Figure 4. H2O2 activation of EGFR does not induce receptor dimerization. A. Serum-starved A549 cells were exposed (or not) to 100 ng/ml EGF or 1 U/ml GO for 15 min. Then, the cross linking reagent EDAC (1 mM) was added and incubation was continued for an additional 15 min. EGFR was IPed from cell lysates, resolved by SDS- PAGE and IBed for total and Tyr-phosphorylated EGFR as indicated; presence of EGFR dimers is indicated. B. Serum-starved NIH-3T3 cells over-expressing wild type EGFR were treated as in A; 50 mg of total cell lysates were IBed for total and Tyr-phosphorylated EGFR as indicated. doi:10.1371/journal.pone.0023240.g004 Moreover, we investigated whether such binding was dependent upon activation of c-Src by ox-stress. Therefore, A549 cells were pre-incubated, or not, with 5 mM PP1 (Src inhibitor) for 45 min. prior to stimulation with 100 ng/ml EGF (15 min) or 1 U/ml GO (30 min.) (Fig. 5B). PP1 inhibition of c-Src activation did not prevent the binding of c-Src to EGFR, and also did not lead to the conventional EGFR dimerization under ox-stress (data not shown). This demonstrates that activation of c-Src is not required for its interaction with EGFR under ox-stress and further supports the notion that an intrinsic conformational change in the EGFR exposed to ox-stress enables its binding to c-Src. Of note is that c-Src is highly activated under ox-stress and thus may also be undergoing degradation. This is presently being investigated in our laboratory. Oxidative stress induces a novel active conformation of EGFR kinase domain In order to support our hypothesis that under ox-stress EGFR acquires a novel active conformation, we tested the binding of a EGFR Activation under Oxidative Stress Figure 5. EGFR directly interacts with c-Src under H2O2 and such interaction is not abolished by Src family kinase inhibitor PP1. A549 cells were incubated (or not) with 5 mM PP1 for 45 min. and then treated (or not) for 15 min. with 100 ng/ml EGF or 30 min. 1 U/ml GO. A. EGFR was IPed from total cell lysates with the mAb 528 and IBed for Y416 phosphorylated c-Src (p-Src) and for total EGFR, as indicated. B. c-Src was IPed from total cell lysates and IBed for total c-Src and EGFR, as indicated. doi:10.1371/journal.pone.0023240.g005 new EGFR monoclonal antibody, a4-2 mAb. This Ab is susceptible to the conformational changes of the activated EGFR kinase domain in non-denaturing conditions [35]. It recognizes amino acids 956–998, epitopes of EGFR that are exposed through the conformational change induced by EGF binding to the receptor. Of note is that same epitopes are also exposed in the constitutively active L858R EGFR mutant, and, therefore, such a mutant also binds efficiently the a4-2 mAb [35]. As shown in figure 6, A549 cells were treated with EGF or GO (Fig. 6A), as before and EGFR was IPed from 300 mg of total cell lysates using 3 mg of either a4-2 Ab or a528 Ab and then IBed with aEGFR (2232) Ab to estimate the total EGFR that was IPed (Fig. 6B). Even though the activation level of EGFR was comparable under EGF or GO treatment, as assessed by the tyrosine phosphorylation level (ap-Y20)(Fig. 6A), the a4-2 Ab pulled down the EGF-stimulated EGFR with an affinity ,3.5 folds higher than that observed under ox-stress (GO) activation (Fig. 6B– C). Control IgG heavy chains of the Abs used in the IPs are also shown (Fig. 6B). Furthermore, we investigated the ability of the a4-2 Ab to bind to the L858R mutant EGFR. For this purpose, we employed NIH-3T3 cells stably over-expressing the mutant [34]. These cells were exposed to EGF or GO and EGFR was IPed with either a4-2 or a528 Ab and IBed with aEGFR (2232) Ab, as described above. Figure 6D confirms that the a4-2 Ab binds efficiently and constitutively to the L858R EGFR MT, as previously reported [35]. However, when cells expressing that mutant were exposed to ox-stress (GO) the affinity of the L858R EGFR for the a4-2 Ab was reduced ,70% in comparison to the untreated or EGF-treated cells (Fig. 6D–E). Therefore, the use of this novel Ab, directly demonstrates that the active conformation of EGFR under ox-stress differs from that of the EGFR stimulated by EGF. Moreover, the conformation of the EGFR mutant L858R under ox-stress is also being altered. H2O2 activation of the EGFR is temperature- dependent We investigated the activation of EGFR by ox-stress (and by EGF) at different temperatures. PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23240