title: The P34G mutation reduces the transforming activity of K-Ras and N-Ras in NIH 3T3 cells but not of H-Ras.
abstract: Ras proteins (H-, N-, and K-Ras) operate as molecular switches in signal transduction cascades controlling cell proliferation, differentiation, or apoptosis. The interaction of Ras with its effectors is mediated by the effector-binding loop, but different data about Ras location to plasma membrane subdomains and new roles for some docking/scaffold proteins point to signaling specificities of the different Ras proteins. To investigate the molecular mechanisms for these specificities, we compared an effector loop mutation (P34G) of three Ras isoforms (H-, N-, and K-Ras4B) for their biological and biochemical properties. Although this mutation diminished the capacity of Ras proteins to activate the Raf/ERK and the phosphatidylinositol 3-kinase/AKT pathways, the H-Ras V12G34 mutant retained the ability to cause morphological transformation of NIH 3T3 fibroblasts, whereas both the N-Ras V12G34 and the K-Ras4B V12G34 mutants were defective in this biological activity. On the other hand, although both the N-Ras V12G34 and the K-Ras4B V12G34 mutants failed to promote activation of the Ral-GDS/Ral A/PLD and the Ras/Rac pathways, the H-Ras V12G34 mutant retained the ability to activate these signaling pathways. Interestingly, the P34G mutation reduced specifically the N-Ras and K-Ras4B in vitro binding affinity to Ral-GDS, but not in the case of H-Ras. Thus, independently of Ras location to membrane subdomains, there are marked differences among Ras proteins in the sensitivity to an identical mutation (P34G) affecting the highly conserved effector-binding loop.
authors.affiliation: Unidad de Biología Celular, Centro Nacional de Microbiología, Instituto de Salud Carlos III (ISCIII), 28220 Majadahonda, Madrid, Spain.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Oliva
authors.forename: Natasha
authors.initials: N
authors.lastname: Zarich
authors.forename: Natalia
authors.initials: N
authors.lastname: Martínez
authors.forename: Rocío
authors.initials: R
authors.lastname: Jorge
authors.forename: Antonio
authors.initials: A
authors.lastname: Castrillo
authors.forename: Marta
authors.initials: M
authors.lastname: Azañedo
authors.forename: Susana
authors.initials: S
authors.lastname: García-Vargas
authors.forename: Silvia
authors.initials: S
authors.lastname: Gutiérrez-Eisman
authors.forename: Angeles
authors.initials: A
authors.lastname: Juarranz
authors.forename: Lisardo
authors.initials: L
authors.lastname: Boscá
authors.forename: J Silvio
authors.initials: JS
authors.lastname: Gutkind
authors.forename: José M
authors.initials: JM
authors.lastname: Rojas
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D015870:Gene Expression; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D041681:NIH 3T3 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology; D014162:Transfection; D050807:ets-Domain Protein Elk-1; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020725:ral GTP-Binding Proteins / Q000378:metabolism; D020726:ral Guanine Nucleotide Exchange Factor / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology
chemical_list: D004268:DNA-Binding Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D010739:Phospholipase D; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D020830:rac1 GTP-Binding Protein; D020725:ral GTP-Binding Proteins; D020726:ral Guanine Nucleotide Exchange Factor; D018631:ras Proteins; C494918:AKT1 protein, human