Figure 1. H2O2 activation of EGFR is ligand-independent. Serum-starved A549 cells were left intact or incubated with 40 nM monoclonal antibody 225 (mAb 225) for 1 hr on ice. Cells were then exposed for 15 min. at 37uC to 100 ng/ml EGF or 1 U/ml GO in the presence or absence of the mAb 225, as indicated. Immuno- precipitation (IP) of EGFR from cell lysates was performed using the mAb 528 and immuno-blotting (IB) for total (EGFR) and Tyr- phosphorylated EGFR (p-EGFR) was carried out as described in ‘‘Material and Methods’’. doi:10.1371/journal.pone.0023240.g001 ligand-independent dimers of EGFR were proven early on to be a step separable from ligand-induced downstream signaling [23]. Our previous studies demonstrated that CS-induced H2O2 generation or direct exposure to H2O2 cause phosphorylation of EGFR with a pattern of phosphorylation sites that differs from the one induced by EGF binding. More specifically, we reported that under H2O2-induced ox-stress EGFR is aberrantly phosphorylated, particularly at tyrosine (Tyr) Y845 and Y1045, which are hyper- phosphorylated and un-phosphorylated, respectively (in comparison to the EGF-stimulated receptor). This ultimately resulted in an active EGFR with impaired trafficking and degradation due to lack of ubiquitination and subsequent strong Src-dependent interaction with phosphorylated caveolin-1 and recruitment into caveolae and not clathrin coated pits. [18,24,25,26]. Additional data suggested a correlation between tumor progression and altered cell redox status, but the underlying mechanisms were far from being understood [27]. Here, we present a novel model of ox-stress-induced EGFR activation which is ligand-independent and does not involve canonical dimerization. It appears to involve a conformational change in the intracellular kinase domain that is also dependent on temperature and membrane cholesterol/ceramide ratio and thus might be affected by changes in membrane structure/fluidity. We previously reported that under H2O2- or cigarette smoke-induced ox-stress membrane ceramide levels are increased [28,29,30,31], which is known to alter membrane fluidity through cholesterol displacement [32]. Consistently, ceramide generation under ox- stress may displace cholesterol in membrane rafts and thus support changes in the EGFR conformation, whereas cholesterol uptake in the plasma membrane could inhibit such ox-stress-induced activation of EGFR. In addition, we show herein that upon exposure to H2O2 active EGFR and active c-Src co-localize with elevated ceramide. Moreover, under such H2O2-induced ox-stress c-Src is physically bound to EGFR; this is not found under EGF treatment. Results H2O2 activates EGFR in a ligand-independent manner A549 cells were serum-starved overnight and, where indicated (Fig. 1), incubated with 40 nM monoclonal antibody (mAb) 225 for 60 min. on ice to block the EGFR ligand binding site. Cells were then exposed to either 100 ng/ml EGF for 15 min. or 1 unit (U)/ml EGFR Activation under Oxidative Stress Figure 2. H2O2-induced EGFR phosphorylation is not inhibited by TKI AG1478 and is inhibited only at tyrosine 845 by Src family kinase inhibitor PP1. Serum-starved A549 cells were incubated (or not) with 1 mM AG1478 or 5 mM PP1 for 30 min. Then, the cells were treated for 30 min. with 100 ng/ml EGF or 1 U/ml GO, as indicated. EGFR was IPed from cell lysates, resolved by SDS-PAGE and IBed for total receptor, total tyrosine phosphorylation (p-EGFR) and specific Tyr-residue phosphorylation level (Y845, Y1068, Y1086, and Y1173). Protein aliquots of the cell lysates were also directly IBed for total and Y416 phosphorylated (active) c-Src (p-Src). doi:10.1371/journal.pone.0023240.g002 glucose oxidase (GO; to generate H2O2) for 30 min. in the absence or presence of the mAb. EGFR was immuno-precipitated (IPed) from cell lysates and immuno-blotted (IBed) for assessing the total and tyrosine (Tyr)-phosphorylated (p-) EGFR levels. Phosphorylation of the EGFR by EGF was blocked by the mAb whereas phosphory- lation by GO could not be inhibited (Fig. 1). This indicates that H2O2-induced EGFR phosphorylation is ligand-independent. H2O2 induces EGFR auto- and trans-phosphorylation Thus far, it appears that H2O2 induces phosphorylation of the EGFR via a mechanism that is different from that induced by its ligand, EGF. One possibility is that H2O2 trans-activates EGFR by a non-receptor tyrosine kinase such as c-Src, which has been shown to be activated by ox-stress and to phosphorylate EGFR at Y845 [18,24]. We, therefore, wondered whether EGFR auto-phosphor- ylation sites were also dependent on c-Src activation by H2O2. To answer this question we examined the effect of PP1, a specific inhibitor of the Src kinase family, on H2O2-induced phosphorylation of EGFR. Pre-treatment of A549 cells with 5 mM PP1 eliminated H2O2-induced trans-phosphorylation of EGFR at Y845 while auto-phosphorylation at Y1068, Y1086, and Y1173 remained unchanged (Fig. 2, last 3 columns vs first 3). These results indicate that H2O2-induced auto-phosphorylation is not dependent on Src-family kinases, which are only responsible for the trans-phosphorylation on Y845 (Fig. 2). H2O2-induced EGFR auto- and trans-phosphorylation are not inhibited by AG1478 in intact cells, but are inhibited in membrane fractions The tyrosine kinase inhibitor (TKI) AG1478 can suppress EGFR auto-phosphorylation under EGF stimulation by reversibly PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23240