Figure 9. H2O2-induced ox-stress does not deplete cellular cholesterol. Serum-starved A549 cells were treated, or not, with 100 ng/ml EGF for 15 min. or 1 U/ml GO for 30 min. or 2% (w/v) MbCD for 1 h or 2 mM MbCD-cholesterol complexes (CC) for 30 min. A. Total cell cholesterol levels were measured after lipid extractions and normalized per protein unit, as described in ‘‘Material and Methods’’; the values in the histogram are reported as % over non treated (NT) cells; St-Devs are indicated and ‘‘*’’ means p,0.05 in respect to control (NT); n = 3. B. Cells were fixed with paraformaldehyde (see ‘‘Material and Methods’’) and stained for cholesterol using the sterol-binding probe filipin (50 mg/ml for 30 min.). doi:10.1371/journal.pone.0023240.g009 leading to a distorted activated conformation. Indeed, the supporting molecular data is as follows: a. EGFR binds c-Src, which does not happen when the receptor is activated by EGF. b. EGFR may dimerize, but because the activated conformation of the receptor is unique, and different than the canonical conformation obtained by EGF induction, the dimer cannot be cross-linked by a conventional cross-linker, such as EDAC. c. Acquiring a non-canonical activated conformation under ox- stress also prevents the inhibition by the tyrosine kinase inhibitor (TKI), AG1478. However, this TKI manages to fully inhibit EGFR phosphorylation in broken cells when the unique ox-stress acquired conformation is demolished. d. Finally, EGFR activated by ox-stress could not bind to a new Ab that recognizes only the EGF-induced active conformation of EGFR (or that of the EGFR activated by somatic mutations, such as the L858R [35]) because the epitopes recognized by this Ab are in the intracellular domain of EGFR, which are exposed only in these active conformations. EGFR Activation under Oxidative Stress EGFR activated under ox-stress, evidently attained a different conformation that did not expose these epitopes, and thus the conformation-specific Ab could not bind EGFR in its unique conformation under ox-stress. Recent data from Chung et al [22], as well as the work of Bublil et al [21], demonstrated that EGFR does not need an extracellular ligand to form dimers. EGFR continuously changes from a monomer to a dimer state, where the interactions between the intracellular domains are as much of importance as that of the extracellular regions of the receptor in forming such dimers. This supports the significance of better understanding the mechanisms involved and the physiological relevance of processes leading to ligand-independent activation of EGFR. In the present study we used A549 adenocarcinoma or NIH- 3T3 cells stably over-expressing either the wild type or the L858R EGFR MT and several biochemical techniques to provide new insight into the mechanism of Tyr-phosphorylation/activation of EGFR under ox-stress. Reports by others demonstrated the inactivation of protein tyrosine phosphatases (PTPs) by H2O2 and suggested that this was responsible for EGFR phosphorylation [43,44,45]. For example, data published by Xu et al indicated that H2O2 induced phosphorylation of EGFR on Y1068 while PTPs activity was reduced to ,70% of control. However, we and others showed that H2O2 did not induce phosphorylation of a kinase-dead EGFR [26,46], demonstrating that the kinase activity of the receptor is required for its activation by H2O2 and that a bulk inactivation of PTPs cannot explain the phenomenon. Here we show that EGFR activation by H2O2 is a ligand- independent event that is not accompanied by ‘‘classical’’ receptor dimerization and is not inhibited by the TKI AG1478, indicating a novel unprecedented active conformation of EGFR that differs from that of the EGF-activated one (Fig. 1–4). Another indication for a unique conformational change of EGFR under ox-stress was supported by the finding that EGFR was strongly associated with c-Src. Moreover, the interaction between EGFR and c-Src was not dependent on the activation of c-Src because it persisted even in the presence of the c-Src kinase inhibitor PP1 (Fig. 5). Consistently, it was reported that c-Src stably interacts with ErbB2, but not with EGFR/ErbB1, because of differences in the kinase domains of the two receptors [47]. Additionally, studies with the L858R EGFR mutant (MT) also demonstrated that this MT could bind c-Src, whereas the wild-type (WT) EGFR could not (under physiological conditions). This MT has been crystal- lized and shown to possess a protein conformation that differs from that of the WT EGFR at the level of the kinase domain, carrying a constitutively open ‘‘activating-loop’’. Interestingly, the L858R EGFR MT was subsequently shown to have a similar functional phenotype to that of the WT EGFR under ox-stress, previously described by our group [24,25,26], i.e. hyper-phosphorylation/ activation, impaired trafficking, lack of ubiquitination and degradation and constitutive interaction with c-Src, without any ligand stimulation [15,33,34,48]. This further supported the idea that H2O2 induces a conformational change in the intracellular kinase domain of EGFR. Accordingly, dimerization of the extracellular domain could not be captured by the EDAC cross linker neither for the wild type EGFR under ox-stress (Fig. 4), nor for the L858R MT (unpublished observations). At the same time, even though EGFR under ox-stress appears to acquire a novel activated conformation, such a conformation seems to be different from that of the L858R EGFR MT, which is known to be sensitive to TKI [49]. PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e23240