Ras Requires ERMs Figure 6. ERM proteins influence full-length SOS GEF activity towards Ras. A, In vivo GEF activity is associated with presence of ERM proteins. NIH 3T3 cells were treated with siERM cocktail, followed by permeation with streptolysin O in the presence of [a32P]-GTP and PDGF treatment. Bound [a32P]-GTP caught in the binding pocket of immunoprecipitated Ras was measured. B In vivo GEF activity is associated with wild type but not R579A ezrin. Dox treatment of dox-inducible ezrin wild type (ezrinWT) or R579A (ezrinRA) NIH 3T3 cells, followed by in vivo GEF activity measurement as described in C. C, In vivo GEF activity is associated with wild type but not R40L ezrin indicating that GEF activity is dependent on Ras binding of ezrin. Dox treatment of dox-inducible ezrin wild type (ezrinWT) or R40L (ezrinRL) NIH 3T3 cells, followed by in vivo GEF activity measurement as described in C. Results represent mean 6 s.d. of at least 3 independent experiments. ONEway ANOVA statistical analysis was performed. D, SOS/wild type ezrin complex has high in vitro GEF activity. (Left panel) SOS/ezrin complexes incubated with Ras preloaded with [a32P]- GTP. The reaction was initiated with excess GTPcS and the loss of Ras-bound radioactive GTP measured. Quantitative results represent mean 6 s.d. of at least 3 independent experiments, **P,0.01 using student’s t-test. (Right panel) Immunoblot shows equal amounts of SOS precipitated from ezrin wild type- and R579A-expressing cells. 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.g006 growth factor-induced Ras activation, strongly argues for a role of the ERM proteins in the control of Ras. In response to the growth factor PDGF we could show that the ERM/actin catalyze the formation of a multiprotein complex consisting of RTK, co-receptor, Grb2, SOS and Ras. In addition we identify ezrin and possibly other ERM proteins as new intracellular scaffold partners for Ras and SOS. Ezrin directly assembles Ras and SOS via separate domains of ezrin, and ezrin mutants unable to interact with Ras or SOS severely compromise Ras activity induced by PDGF or EGF. Moreover the PDGF- induced stimulation of guanine nucleotide uptake by Ras in permeated cells is specifically aborted by siRNA down-regulation of the ERM proteins or expression of dominant-negative ezrin mutants. These data suggest that functional ezrin directly assembling Ras and SOS is required for the PDGF-dependent stimulation of nucleotide exchange on Ras. Beyond scaffolding function ezrin could be a new intracellular partner required for SOS regulation. SOS, like other GEFs, is a large protein with autoinhibitory domains. According to mutant data [23], SOS requires to be unfolded in order to enable the access of Ras to an allosteric regulatory site which then releases the catalytic activity. The catalytic domain then promotes the exchange of GTP for GDP on another Ras molecule. An in vitro Ras activation assay performed with purified SOS isolated from cells demonstrated that its GEF activity is enhanced only when associated with ezrin. We propose that ezrin not only assembles PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27511