EGFR Activation under Oxidative Stress Figure 10. Ox-stress induces sustained increase of ceramide levels and activation of EGFR and c-Src. A549 cells were seeded on cover- glasses, serum starved for 1 h and treated (or not) with 1 U/ml GO as indicated. After treatments, ceramide, EGFR phosphorylated on Y1173 and Y416 phosphorylated c-Src were localized in situ by IF as indicated in ‘‘Material and Methods’’; nuclei were stained by DAPI. doi:10.1371/journal.pone.0023240.g010 Figure 11. Under H2O2-induced ox-stress elevated ceramide co-localizes with active EGFR and c-Src. A549 cells were seeded on cover-glasses, serum starved for 1 h and treated (or not) with 1 U/ml GO as indicated. After treatments, ceramide (A and B), EGFR phosphorylated on Y1173 (A) and Y416 phosphorylated c-Src (B) were localized in situ by IF as indicated in ‘‘Material and Methods’’; nuclei were stained by DAPI. White arrows indicate regions where p-Y1173 EGFR and ceramide (A) or p-Y416 c-Src (p-Src) and ceramide (B) co- localized under ox-stress (GO). Z-stack sections of cells have been discriminated by confocal microscopy: the panels show the merge of all of the Z-stack sections. doi:10.1371/journal.pone.0023240.g011 Consistently, by employing a novel EGFR antibody (a4-2 mAb) that is susceptible to the conformational changes induced by EGF binding to the receptor [35] or by the somatic mutation L858R, we confirmed that H2O2 induces a unique active conformation of the receptor (Fig. 6), which is different from that of both EGF- induced EGFR and the L858 MT. Hetero-dimerization of EGFR with other members of the ErbB family could possibly be different under ox-stress exposure. However, no high molecular dimers are observed, what so ever, in figure 4. At the same time, the cross linker EDAC used in our study had been shown by others [50] to be able to crosslink the hetero-dimer EGFR-ErbB2. Therefore, the likelihood of ErbB2 functioning as the activating kinase that phosphorylates EGFR under exposure to ox-stress is being excluded. Furthermore, we repeated all our studies in NIH-3T3 cells stably transfected with EGFR (not shown). It was previously reported by others that these cells ‘‘do not express’’ or ‘‘do not express significant amount of’’ ErbB family members [51,52]. However, following ox-stress exposure, the same aberrant phenotypes of EGFR were obtained, indicating that EGFR aberrant phosphorylation, lack of dimer- ization, novel active conformation and the subsequent resistance to TKI AG1478 cannot be attributed to any irregular association of EGFR with any other member of the ErbB family under ox-stress exposure. Since EGFR phosphorylation by H2O2 is shown to be temperature dependent, suggesting a requirement for membrane involvement, it was not surprising to find that the TKI AG1478 was ineffective in quenching EGFR phosphorylation by H2O2 in living cells, but capable of inhibiting it in a crude membrane fraction, where the membrane structure was destroyed. This supports our novel suggestion that the fluidity/structure of the membranes may be involved in either inducing or stabilizing the new H2O2-induced active conformation of EGFR. Therefore, we further investigated the participation of membrane constituents in EGFR activation by H2O2. It was previously reported that disruption of cholesterol- enriched lipid rafts causes ligand-independent activation of EGFR [20]. One of the widespread explanations was that a population of PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e23240