The Src kinase controls aspects of cell biology and its activity is regulated by intramolecular structural changes induced by protein interactions and tyrosine phosphorylation. Recent studies indicate that Src is additionally regulated by redox-dependent mechanisms, involving oxidative modification(s) of cysteines within the Src protein, although the nature and molecular-level impact of Src cysteine oxidation are unknown. Using a combination of biochemical and cell-based studies, we establish the critical importance of two Src cysteine residues, Cys-185 and Cys-277, as targets for H2O2-mediated sulfenylation (Cys-SOH) in redox-dependent kinase activation in response to NADPH oxidase-dependent signaling. Molecular dynamics and metadynamics simulations reveal the structural impact of sulfenylation of these cysteines, indicating that Cys-277-SOH enables solvent exposure of Tyr-416 to promote its (auto)phosphorylation, and that Cys-185-SOH destabilizes pTyr-527 binding to the SH2 domain. These redox-dependent Src activation mechanisms offer opportunities for development of Src-selective inhibitors in treatment of diseases where Src is aberrantly activated.
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...
The reversible oxidation of protein cysteine residues (Cys-SH) is a key reaction in cellular redox signaling involving initial formation of sulfenic acids (Cys-SOH), which are commonly detected using selective dimedone-based probes. Here, we report that significant portions of dimedone-tagged proteins are susceptible to cleavage by DTT reflecting the presence of perthiosulfenic acid species (Cys-SSOH) due to similar oxidation of hydropersulfides (Cys-SSH), since Cys-S-dimedone adducts are stable toward DTT. Combined studies using molecular modeling, mass spectrometry, and cell-based experiments indicate that Cys-SSH are readily oxidized to Cys-SSOH, which forms stable adducts with dimedone-based probes. We additionally confirm the presence of Cys-SSH within protein tyrosine kinases such as EGFR, and their apparent oxidation to Cys-SSOH in response NADPH oxidase activation, suggesting that such Cys-SSH oxidation may represent a novel, as yet uncharacterized, event in redox-based signaling.
Protein kinases are essential mediators of cellular signal transduction and are often dysregulated in disease. Among these, protein tyrosine kinases (PTKs) have received specific interest due to their common roles in various diseases including cancer, and emerging observations indicating that PTK signalling pathways are susceptible to regulation by reactive oxygen species (ROS), which are also frequently implicated in disease pathology. While it is well recognized that ROS can impact on tyrosine kinase signalling by inhibiting tyrosine phosphatases, more recent studies highlight additional modes of redox-based regulation of tyrosine kinase signalling by direct redox modification of non-catalytic cysteines within tyrosine kinases or other protein components of this signalling pathway. In this review, we will present recent advancements with respect to redox-based mechanisms in regulating PTK signalling, with a specific focus on recent studies demonstrating direct redox regulation of Src-family kinases and epidermal growth factor receptor kinases. Importantly, redox-based modulation of tyrosine kinases may be relevant for many other kinases and has implications for current approaches to develop pharmacological inhibitors for these proteins.
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