title: A distinct class of dominant negative Ras mutants: cytosolic GTP-bound Ras effector domain mutants that inhibit Ras signaling and transformation and enhance cell adhesion.
abstract: Cytosolic GTP-bound Ras has been shown to act as a dominant negative (DN) inhibitor of Ras by sequestering Raf in non-productive cytosolic complexes. Nevertheless, this distinct class of DN mutants has been neither well characterized nor extensively used to analyze Ras signaling. In contrast, DN Ras17N, which functions by blocking Ras guanine nucleotide exchange factors, has been well characterized and is widely used. Cytosolic GTP-bound Ras mutants could be used to inhibit particular Ras effectors by introducing additional mutations (T35S, E37G or Y40C) that permit them to associate selectively with and inhibit Raf, RalGDS, or phosphoinositide 3-kinase, respectively. When the wild-type Ras effector binding region is used, cytosolic Ras should associate with all Ras effectors, even those that are not yet identified, making these DN Ras mutants effective inhibitors of multiple Ras functions. We generated cytosolic GTP-bound H-, N-, and K-Ras, and we assessed their ability to inhibit Ras-induced phenotypes. In fibroblasts, cytosolic H-, N-, and K-Ras inhibited Ras-induced Elk-1 activation and focus formation, induced a flattened cell morphology, and increased adhesion to fibronectin through modulation of a beta(1)-subunit-containing integrin, thereby demonstrating that DN activity is not limited to a subset of Ras isoforms. We also generated cytosolic GTP-bound Ras effector domain mutants (EDMs), each of which reduced the ability of cytosolic GTP-bound Ras proteins to inhibit Elk-1 activation and to induce cell flattening, implicating multiple pathways in these phenotypes. In contrast, Ras-induced focus formation, platelet-derived growth factor (PDGF)-, or Ras-induced phospho-Akt levels and cell adhesion to fibronectin were affected by T35S and Y40C EDMs, whereas PDGF- or Ras-induced phospho-Erk levels were affected only by the T35S EDM, implying that a more limited set of Ras-mediated pathways participate in these phenotypes. These data constitute the first extensive characterization of this functionally distinct class of DN Ras inhibitor proteins.
authors.affiliation: Department of Radiation Oncology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: James J
authors.initials: JJ
authors.lastname: Fiordalisi
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Holly
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Johnson
authors.forename: Leslie V
authors.initials: LV
authors.lastname: Parise
authors.forename: Adrienne D
authors.initials: AD
authors.lastname: Cox
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002448:Cell Adhesion; D003600:Cytosol / Q000378:metabolism*; D004268:DNA-Binding Proteins; D005353:Fibronectins / Q000378:metabolism; D005799:Genes, Dominant; D006160:Guanosine Triphosphate / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C494113:Elk1 protein, mouse; D005353:Fibronectins; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D006160:Guanosine Triphosphate; D018631:ras Proteins