Molecular Basis for Redox Regulation of the Src Kinase

The non‐receptor tyrosine kinase Src facilitates signal transduction processes that govern a broad range of cellular functions. While it is well established that Src activity is controlled in tyrosine phosphorylation‐dependent mechanisms, recent evidence supports the notion that Src is also redox regulated involving oxidation of Src cysteine residues. However, current reports are inconsistent with respect to which of the nine Src cysteine residues are susceptible to oxidation and the molecular nature by which they contribute to Src kinase activation or inactivation. We have previously observed in cell‐based studies that the NADPH oxidase DUOX1 generates H 2 O 2 to promote Src activation as a critical mechanism in lung epithelial wound responses, which is associated with DUOX1‐dependent oxidation of Src cysteines to sulfenic acids (‐SOH). This present study aims to characterize the molecular details of Src redox regulation from biochemical and cellular experiments as well as structural insights from molecular dynamic simulations. First, the identity of oxidized Src cysteine residues was assessed from mass spectrometry (MS) experiments with purified recombinant Src oxidized by H 2 O 2 in the presence of dimedone to trap transiently formed cysteine sulfenic acids (‐SOH). These MS experiments confirmed that H 2 O 2 promotes the sulfenylation of C185, C277, and C498. To assess the importance of the oxidation of these cysteines, we found that Src tyrosine kinase activity is indeed enhanced by H 2 O 2 in WT but not in the C185A or C277A variants confirming a role for these cysteine residues in regulating Src activity upon oxidation. To confirm the importance of C185 in a cellular context, we expressed FLAG‐tagged WT and C185A Src in human mucoepidermoid H292 cells and confirm that ATP‐induced DUOX1‐mediated activation of Src by phosphorylation of Y416 is significantly suppressed in C185A Src compared to WT Src. These experiments are particularly insightful since reports have previously implicated C277 oxidation in Src redox regulation, but the potential role of oxidation of C185 has not been addressed previously. Since Src activity is controlled by changes within the Src protein structure, we assessed the impact of oxidized cysteine sulfenic acids (‐SOH) compared to reduced thiols (‐SH) at C185 and C277 on the structure of Src with molecular dynamics (MD) simulations. Starting from the X‐ray crystal structure of inactive Src, MD simulations reveal that oxidation of C185 and C277 induce structural perturbations that activate Src in distinctive mechanisms. Specifically, oxidation of C185 impacts the binding of pY527 to the SH2 domain leading to enhanced Src activation and target binding, while oxidation of C277 promotes unfolding of the activation loop allowing for enhancing substrate and ATP accessibility to the catalytic active site. These results definitively characterize the molecular basis of the redox regulation of Src and present a more complete understanding of oxidative mechanisms of Src‐mediated signaling relevant to physiology and in numerous pathological conditions. Support or Funding Information Supported by NIH Grants (R01HL085646, F32HL129706, P20GM103449), and ANTON1 Supercomputer Allocation (PSCA15029P).