blocking the ATP binding site of the EGFR kinase domain. Thus, we decided to test the efficacy of this TKI on H2O2-induced phosphorylation of EGFR. Serum-starved A549 cells were incubated (or not) with 1 mM AG1478 for 30 min. and then treated (or not) with 100 ng/ml EGF or 1 U/ml GO for additional 30 min. Cells were lysed, EGFR was IPed and IBed for total receptor, total Tyr- phosphorylation and site specific Tyr-phosphorylation, as indicat- ed in figure 2. EGF-stimulated cells showed a marked increase in phosphorylation compared to un-stimulated cells at Y1068, Y1086 and Y1173, known EGFR auto-phosphorylation sites, while trans- phosphorylation of Y845 was comparable to controls (Fig. 2). In contrast, treatments with 1 U/ml GO for 30 min. resulted in marked phosphorylation of Y845, Y1173, Y1068 and Y1086 compared to un-treated cells. Identical results were obtained when a concentration of 10 mM AG1478 was used (not shown). Surprisingly, while pre-incubation with AG1478 was able to quench EGFR auto-phosphorylation upon EGF stimulation, it was not effective in the GO treated cells (Fig. 2, fifth vs sixth column). Since we previously showed that the kinase activity of EGFR is necessary for auto-phosphorylation of the receptor under ox-stress [26], this evidence suggested a conformational change in the kinase domain of EGFR under ox-stress, which prevents the inhibition of AG1478. We further investigated the ability of the TKI AG1478 to suppress EGFR phosphorylation under ox-stress using crude membrane fractions of A549 cells, prepared as described in ‘‘Material and Methods’’. Membrane fractions of equal protein content (10 mg each) were incubated (or not) with 1 mM AG1478 for 30 min. and then treated (or not) for additional 30 min. at 37uC with 100 ng/ml EGF or 300 mM H2O2. Samples were then separated by SDS-PAGE and IBed for total and Tyr-phosphor- ylated receptor (Fig. 3). AG1478 was able to completely inhibit both EGF- and H2O2-dependent EGFR phosphorylation (Fig. 3). In addition, AG1478 could inhibit both the EGF- and the H2O2- induced EGFR phosphorylation when crude broken cells were used instead of membrane preparations for the above in vitro phosphorylation assay (not shown). The ability of AG1478 to inhibit H2O2-induced EGFR kinase activity in crude membrane fractions or in broken cells, but not in intact cells (Fig. 3 vs Fig. 2) further suggests that activation of EGFR by H2O2 involves a change in EGFR conformation that is different from the sequence of events following ligand binding and also implicates that the membrane integrity could be mediating or involved in stabilizing such a newly acquired conformation. EGFR phosphorylation by ox-stress is not accompanied by canonical receptor dimerization To examine whether ox-stress can induce EGFR dimerization, serum-starved A549 cells were left untreated or were exposed to 100 ng/ml EGF or 1 U/ml GO for 15 min. Then, the cross- linking agent, EDAC (1 mM), was added (or not) for an additional 15 min. EGFR was IPed from protein extracts, separated by SDS- PAGE and IBed for total and Tyr-phosphorylated EGFR (Fig. 4A). EGF treatment resulted in the formation of phosphorylated EGFR dimers, as seen in figure 4A at about 340 kDa. In contrast, ox- stress did not induce formation of EGFR dimers, both in the absence and presence of the cross-linking EDAC (Fig. 4A). In addition, NIH-3T3 cells over-expressing wild type EGFR were treated as above and total protein lysates were IBed for total and Tyr-phosphorylated EGFR as indicated in figure 4B. Again, EGF stimulation generated an EGFR dimer at ,340 kDa, but ox-stress exposure (GO) did not (Fig. 4B), further supporting the idea that EGFR Activation under Oxidative Stress Figure 3. H2O2-induced EGFR phosphorylation is inhibited by TKI AG1478 in crude membrane fractions. A549 crude membrane fractions were prepared as indicated in ‘‘Material and Methods’’. Membrane fractions were incubated with 1 mM AG1478 for 30 min. at 4uC and then incubated for 30 min. with 100 ng/ml EGF or 300 mM H2O2 at 37uC. A. Membrane proteins were separated by SDS-PAGE and IBed for total and Tyr-phosphorylated EGFR, as indicated. B. The histogram represents the levels of Tyr-phosphorylation of EGFR under different conditions, reported as fold-increase/decrease of the non treated (NT) sample; St-Deviations are indicated. doi:10.1371/journal.pone.0023240.g003 the ox-stress-activated EGFR acquires a conformation that differs from that of the EGF-stimulated one. c-Src binds activated-EGFR under ox-stress Since we could not detect EGFR dimers under ox-stress using a plasma membrane impermeable zero spacer length cross-linking agent (EDAC) (Fig. 4), we wondered whether there was a conformational change within the intracellular domain of the EGFR that allowed its phosphorylation. Of note is that wild type (WT) EGFR does not bind c-Src. However, it has been previously shown that a conformational change in the kinase domain of the EGFR due to somatic mutation (L858R) can result in a constitutive interaction with c- Src [33,34]. Therefore, we investigated whether WT- EGFR could bind c-Src under ox-stress. Serum-starved A549 cells were treated (or not) with 100 ng/ml EGF for 15 min or 1 U/ml GO for 30 min. Then, EGFR was IPed, separated by SDS-PAGE and IBed for total EGFR and active (p-Y416) c-Src (Fig. 5A). At the same time an IP was carried out from the same samples using an Ab specific for total c-Src and respective IBs were performed against total EGFR and total c-Src (Fig. 5B). The results shown in figure 5 demonstrate that EGFR is bound by c-Src (active c-Src) under ox-stress (Fig. 5A and B). PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23240