catalyzed by the ERM proteins to the activation of Ras. Growth factor induced Ras activation is severely inhibited or even abolished by either the disruption of the interaction of the ERM proteins with co-receptors, by siRNA dependent down-regulation of ERM proteins, by the expression of dominant-negative mutants of ezrin, or by the disruption of F-actin. The actin-associated ezrin carries independent binding sites for the co-receptor, for Ras and for the autoinhibitory Dbl homology (DH) domain of the GEF Son of sevenless (SOS), stabilizing a complex consisting of receptor, co- receptor, Grb2, SOS, ezrin-actin and Ras. Mutations of these binding sites destroy the interactions and inhibit the activation of Ras. Furthermore, ezrin (used as an ERM protein prototype) not only serves as a scaffold assembling the partners Ras and SOS, but also appears to be an important regulator of SOS activity. Thus, our data reveal a novel aspect of Ras signaling that may be relevant to normal physiology as well as human cancer. Results Ezrin domains required for the catalysis of signal transduction We established the requirement of the ERM proteins for Ras signaling by three independent methods and in several cell lines. First, we overexpressed a soluble peptide – the cytoplasmic tail of the co-receptor CD44 – that sequesters the ERM proteins [10,15,16]. As a measure of Ras activity, we used Erk phosphorylation. The wild type peptide, but not a mutant defective in ERM protein binding (Figure S1A), strongly reduced the basal and platelet-derived growth factor (PDGF)-induced phosphorylation of Erk (Figure S1B). Kinetic resolution of PDGF signaling revealed attenuated Erk activation characterized by a shifted phosphorylation maximum (from 1 to 5 minutes) and lower fold-induction (from 19 to 5 times; Figure S1C, D). Thus, removal of the ERM proteins from the co-receptor interferes with signaling. Second, down-regulation of the ERM proteins by two independent siRNA cocktails directed against all three ERM proteins lowered PDGF-induced Erk phosphorylation, but did not impair the activation of the PDGF receptor (PDGFR) itself (Figure 1A; Figure S1E, F). As will be shown below, each of the three ERM proteins contribute to activation of signaling, at least in NIH3T3 cells (Figure 1F). Importantly, overexpression of all three human ERM proteins or of ezrin alone rescued the mouse-siRNA- dependent inhibition (Figure 1A). We conclude that the ERM proteins are essential for PDGF signaling towards Erk. Third, we interfered with ERM protein functions by mutations in ezrin. Thereby, we also defined ezrin domains involved in Ras activation. Ezrin mutants were overexpressed to compete with endogenous ERM proteins for cellular interactions. We first constructed ezrin with deletions in the C-terminus (Figure 1B; Nterm and DABD deleting the actin-binding domain). These mutant proteins are still able to bind to co-receptors at the plasma membrane, but cannot interact with F-actin. Stable overexpres- sion of these deletion mutants (as GFP fusions) abolished PDGF- induced Erk phosphorylation (Figure 1C, left panel). Because the deletions might abolish more than just the binding of ezrin to actin, we used ezrin R579A (see Figure 1B), a mutant that is completely defective in F-actin binding, but is otherwise functionally intact [14]. Overexpression of this mutant, but not of wild type ezrin, reduced PDGF-dependent and IL-6-dependent Erk phosphorylation (Figure 1C, right panel; Figure S2A, B). Of note, ezrin R579A was specific for Ras-dependent signaling in as much as it did not interfere with PKC-induced Erk phosphory- lation (Figure S2B) nor with IL-6-dependent STAT3 activation, Ras Requires ERMs which is independent of Ras (Figure S2B). In conclusion, F-actin binding or an unknown step linked to the ezrin/F-actin interaction is crucial for PDGF signaling to Erk, possibly at the level of or upstream of Ras. Ezrin R579A prevents Ras activation To address whether the mutants directly interfere with the activation of Ras, we measured the PDGF-dependent loading of GTP onto Ras. Overexpression of ezrin R579A (Figure 1C,D) inhibited the activation of Ras. As a consequence of reduced Ras activation, doxycycline-induced expression of ezrin R579A inhibited agar colony formation of RT4 cells, which are driven by mutated ErbB2 (Figure 1G). Interestingly, the effect of ezrin was not limited to the signaling by the PDGFR, but was important for other RTKs too. Epithelial growth factor (EGF)-induced Ras activation was also abolished (Figure 1D), suggesting a general requirement for the ERM proteins in RTK-induced Ras activation. In addition, the contribution of the individual ERM proteins to signal transduction was examined using the corre- sponding mutants radixin R576A and moesin R570A. Overex- pression of each mutant in NIH 3T3 cells inhibited spontaneous and PDGF-induced Ras activation (Figure 1H). Specific siRNA down-regulations (Figure 1F) showed that single knockdowns had partial inhibitory effects on Ras activation, with the combination of all three siRNAs being the most effective. Thus, all three ERM proteins participate in catalyzing the signal transduction to Ras and the overexpression of any one of the mutants competes with the functions of all three wild type proteins. Because ezrin R579A is defective in binding to F-actin, we investigated whether actin itself has a role in Ras activation. Latrunculin B, an inhibitor of actin polymerization (Spector et al, 1983), caused a reduction of stress fibers within minutes (Figure 2A). It also inhibited PDGF-induced Ras activation and downstream signaling to Erk (Figure 2B), while phosphorylation of the receptor (Figure 2B) and its association with Grb2 (not shown) were not affected. Interestingly, its effect on signaling was not indiscriminate. While latrunculin B specifically interfered, in addition to PDGF signaling, with the lysophosphatidic acid- induced phosphorylation of CREB (Figure 2C) and of Erk (not shown), and the IL-6-induced phosphorylation of Erk (Figure 2D), it did not inhibit the activation of STAT3 in response to IL-6 (Figure 2D). Additionally, PKC-dependent Erk phosphorylation in response to phorbol ester, which bypasses Ras, was not affected (Figure 2E). Thus, despite the general disruption of the actin cytoskeleton caused by this inhibitor (which is likely to be lethal over a longer observation period), it exerts, at least at this early time following growth factor stimulation, a very specific effect on signal transduction through the Ras-Erk pathway. Ezrin engages SOS and Ras Because the actin-binding function of ezrin is required for Ras activation, we speculated on the existence of an actin-associated protein complex involving ezrin. Such a multiprotein complex was indeed confirmed and its composition defined by immunoprecip- itation. Optimal isolation of the complex including the PDGFR was obtained using mild lysis conditions with Lubrol, a detergent used for the solubilization of transmembrane protein complexes [17,18,19]. Antibodies against the PDGFR co-precipitated both the co-receptor b1-integrin and the ERM proteins following PDGF stimulation (Figure 3A). In addition, the PDGF-activated complex also contained Ras (Figure 3D), suggesting that SOS, being a Ras regulator, may also be part of the complex. Indeed, antibodies against SOS, as well as antibodies against the PDGFR, precipitated the multiprotein complex including either endoge- PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27511