EGF Receptor Exposed to Oxidative Stress Acquires Abnormal Phosphorylation and Aberrant Activated Conformation That Impairs Canonical Dimerization . . Simone Filosto , Elaine M. Khan , Emiliana Tognon, Cathleen Becker, Majid Ashfaq, Tommer Ravid, Tzipora Goldkorn* Center for Comparative Respiratory Biology and Medicine, Genome and Biomedical Sciences Facility, University of California School of Medicine, Davis, California, United States of America Abstract Crystallographic studies have offered understanding of how receptor tyrosine kinases from the ErbB family are regulated by their growth factor ligands. A conformational change of the EGFR (ErbB1) was shown to occur upon ligand binding, where a solely ligand-mediated mode of dimerization/activation was documented. However, this dogma of dimerization/activation was revolutionized by the discovery of constitutively active ligand-independent EGFR mutants. In addition, other ligand- independent activation mechanisms may occur. We have shown that oxidative stress (ox-stress), induced by hydrogen peroxide or cigarette smoke, activates EGFR differently than its ligand, EGF, thereby inducing aberrant phosphorylation and impaired trafficking and degradation of EGFR. Here we demonstrate that ox-stress activation of EGFR is ligand-independent, does not induce ‘‘classical’’ receptor dimerization and is not inhibited by the tyrosine kinase inhibitor AG1478. Thus, an unprecedented, apparently activated, state is found for EGFR under ox-stress. Furthermore, this activation mechanism is temperature-dependent, suggesting the simultaneous involvement of membrane structure. We propose that ceramide increase under ox-stress disrupts cholesterol-enriched rafts leading to EGFR re-localization into the rigid, ceramide-enriched rafts. This increase in ceramide also supports EGFR aberrant trafficking to a peri-nuclear region. Therefore, the EGFR unprecedented and activated conformation could be sustained by simultaneous alterations in membrane structure under ox-stress. Citation: Filosto S, Khan EM, Tognon E, Becker C, Ashfaq M, et al. (2011) EGF Receptor Exposed to Oxidative Stress Acquires Abnormal Phosphorylation and Aberrant Activated Conformation That Impairs Canonical Dimerization. PLoS ONE 6(8): e23240. doi:10.1371/journal.pone.0023240 Editor: David Holowka, Cornell University, United States of America Received April 20, 2011; Accepted July 8, 2011; Published August 10, 2011 Copyright: 2011 Filosto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Institutes of Health (HL-71871 and HL-66189 to T.G.) and from the Tobacco-Related Disease Research Program (TRDRP) (17RT-0131 to T.G.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ttgoldkorn@ucdavis.edu . These authors contributed equally to this work. Introduction The epidermal growth factor receptor (EGFR, ErbB1) is a member of the ErbB family of receptor tyrosine kinases, which also includes ErbB2, ErbB3, and ErbB4. While these receptors have a critical role in normal cellular processes such as cell division, differentiation, and migration, their over-expression or dysregula- tion have been linked to a variety of human cancers, including breast, head and neck, lung, and ovarian [1,2,3]. As such, the activation of these receptors, particularly the EGFR, has been a subject of intense study. A paradigm of EGFR activation has been established wherein ligand binding induces receptor dimerization, leading to the activation of its intrinsic tyrosine kinase activity, auto-phosphor- ylation, and subsequent phosphorylation of downstream signaling molecules [4,5,6,7]. Recent advancements in crystallographic studies have demonstrated that EGFR dimerization occurs upon the binding of one EGF molecule to one EGFR, which releases the extracellular portion of the receptor from its ‘‘tethered’’ confor- mation. This exposes the otherwise buried ‘‘dimerization arm’’, which can then interact with its dimerization partner in an entirely receptor-mediated back-to-back orientation [8,9,10,11]. However, the dogma of EGFR dimerization/activation upon ligand binding has been challenged by the discovery of the L858R and other somatic mutations of EGFR that affect receptor conformation and sensitivity to tyrosine kinase inhibitors (TKIs), supporting the idea that EGFR can be activated without its ligand and without ligand-supported dimerization [12,13,14,15,16,17]. Indeed, besides EGFR mutations, a few ligand-independent activations of EGFR have been described, such as via cigarette smoke [18], cell to cell interaction [19] or upon cholesterol- enriched lipid rafts disruption [20]. The physiological relevance of such poorly understood mechanisms has been recently brought to attention again by the work of Bublil et al [21], who demonstrated that EGFR exists in a ‘‘quasi-dimer’’ state that is stabilized by both extracellular and intracellular receptor-receptor interactions, whose formation does not require extracellular ligand. Also, Chung et al [22] demonstrated that unligated EGFR changes constantly between monomer and dimer states and pre-created dimers are ready for ligand binding and signaling. Consistently, PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23240