Volume 89, Issue 3, Pages 457-467 (May 1997) Role of Phosphoinositide 3-OH Kinase in Cell Transformation and Control of the Actin Cytoskeleton by Ras  Pablo Rodriguez-Viciana, Patricia H Warne, Asim Khwaja, Barbara M Marte, Darryl Pappin, Pamela Das, Michael D Waterfield, Anne Ridley, Julian Downward  Cell  Volume 89, Issue 3, Pages 457-467 (May 1997) DOI: 10.1016/S0092-8674(00)80226-3

Figure 1 In Vitro Binding of Ras Mutants to Effectors (A) Mutant Ras proteins were loaded with GTP (T) or GDP (D) and bound to GST-fusion proteins of the effectors Ral.GDS (residues 1–97), c-Raf-1 (residues 1–259), and PI 3-kinase (full-length p110). Bound Ras was visualized by immunoblotting with monoclonal antibody Y13-259. In the control lane (cont), GST was substituted for effector fusion protein. All mutants were in a V12 background. (B) Ras mutant interaction with effectors was measured by scintillation proximity assay. GST-fusion proteins spanning the Ras-binding site in Ral.GDS and Raf were used. The means and standard errors of triplicate assays are shown. Cell 1997 89, 457-467DOI: (10.1016/S0092-8674(00)80226-3)

Figure 2 Effects of Ras Mutants on Effector Activity in COS Cells Ras mutants (in a V12 background) were transfected into COS-7 cells. For PI 3-kinase, Ras mutants were transfected along with wild-type p110α or alone as indicated. p110* is the activated PI 3-kinase mutant K227E p110α. PIP3 levels were measured in whole cells 48 hr after transfection. Raf activity was measured by cotransfecting Ras mutants with epitope-tagged wild-type c-Raf-1. After 48 hr, the Raf was immunoprecipitated and assayed in a coupled assay for its ability to activate MEK. The means and standard errors of duplicate assays are shown. Representative of at least three experiments. Cell 1997 89, 457-467DOI: (10.1016/S0092-8674(00)80226-3)

Figure 3 Transformation of NIH 3T3 by Ras Mutants and Effectors (A) Ras mutants (in a V12 background) were transfected into NIH 3T3 cells either alone or in combinations. Focus formation assays were performed over two weeks. The following Ras effectors and other signaling proteins were assayed for transformation alone and in combination: Δ1–295 Raf, K227E p110α, V12 Rac, and V14 Rho. Assays were performed using 20 ng of V12 Ras plasmid and 1.5 μg of every other construct. (B) Ras effector mutants in a V12 background were assayed for transformation in combination with Δ1–295 Raf, V12 Rac, V14 Rho, and K227E p110α. Assays were performed using 20 ng of V12 Ras plasmid and 1.5 μg of every other construct. Cell 1997 89, 457-467DOI: (10.1016/S0092-8674(00)80226-3)

Figure 4 Inhibition of Ras Transformation of NIH 3T3 Cells by Dominant-Negative PI 3-Kinase V12 Ras or v-Src was transfected into NIH 3T3 cells along with the indicated constructs, including p85-derived dominant-negative PI 3-kinase constructs, dominant-negative Rac and MEK, and the SH2 and SH3 domains of p120GAP. Focus formation was quantitated after two weeks. Cell 1997 89, 457-467DOI: (10.1016/S0092-8674(00)80226-3)

Figure 5 Effect of Ras Mutants and Effectors on Actin Rearrangement in Porcine Aortic Endothelial Cells (A) PAE cells were microinjected with the indicated plasmids (p110* is the activated PI 3-kinase K227E p110α) and actin visualized after 5 hr by immunofluorescence using TRITC-phalloidin. (B) Cells were microinjected with V12 Ras protein or K227E p110α plasmid, along with dominant-negative Ras, Rac, or Cdc42 protein or plasmid, respectively. Cell 1997 89, 457-467DOI: (10.1016/S0092-8674(00)80226-3)

Figure 6 Effect of Inhibitors on Ras-Induced Actin Rearrangement in Porcine Aortic Endothelial Cells (A) PAE cells were microinjected with plasmids encoding activated Ras, PI 3-kinase, Rac, or V12 C40 Ras. Four hours after nuclear injection of the constructs, LY294002 (LY) was added to the cells for another 90 min. (B) p85-based dominant-negative PI 3-kinase constructs were microinjected along with V12 Ras or V12 Rac constructs. Actin was visualized after 5 hr. (C) Cells were injected with either V12 Ras or V12 Rac protein, or activated PI 3-kinase plasmid, or were treated with PDGF. Cells were preincubated for 1 hr with 250 nM Calphostin C (CAL.) or 10 mM Bisindoylmaleimide 1/GF 109203X (BIM.) before injection of the V12 Ras or V12 Rac proteins. The cells were then incubated for a further 60 and 30 min, respectively, before fixing. Where indicated, cells were stimulated with 25 ng/ml PDGF for 4 min. For PI 3-kinase, cells were microinjected with the plasmid 5 hr before treatment with the inhibitors for 1 hr. Cell 1997 89, 457-467DOI: (10.1016/S0092-8674(00)80226-3)

Figure 7 Effect of LY294002 and p85 Overexpression on Ras- Induced Cellular PIP3 Levels (A) PIP3 levels were measured in metabolically labeled, serum-starved NIH 3T3 cells. Cells were treated with LY294002 for 90 min before lysis. PDGF (50 ng/ml) treatment was for 3 min before lysis. V12 Ras was expressed by retroviral infection for 48 hr before labeling. (B) V12 Ras and PI 3-kinase constructs, including wild-type p110, were transfected into COS-7 cells. PIP3 levels were measured in whole cells 48 hr after transfection. Cell 1997 89, 457-467DOI: (10.1016/S0092-8674(00)80226-3)