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 H 2 O 2 but not by GSSG, indicating that the intermediate sulfenylation is the activating modification. H 2 O 2 -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 redoxdependent EGFR kinase activation involves a dynamic and reversible cysteine oxidation mechanism and that this activation mechanism variably involves DUOX1 and NOX2.The epidermal growth factor receptor (EGFR 2 ; HER1; ErbB1) is the principal member of the human ErbB receptortyrosine kinase family that facilitates diverse signal transduction pathways to regulate imperative cellular functions including growth, development, differentiation, and migration (1). A transmembrane protein expressed on the cell membrane, EGFR consists of an extracellular ligand binding domain, a single transmembrane-spanning sequence, and an intracellular kinase domain containing a C-terminal peptide tail with several tyrosine residues targeted for autophosphorylation or phosphorylation by related tyrosine kinases (2). Activation of EGFR is enabled though binding of one of seven ligands to its extracellular domain, thereby promoting receptor homo-or heterodimerization and autophosphorylation of several C-terminal tail tyrosines (e.g. Tyr-1068) and activation of downstream signals. In addition, it has become apparent that EGFR can also be activated in response to stimulation of a large class of G-protein coupled receptors (GPCRs), which involves activation of membrane-associated EGFR ligands by matrix metalloproteases or ADAM (a disintegrin and metalloprotease)-family sheddases, a process known as "triple-membrane-passing-signaling," as well as ligand-independent mechanisms me...

DUOX1 mediates persistent epithelial EGFR activation, mucous cell metaplasia, and airway remodeling during allergic asthma

“…Although the present studies focused primarily on sulfenylation as a mechanism of oxidative EGFR activation, based on recent findings (25, 56), we cannot rule out the potential contribution of protein glutathionylation within EGFR or in other bystander proteins in mediating DUOX1-related allergic inflammation (e.g., ref. 57,58). Likewise, it is plausible that H 2 O 2 gives rise to production of other (secondary) reactive intermediates that would expand the range of potential oxidative posttranslational modifications that affect protein function.…”

The role of sulfenic acids in cellular redox signaling: Reconciling chemical kinetics and molecular detection strategies

“…[39–41]), and the activation of NOX enzymes within such signaling scaffolds generates ROS within a local environment that is highly dissimilar from homogeneous aqueous solutions. [42, 43] Moreover, recruitment of redox-sensitive protein Cys-SH targets towards such NOX-containing signaling complexes during activation, as demonstrated in several experimental studies [25, 32, 44, 45], would enhance the competitiveness of such Cys-SH targets compared to bulk concentrations of e.g. GSH or other reactive cell targets for locally generated H 2 O 2 , even if it needs to first pass a membrane through an aquaporin channel.…”

“…oxidized PTP1B. [69, 70] Such sulfenyl amide formation not only minimizes susceptibility to more irreversible oxidation (to Cys-SO 2 H and Cys-SO 3 H) but may also induce or eliminate structural alterations that may be relevant in a redox-dependent response, such as redox-dependent regulation of tyrosine kinase activity [25]. The molecular factors that control formation of sulfenyl amides are still unclear, but recent computational studies suggest that a constrained cysteine conformation within a β-loop-helix fold motif favors sulfenyl amide formation from initially generated Cys-SOH.…”

“…Indeed, NOX-dependent S -glutathionylation of target proteins is commonly preventable in the presence of dimedone (e.g. [25, 78, 79]), and accompanied by formation of detectable dimedone adducts (e.g. [80]).…”

“…Since intermediate formation of Cys-SOH is being recognized as a functionally critically intermediate in redox-dependent regulation of e.g. tyrosine kinases [24, 25], it is important to reconcile this issue. In the following paragraphs, we will briefly discuss some of the concerns regarding the selectivity of certain derivatization strategies [26, 27], and point out important considerations with respect to extrapolating experimental findings from biochemical kinetic studies with isolated proteins conducted in the test tube to intact cellular contexts.…”