Activation of Ras Requires the ERM-Dependent Link of Actin to the Plasma Membrane Tobias Sperka1,2, Katja J. Geißler1, Ulrike Merkel1, Ingmar Scholl1, Ignacio Rubio3, Peter Herrlich2, 1 Helen L. Morrison * 1 Morrison Laboratory, Leibniz Institute for Age Research – Fritz Lipmann Institute (FLI), Jena, Germany, 2 Herrlich Laboratory, Leibniz Institute for Age Research – Fritz Lipmann Institute (FLI), Jena, Germany, 3 Institute of Molecular Cell Biology, Centre for Molecular Biomedicine, Friedrich-Schiller-University, Jena, Germany Abstract Background: Receptor tyrosine kinases (RTKs) participate in a multitude of signaling pathways, some of them via the small G-protein Ras. An important component in the activation of Ras is Son of sevenless (SOS), which catalyzes the nucleotide exchange on Ras. Principal Findings: We can now demonstrate that the activation of Ras requires, in addition, the essential participation of ezrin, radixin and/or moesin (ERM), a family of actin-binding proteins, and of actin. Disrupting either the interaction of the ERM proteins with co-receptors, down-regulation of ERM proteins by siRNA, expression of dominant-negative mutants of the ERM proteins or disruption of F-actin, abolishes growth factor-induced Ras activation. Ezrin/actin catalyzes the formation of a multiprotein complex consisting of RTK, co-receptor, Grb2, SOS and Ras. We also identify binding sites for both Ras and SOS on ezrin; mutations of these binding sites destroy the interactions and inhibit Ras activation. Finally, we show that the formation of the ezrin-dependent complex is necessary to enhance the catalytic activity of SOS and thereby Ras activation. Conclusions: Taking these findings together, we propose that the ERM proteins are novel scaffolds at the level of SOS activity control, which is relevant for both normal Ras function and dysfunction known to occur in several human cancers. Citation: Sperka T, Geißler KJ, Merkel U, Scholl I, Rubio I, et al. (2011) Activation of Ras Requires the ERM-Dependent Link of Actin to the Plasma Membrane. PLoS ONE 6(11): e27511. doi:10.1371/journal.pone.0027511 Editor: Efthimios M. C. Skoulakis, Alexander Flemming Biomedical Sciences Research Center, Greece Received August 9, 2011; Accepted October 18, 2011; Published November 21, 2011 Copyright: 2011 Sperka 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: The authors had three grants that funded this work: 1) Krebshilfe 107089 www.krebshilfe.de, 2) Deutsche Forschungsgemeinschaft (He551/10.3), 3) SFB604 Multi functional signalling proteins (from DFG) www.dfg.de/index.jsp. 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: helen@fli-leibniz.de Introduction The small G-protein Ras functions as a molecular switch relaying extracellular stimuli to diverse intracellular effector pathways, which are responsible for controlling proliferation, motility and differentiation. Because of this central role Ras activity and its downstream signaling pathways must be tightly regulated. At the level of Ras the major determinants currently known are guanine nucleotide exchange factors (GEFs), which catalyze the loading of Ras with GTP replacing tightly bound GDP, and GTPase-activating proteins (GAP), which down- regulate the activity state by enhancing Ras-bound GTP hydrolysis. Specificity of GEF activity e.g. Son of sevenless (SOS) is linked not only to active RTKs through the adaptor protein, Growth factor receptor-bound protein 2 (Grb2), but is also influenced in its activity through interaction with membrane lipids [1,2,3]. Further but less well understood complexity of the Ras pathway has been created by the identification in the plasma membrane of nanoclusters of proteins and lipids which are thought to concentrate the components of effector cascades [4]. Also by the finding of scaffold proteins (e.g. kinase suppressor of Ras, KSR, and sprouty-related proteins, (spred) thought to coordinate kinetics of the downstream signaling components and preventing activation of physiologically inappropriate signals [5,6]. We discovered previously an additional level of regulation of the Ras dependent MAP kinase pathway: Co-receptors specific for a given RTK focus the MAP kinase activation to this receptor [7,8]. Our observations triggered our interest in defining at what level this control was exerted. Most RTKs require co-receptors such as integrins or other cellular adhesion molecules [7,8,9]. On the extracellular side, one of the functions of co-receptors appears to be the local enrichment or proper presentation of receptor ligands [10,11]. On the intracellular side, the cytoplasmic domains of co- receptors are required for RTK-dependent signaling [12,13,14]. Moreover, we identified a new component required for MAP kinase activation – the filamentous actin (F-actin)-binding protein ezrin (or other members of the ezrin-radixin-moesin (ERM) family) that connects the actin cytoskeleton with the plasma membrane. Initial evidence suggests that the ERM proteins bind to the cytoplasmic domain of the co-receptor and from this location required for growth factor induced Ras-MAP kinase activation. However, the precise mechanism of their action has remained elusive to date. In the present study, we explore how the ERM proteins precisely affect the MAP kinase pathway. We localize the step PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27511