Ras Requires ERMs Figure 5. Ezrin interacts with the DH-PH domains of SOS. A, Schematic representation of the architecture of used SOS constructs. B, ERM proteins interact with the DH-PH domains of SOS. Incubation of His-SOS DH-PHcat- or His-SOScat with RT4 lysates. His pull-downs were immunoblotted as indicated. C, ERM proteins interact with the DH-PH domain of SOS. NIH 3T3 cells were transfected with constructs expressing Myc- tagged fragments of SOS (specific band marked with a red asterisk) or an empty vector control. Co-precipitated proteins were immunoblotted with antibodies against the ERM proteins. D, Full-length SOS interacts with ezrin in vitro only in the presence of F-actin. SOS was incubated with ezrin wild type- or R579A-His in the presence of filamentous (F) or globular (G) actin. The His pulldown was immunoblotted as indicated. The results are representative of at least three independent assays and each panel represents experiments from the same blot and the same exposure. doi:10.1371/journal.pone.0027511.g005 into permeated cells and the loading of [a32P]-GTP onto Ras. The nucleotide exchange activity in resting cells was high; however, it was insensitive to ERM protein knockdown and also to the expression of ezrin mutants R579A or R40L, indicating that basal SOS activity does not depend on ERM proteins (Figure 6A–C). At present, we do not have an explanation for this high SOS activity in resting cells, which has also been reported by another group [24]. In marked contrast to the basal SOS activity, the PDGF- sparked acceleration of nucleotide uptake onto Ras was completely abrogated by ERM protein knockdown and also by the expression of ezrin R579A or R40L (Figure 6A–C). The disrupting effect of these ezrin mutants reflects an essential contribution of the ezrin/ F-actin and ezrin-Ras complexes to the growth factor-dependent enhancement of SOS activity. We conclude that the ERM proteins are targeted to the plasma membrane/F-actin interface where they assemble SOS and Ras. This action possibly contributes to the release of SOS autoinhibition, thus enabling the presentation of Ras to the allosteric regulatory site of SOS. Ezrin controls SOS activity in vitro Based on these in vivo data, we investigated the effect of ezrin on SOS activity in vitro using precipitated complexes. PDGF- induced protein complexes were collected by GST-Grb2 pull- downs (the stoichiometric interaction of Grb2 with SOS ascertained equal SOS levels) of total lysates of cells expressing wild type or R579A ezrin. The complexes were incubated with recombinant Ras preloaded with radioactive GTP; the exchange reaction was initiated by adding an excess of non-radioactive GTPcS. Despite the presence of equal amounts of SOS (Figure 6D, right panel), a much higher exchange activity was observed in samples containing the SOS-ezrin wild type complex compared with the complexes precipitated from ezrin R579A- expressing cells (Figure 6D). These latter precipitates contained no ezrin (Figure 6D, right panel). This in vitro experiment demonstrates that SOS exerted significant nucleotide exchange activity only when bound to wild type ezrin. Discussion We show here that the activation of Ras is linked to actin dynamics and the presence of the ERM proteins. The classic model for the regulation of Ras by the GEF SOS involves the local assembly of SOS through adaptor proteins upon ligand stimula- tion of a RTK. One of these adaptor proteins, Grb2, brings SOS into proximity with the receptor where it is thought to engage Ras. However, we can now demonstrate a novel step in the control of Ras where regulators of the plasma membrane-cytoskeleton interphase, the ERM proteins, are essential for Ras activation downstream of RTK activity. We show that the ERM proteins in conjunction with F-actin associate with co-receptors, such as b1- integrin, focusing the ERM proteins to relevant sites of RTK activity with which the co-receptors are linked. Our observation that disruption of the interaction of the ERM proteins with co- receptors (by sequestering, by siRNA down-regulation or expres- sion of dominant-negative mutants of the ERM proteins) abolishes PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e27511