EGFR is strongly associated with lipid rafts and their disruption by a cholesterol sequestering agent, such as MbCD, would cause the re-localization of EGFR in non-raft portions of the plasma membrane where it could be activated due to its release from raft- associated inhibiting factors [53]. However, recent studies suggested the existence of at least two kinds of raft populations, the cholesterol-enriched- and the ceramide-enriched-rafts. The ceramide-enriched rafts are typically generated through choles- terol displacement by ceramide and are ‘‘less fluid’’ [54]. Therefore, we are now proposing a new role for ceramide generation under ox-stress exposure in the process of EGFR aberrant activation and subsequent trafficking via caveolae to a peri-nuclear region, where it remains active (Fig. 10–11). Over the last ten years our group has been investigating the mechanism of ceramide generation under ox-stress in human airway epithelial (HAE) cells, showing that ceramide levels are increased not only in the membranes of HAE cells, but also in the lungs of mice exposed to H2O2 generated by cigarette smoke [28,29,31,40,41,55]. At the same time others have shown that ceramide alters membrane fluidity and causes cholesterol displacement from lipid rafts. Finally, it was suggested that ceramide can induce the merging of lipid rafts in bigger signaling ceramide-enriched membrane platforms [32,38,39,54,56,57]. Since we were not able to demonstrate cholesterol depletion induced by ox-stress (Fig. 8), we suggest that cellular ceramide generated (as a result of exposure to ox-stress) may just displace cholesterol from the rafts, thereby disrupting cholesterol-enriched rafts and leading to EGFR re-localization to the more rigid ceramide-enriched rafts. We show here for the first time that ox-stress-activated EGFR, as well as activated c-Src, co-localize within such ceramide enriched regions. At early time points of ox-stress exposure (15 min.) active EGFR and elevated ceramide co-localize primarily in the plasma membrane of the cells. Later on (30 min), the co-localization is observed mainly in a peri-nuclear region of the cells. Interestingly, we have previously shown that ox- stress activated EGFR, unlike the EGF-stimulated receptor, is not internalized via clathrin-coated pits; while it is not degraded and remains active, it can then traffic via caveolae to the peri-nucleus because of strong association with phosphorylated caveolin-1 (Cav-1) under ox-stress [26]. Moreover, the ox-stress activated EGFR co-localized with the early endosomes marker EEA-1 [25,26] and the recycling endosome marker rab-11 (unpublished observation). Taken together, the generation of ceramide, followed by cholesterol displacement may have a role in the aberrant trafficking of EGFR via caveolae to the peri-nuclear region under ox-stress exposure. Consistent with our hypothesis, it has been recently shown that ceramide generation increases the recruitment of Cav-1 into caveolae [58,59], Given the well established patho- physiological function of EGFR and ceramide in carcinogenesis and severe lung injury, respectively [55,60,61,62,63,64,65,66,67], this evidence may have far reaching implications, which will drive further investigations in this direction. In conclusion, data presented herein demonstrate that EGFR in cells exposed to ox-stress is not only aberrantly phosphorylated but it also acquires a novel conformation, which supports a ligand- independent mechanism of activation that does not require conventional receptor dimerization and is not inhibited by the TKI AG1478. These alterations in EGFR conformation are accompanied by c-Src binding to the receptor (Fig. 5). Whether the change in EGFR conformation under ox-stress occurs as a result of simultaneous alterations in the membrane structure, or happens independently and is only being stabilized by the EGFR Activation under Oxidative Stress simultaneous membrane changes requires additional studies (see model in Fig. 12). Materials and Methods Cell culture, treatments and reagents A549 adenocarcinoma (ATCC) and NIH-3T3 cells have been employed in this study. A549 cells were cultured in F12K medium (GIBCO) supplemented with 10% FBS (GIBCO) and 1% pen/ strep (GIBCO). NIH-3T3 cells were stably transfected with either the wild type (WT) EGFR or with the L858R EGFR mutant (these cells were kindly provided by Dr. H. Band, University of Nebraska Medical Center [34]) and were cultured in DMEM (GIBCO) medium supplemented with 10% FBS and 1% pen/strep. DMEM high glucose medium (GIBCO) was used for the treatments with no FBS supplementation. EGF was added directly into the treatment medium at a final concentration of 100 ng/ml. Glucose oxidase (GO, from Sigma) was used to generate H2O2, under conditions that were previously optimized [18,24,26]: briefly, DMEM medium was incubated for 15 min. at 37uC with 1 U/ml GO prior to being added on top of sub-confluent (,70%) cells for an additional 15 min. For longer treatments the GO-medium was replaced every 15 min. 1-Ethyl-3-(3-dimethylaminopropyl) carbo- diimide hydrochloride (EDAC) cross linker agent (Thermo Scientific) was dissolved in phosphate buffer saline (PBS) and then added to the treatment medium at a final concentration of 1 mM. Methyl-beta-cyclodextrin (MbCD, from Sigma) was dissolved directly in treatment medium at a final concentration of 2% (w/v), prior to putting such treatment medium on top of sub-confluent cells. PP1, a Src family inhibitor (Enzo Life Sciences), was dissolved in DMSO and added to the treatment medium at a final concentration of 5 mM. Tyrosine kinase inhibitor (TKI) AG1478 (Cell Signaling) was dissolved in DMSO and then added to the treatment medium at a final concentration of 1 mM. For MbCD- cholesterol complex (CC) preparation, 9% (w/v) MbCD was dissolved in PBS and heated while mixing at 80uC; cholesterol (Matreya) was slowly added to the heated solution until the MbCD was completely saturated by cholesterol (cholesterol is no longer solubilized); then the solution was filtered through 0.2 mm pores and added to the treatment medium with a final concentration of ,2 mM MbCD-cholesterol complexes. PBS or DMSO was added at the appropriate concentration to the control-untreated cells where needed. Cells were collected by scraping in either PBS or directly in the lysis buffer: 1% NP-40 (Igepal, from Sigma), 50 mM Tris, 10% Glycerol, 0.02% NaN3, 150 mM NaCl, pH 7.4, containing a cocktail of phosphatase and protease inhibitors (Sigma) as well as 1 mM NaF and Na3VO4. Lysates were passed 5 times through a 30 gauge needle prior to centrifugation and further processing of the samples (either by immuno-precipitation or immuno-blotting). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 5, 6, 8, 10 or 12% acrylamide gels were prepared following common procedures (not described) and run via a 2 Cell system (BioRad) for 1–4 h at 100 V at room temperature (RT). Immuno-precipitation (IP) 200–400 mg of total protein extracts were incubated overnight with 2–4 mg of antibodies (Abs): a528 (anti (a) EGFR Ab), ac-Src (Santa Cruz Biotech) or a4-2 (aactive-EGFR [35], kindly provided by Dr. K. Omi, Fujirebio Inc., Tokyo, Japan). 50 ml of 50% protein A-agarose bead complexes (Repligen) were added to the samples and incubated for 90 min. Four washes (by sequential PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23240