dependent activation of EGFR causes the activation of NOX2 that depends on tyrosine kinase activity. A, Western blotting analysis of EGFR and Src phosphorylation and sulfenylation as a function of EGFR-tyrosine kinase inhibitors AG-1478, erlotinib, and Src inhibitor PP2 (1 μm) in MTE cells. B, effects of hydrogen peroxide generation from H292 cells upon inhibition of EGFR with AG-1478 (1 μm). Data represent the mean ± S.D. from four-six replicates of two-three independent experiments. Western blots are representative of two-three independent experiments. Two-way ANOVA: n = 4–6; *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001.

FIGURE 7.

Cysteine 797 resides within the kinase domain of EGFR near the site of ATP binding. A, crystal structure of the EGFR kinase domain (PDB code 2ITX) with bound AMP-PNP (adenosine 5'-(β,γ-imino)triphosphate) highlighting cysteine 797. B, active site view showing proximity of Cys-797 to AMP-PNP.

FIGURE 8.

Molecular mechanisms of ATP-dependent transactivation and EGF-dependent direct activation of EGFR. A, ATP-mediated stimulation of P2Y₂R activates DUOX1-dependent H₂O₂ production leading to cysteine oxidation and activation of Src, which promotes shedding EGF of ligands from the cell surface in an ADAM17-dependent fashion, and the activation of EGFR in addition to cysteine oxidation resulting in phosphorylation of ERK1/2 and STAT3. B, EGF-mediated direct activation of EGFR activates NOX2-dependent ROS generation through Src activation that leads to cysteine oxidation and activation of Src and EGFR and phosphorylation of STAT3.