Figure 7. H2O2 activation of EGFR is temperature-dependent. Serum-starved A549 cells were left intact or incubated for 30 min. with 1 U/ml GO or 15 min. 100 ng/ml EGF at the indicated temperatures (T). EGFR was IPed from cell lysates and IBed for total and Tyr- phosphorylated EGFR, as indicated. doi:10.1371/journal.pone.0023240.g007 The results show that cholesterol uptake (CC) significantly reduced the EGFR activation/phosphorylation under ox-stress exposure (GO) (Fig. 8B). At the same time, EGFR activation by EGF was just slightly changed by the addition of the MbCD-cholesterol complex (Fig. 8B). Interestingly, the uptake of cholesterol (CC) also inhibited the activation of c-Src under ox-stress (Fig. 8C), supporting the idea that not only EGFR activation under ox-stress could be regulated by membrane structure, but that also c-Src activity could be affected by such changes in the membrane. Finally, we measured the total cholesterol levels after the various treatments in cell lipid extracts (Fig. 9A), and also, by fluorescence microscopy (using the sterol-binding fluorescent dye filipin) in whole cells (Fig. 9B). As shown in figure 9, we confirmed that cholesterol was depleted from PM by MbCD treatment and that MbCD-cholesterol complexes substantially increased the amount of cholesterol in the PM. However, total cholesterol levels did not appear to be affected by GO treatment (nor by EGF). H2O2 elevates ceramide cellular levels, leading to both c-Src and EGFR co-localization with ceramide Elevated ceramide levels have been shown to alter cell membrane structure and fluidity through displacement of cholesterol [32,38,39], and thus ceramide may act by physically changing the structure and properties of membrane lipid rafts. Since we have being studying the mechanisms of ceramide generation under exposure to ox-stress [28,29,30,40,41,42], we wondered whether ceramide levels were changed in our present studies and whether this alteration was coordinated within the same time frame of c-Src and EGFR activation under ox-stress exposure. Serum-starved A549 cells were treated with 1 U/ml GO, as before, and then subjected to IF analyses by staining for ceramide, p-Y1173 EGFR or p-Y416 c-Src (as described in ‘‘Material and Methods’’). As shown in figure 10, increasing time points of exposure to GO elevated not only ceramide levels, but also the activation of EGFR (p-Y1173) and c-Src (p-Src). However, while under the addition of EGF a rapid activation of EGFR was followed by its rapid internalization via clathrin coated pits and lysosomal degradation [24], the activation of EGFR under ox-stress was sustained and increased over time (Fig. 10). Subsequently, we aimed to elucidate the kinetics of co-localization between the generated ceramide and the ox-stress activated EGFR. Using confocal microscopy, we demonstrated (Fig. 11) that after 15 min. of exposure to ox-stress (GO), active EGFR (p-Y1173) and the elevated ceramide levels were localized to the plasma membrane, where the two signals EGFR Activation under Oxidative Stress Figure 8. Cholesterol levels modulate EGFR activation by H2O2. A. Serum-starved A549 cells were treated, or not, with 100 ng/ml EGF for 15 min. or 2% (w/v) MbCD for 1 h and cell lysates were IBed for total and Tyr-phosphorylated EGFR. B and C. Cells were treated with EGF as in A or with 1 U/ml GO for 30 min. in the absence or presence of 2 mM MbCD-cholesterol complex (CC), prepared as described in ‘‘Material and Methods’’. Cell lysates were separated by SDS-PAGE and IBed for total and Tyr-phosphorylated EGFR (B) or total and Y416 phosphorylated Src (C). doi:10.1371/journal.pone.0023240.g008 merged (white arrows in Fig. 11A). However, after 30 min. of GO exposure, the elevated ceramide and the activated EGFR were observed in a ‘‘peri-nuclear’’ cell region (Fig. 11A). In addition, at that time point, the co-localization between active EGFR and ceramide was mainly observed at the peri-nuclear region (arrows in Fig. 11A). Furthermore, at that time point the GO-activated c- Src (p-Src) also co-localized with ceramide mainly in the peri- nuclear region (arrows in Fig. 11B). Since we have shown before [26] that under ox-stress activated EGFR traffics via caveolae to a peri-nuclear region, we suggest there is a role for ceramide generation in facilitating the recruitment of H2O2-activated EGFR into caveolae trafficking to peri-nuclear ceramide-enriched membrane domains as discussed below. Discussion We have shown before that ox-stress exposure of lung epithelial cells induces aberrant EGFR phosphorylation, resulting in its lack of ubiquitination by c-Cbl and its impaired degradation. Subsequently the stabilized ox-stress activated EGFR remains plasma membrane-bound, while a portion of it is trafficked via caveolae to the peri-nucleus. In the present paper, we are starting to dissect the upstream molecular alteration in the structure/ conformation of EGFR itself following exposure to ox-stress. Data presented herein substantiate the findings that EGFR activation/phosphorylation under ox-stress is aberrant, thereby PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23240