摘要
A growing body of evidence suggests that reactive oxygen species are critical components of cell signaling pathways, in particular, regulating protein phosphorylation events. Herein, we show that oxidative stress in response to hydrogen peroxide treatment of human epithelial cells induces robust tyrosine phosphorylation on multiple proteins. Using an anti-phosphotyrosine purification and LC-MS/MS approach, we have identified numerous of these H2O2-induced tyrosine phosphorylated proteins. Importantly, we show that EGFR and Src are the primary upstream kinases mediating these events, through their redox activation. The finding that many of the identified proteins have functions in cell adhesion, cell-cell junctions and the actin cytoskeleton, prompted us to examine stress-induced changes in adhesion. Immunofluorescence analysis showed that H2O2 alters cell adhesion structures and the actin cytoskeleton causing loss of adhesion and apoptosis. Remarkably, these cellular changes could be attenuated by inhibition of EGFR and Src, identifying these kinases as targets to block oxidative damage. In summary, our data demonstrate that EGFR and Src together play a central role in oxidative stress-induced phosphorylation, which in turn results in loss of adhesion, morphological changes and cell damage in epithelial cells. These data also provide a general model for redox signalling in other cell systems.