58 - DUOX1-Dependent Disulfide Bonding of Src Kinase in the Airway Epithelium

Abstract

The non-receptor tyrosine kinase Src is ubiquitously expressed and is involved in regulation of cell proliferation, differentiation, and motility. Strongly linked with cancer progression, Src also contributes to non-malignant diseases such as allergic asthma. Regulation of Src activity is incompletely understood, but critically involves tyrosine (de)phosphorylation events that mediate Src “unclamping” and interactions with regulatory or substrate proteins as well as kinase activation. In addition, more recent studies indicate that Src-dependent signaling is also regulated by redox-dependent mechanisms involving reversible oxidation of one or more conserved cysteine residues. Indeed, our previous studies indicate that epithelial wound responses or innate response to airborne allergens are mediated by oxidant-mediated activation of Src by the NADPH oxidase DUOX1. However, the precise mechanisms by which cysteine oxidation regulates Src activity are still not clear, and diverse cysteine residues have been implicated. In addition to its potential role in regulating Src tyrosine kinase activity, evidence is emerging that cysteine oxidation within Src can promote intermolecular disulfide bonding, e.g. between Src monomers, with its negative regulator C-terminal Src Kinase (CSK), or its substrate cortactin. The aim of the present study was to determine whether DUOX1-dependent activation of Src oxidation in airway epithelial cells results in intermolecular disulfide cross-linking with other proteins, and to characterize Src interacting partners. Using diagonal electrophoresis to visualize intermolecular disulfides, we observed that stimulation of H292 epithelial cells with H2O2 or with ATP, which activates DUOX1, induces disulfide-based cross-linking of Src with a number of proteins. This was confirmed after immunoprecipitation of Src, and interacting proteins are being identified using mass spectrometry. We also used recombinant Src and various Cys-to-Ala variants to determine oxidant-mediated cross-linking between Src monomers, with indicated intermolecular crosslinking between Src monomers, which was diminished with Cys mutants. We seek to extend these findings in studies with epithelial cells expressing different Src Cys-to-Ala variants to gain further insight into the significance of specific cysteine oxidation events in Src-dependent activation of various signaling pathways. Supported by NIH R01 ES021476, NIH R01 HL085646, and NIH F32 HL129706.