Ras Requires ERMs Figure 4. Ezrin interacts directly with Ras. A, EzrinNterm interacts with Ras. RT4 cell lysates were incubated with GST-Nterminal ezrin or GST alone. GST pull-downs were immunoblotted as indicated. B, Direct interaction between Ras and ezrin determined by GST pull-down. Purified GST-Ras or GST agarose alone was incubated with purified recombinant N-terminal or C-terminal half of ezrin. The pull-down was subjected to SDS-PAGE, followed by colloidal Coomassie staining. C, Ezrin binds to GDP-loaded Ras. Recombinant Ras loaded with GDP or non-hydrolysable GTPcS was incubated with His-ezrin wild type. The His pull-down was immunoblotted as indicated. D, Ezrin binds fluorescent GDP-Ras in solution. Bacterially expressed Ras was loaded with fluorescent mantGDP. 1 mM mantGDP-Ras was then incubated with increasing amounts of recombinant ezrin wild type and fluorescence intensity was measured. E, Ezrin R40L cannot bind Ras. NIH 3T3 lysates of cells expressing dox-inducible ezrin wild type-VSVg or ezrin R40L-VSVg were incubated with GDP- or GTPcS-loaded GST-Ras agarose or GST agarose alone (control). The GST pull-down was immunoblotted as indicated. F, Ezrin R40L inhibits PDGF-dependent Ras activation. NIH 3T3 cells expressing dox-inducible ezrin wild type or R40L mutant were plated at a low density, serum starved overnight prior to treatment with PDGF for 3 min. Ras activity was determined as in 1D. Lysates were immunoblotted as indicated. G, Ezrin R40L, but not wild type ezrin, inhibits BrdU incorporation as quantified with a fluorescein-coupled BrdU antibody. Dox-inducible ezrin wild type- or mutant-expressing NIH 3T3 cells were generated and placed in soft agar (2 and + dox). Quantitative results represent mean 6 s.d. of at least three independent experiments, **P,0.01 using student’s t-test. 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.g004 purified recombinant proteins form a trimetric complex of ezrin with SOS (full length) and F-actin, not G-actin (Figure 5D). Wild type ezrin engaged SOS (full length) only in the presence of F- actin, the mutant defective in F-actin binding (R579A) did not (pulldown with His-tagged ezrin). Thus, interestingly, ezrin in conjunction with F-actin appears to adopt a specific SOS-binding conformation, enabling the interaction with the DH/PH domain of SOS. Ezrin interaction with the allosteric site of SOS enhances SOS activity The GEF SOS is itself subject to complex regulation. The DH- PH domains decrease catalytic activity by folding back onto the catalytic domain and restricting the accessibility to a second Ras- binding site distal to the catalytic one. This allosteric Ras-binding site is absolutely required for the full activation of SOS, implicating Ras itself as an essential determinant of SOS regulation [23]. While we identified ezrin as a scaffold protein assembling for both Ras and SOS, we considered whether ezrin might, in addition, help to activate SOS, perhaps by removing this steric block and presenting Ras-GDP to the allosteric site on SOS, essential for SOS activity [23]. This interesting possibility encouraged us to measure cellular SOS activity in the presence or absence of ezrin. Furthermore, in vitro experiments comparing SOS with SOS in complex with ezrin were subsequently performed. First, we tested whether ezrin is required for SOS nucleotide exchange activity in vivo by following the uptake of [a32P]-GTP PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27511