Ras Requires ERMs Figure 3. Ezrin engages in a complex with SOS and Ras. A, Endogenous ERM proteins associate with the PDGFR and its co-receptor b1- integrin. RT4 cells were plated at low density, serum starved overnight and treated with PDGF for 1 min. Proteins co-immunoprecipitated (CoIP) with PDGFR were immunoblotted, rabbit lgG was used for control IP. B, Endogenous ERM proteins associate with SOS and Ras. RT4 cells treated as in A. SOS was immunoprecipitated by a rabbit SOS-specific antibody, rabbit lgG was used for control IP. Coimmunoprecipitated proteins were immunoblotted. C, Ezrin R579A cannot associate with SOS and Ras. RT4 cells expressing dox-inducible ezrin wild type or R579A mutant were plated at low density, serum starved overnight and incubated with dox prior to treatment PDGF for 1 min. Proteins co-immunoprecipitated with SOS (as in B) were immunoblotted as indicated. D, Ezrin R579A cannot associate with the PDGFR and Ras. Cells were treated as in C followed by PDGFR-CoIP (as in A). Proteins co-immunoprecipitated with PDGFR were 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.g003 Direct interaction of ezrin and Ras is essential for Ras activation Because the association of Ras with SOS should be transient and not stable enough for immunoprecipitation, the co-immunoprecip- itation data might indicate that ezrin forms a stabilizing scaffold that assembles Ras and SOS. Employing three independent techniques, we provide evidence for a direct interaction between Ras and ezrin (Figure 4). First, a fusion protein of the N-terminus of ezrin and GST, but not GST alone, brought down Ras from cell lysates (Figure 4A). Furthermore, a fusion protein of GST and Ras precipitated the purified N-terminus of ezrin, but not the C- terminus (Figure 4B). Finally, purified His-tagged ezrin precipitated purified Ras (Figure 4C). Second, if the interaction of ezrin and Ras serves to stabilize the complex with SOS prior to nucleotide exchange, then Ras-GDP should be the preferred interaction partner of ezrin. Indeed, it was Ras-GDP that was pulled down with His-tagged ezrin (Figure 4C). Another technique using Ras loaded with fluorescent-labeled GDP (mantGDP-Ras) also demonstrated interaction. The fluorescence intensity increased in a concentration-dependent manner when ezrin was added to mantGDP-Ras (Figure 4D), indicating that Ras- GDP and ezrin do indeed interact. Finally, ezrin carries a motif in its N-terminal domain which resembles the Ras-binding domain of Raf1 kinase [20]. To test whether this motif is involved in the ezrin-Ras interaction, we mutated several amino acids individually within this motif [21,22]. Of these mutants, ezrin R40L lost the ability to bind Ras most strongly (Figure 4E). Expression of ezrin R40L abolished PDGF- induced activation of Ras (Figure 4F) and inhibited serum- dependent incorporation of BrdU (Figure 4G). Therefore, the most straightforward explanation of these findings is that Ras and ezrin interact directly and that this interaction is part of the scaffolding function of ezrin required for Ras activation. Ezrin interacts with the Dbl homology (DH)-pleckstrin homology (PH) domains of SOS The ezrin mutants identify ezrin as an organizer of both SOS and Ras. The ability of ezrin to act as a scaffold for SOS was further suggested from mapping the domains of SOS interacting with ezrin (SOS constructs are illustrated and described in Figure 5A). Co-immuno and co-affinity precipitation experiments identified the DH-PH tandem domains as an interaction site for ezrin (Figure 5B,C). As suggested from the co-IP experiments of Figure 3 and ezrin mutant R579A lacking binding to actin, PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27511