Ras Requires ERMs Figure 1. ERM proteins are necessary for PDGFR signaling. A, Down-regulation of ERM proteins reduces PDGF-dependent Erk phosphorylation. NIH 3T3 cells plated at low density were treated with a combination of siRNA SMARTpools against mouse ERM proteins for 24 hours. For exogenous reconstitution of human ERMs, cells were transfected with plasmid DNA coding for human ezrin-VSVg, radixin-Flag and untagged moesin or ezrin-VSVg alone. Cells were serum starved overnight prior to treatment with PDGF for 5 min. Lysates were immunoblotted as indicated. B, Schematic representation of the architecture of ezrin mutants. C, Ezrin mutants inhibit PDGF-dependent Erk phosphorylation. RT4 cells at low density were transfected with either empty vector (control) or ezrin mutants (ezrinNterm-GFP or ezrin deleted in the Actin-Binding-Domain ezrinDABD-GFP) (left panel) ezrin wild type or ezrin mutant R579A (right panel). Cells with high GFP expression were sorted by FACS, replated at low cell density and serum starved overnight prior to induction with PDGF for 5 min. Lysates were immunoblotted as indicated. D, NIH 3T3 cells were plated at a low density, co-transfected in a 5:1 ratio with constructs encoding either ezrin wild type-GFP or ezrin mutant-GFP and with a hygromycin resistance construct, selected by hygromycin for 1 day, and serum starved overnight prior to treatment with PDGF or EGF for 3 min. For Ras-GTP levels, lysates were treated with GST-Raf1-RBD (Ras-binding domain, RBD). Co-precipitated proteins were immunoblotted with antibodies against Ras. Lysates were immunoblotted as indicated. E, Ezrin R579A also inhibits PDGF-dependent Ras activation in RT4 cells. Experiment as in D, except ezrin constructs encoding either ezrin wild type-VSVg or ezrin mutant-VSVg were used. F, Down-regulation of individual ERM proteins using a cocktail of specific siRNAs reduces PDGF-dependent Ras activation in NIH 3T3 cells. Ras activity was determined as in D. G, Ezrin mutant, but not wild type ezrin, inhibits agar colony formation in RT4 cells. Dox-inducible ezrin wild type- or mutant-expressing cells were generated and placed in soft agar (2 and + dox). Results represent mean absolute colony number 6 s.d. of at least three independent experiments, **P,0.01 using student’s t-test. H, The F- actin-binding mutants of moesin (R570A, middle panel) and radixin (R576A, right panel) inhibit PDGF-dependent Ras activation in NIH 3T3 cells. Experiment set up as in D. In all panels lysates 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.g001 nous or overexpressed ERM protein(s) (Figure 3B,C,D). These results demonstrate that both SOS and Ras are part of the complex. Ezrin R579A, however, could not be co-precipitated with SOS or the PDGFR (Figure 3C,D), and, consequently, expression of ezrin R579A blocked the formation of the PDGFR complex. Figure 2. Latrunculin B mimics the ezrin mutants. A Reduction in actin filaments by treatment with latrunculin B. The parental schwannoma cells RT4 were plated at low density and treated with latrunculin B (1.25 mM, 10 min). Cells were processed as described in material and methods (scale bar 10 mm). B Latrunculin B inhibits signaling. RT4 cells at low density were serum starved overnight, then treated with latrunculin B (1.25 mM, 5 min) prior to treatment with PDGF (10 ng/ml, 5 min). Lysates were treated with GST-Raf1-RBD (to pulldown Ras-GTP). Co-precipitated proteins were immunoblotted with antibodies against Ras. Lysates immunoblotted as indicated. C, D, E Specific interference with signaling by latrunculin B. RT4 cells prepared and treated with latrunculin B as in panel A, then stimulated with LPA (20 mM, 5 min, panel B), IL-6 (1 ng/ml, 5 min, panel C) or TPA (100 ng/ml, 5 min, panel D). Lysates 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.g002 PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27511