Major role of epidermal growth factor receptor and Src kinases in promoting oxidative stress-dependent loss of adhesion and apoptosis in epithelial cells.
A growing body of evidence suggests that reactive oxygen species are critical components of cell signaling pathways, in particular regulating protein phosphorylation events. Here, 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 liquid chromatography-tandem mass spectrometry approach, we have identified many of these H(2)O(2)-induced tyrosine-phosphorylated proteins. Importantly, we show that epidermal growth factor receptor (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 H(2)O(2) 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 signaling in other cell systems.
Chan
Hong-Lin
HL
Elizabeth Garrett Anderson Institute for Women's Health, University College London, WC1E 6BT London, United Kingdom.
Chou
Hsiu-Chuan
HC
Duran
MaCarmen
M
Gruenewald
Jana
J
Waterfield
Michael D
MD
Ridley
Anne
A
Timms
John F
JF
D017209:Apoptosis / Q000187:drug effects*; D002448:Cell Adhesion / Q000187:drug effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002853:Chromatography, Liquid; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D015151:Immunoblotting; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D018384:Oxidative Stress / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D053719:Tandem Mass Spectrometry; D019061:src-Family Kinases / Q000378:metabolism*
D006861:Hydrogen Peroxide; D066246:ErbB Receptors; D019061:src-Family Kinases