C3G-mediated suppression of oncogene-induced focus formation in fibroblasts involves inhibition of ERK activation, cyclin A expression and alterations of anchorage-independent growth.
We showed previously that exogenous overexpression of C3G, a guanine nucleotide releasing factor (GEF) for Rap1 and R-Ras proteins, blocks the focus-forming activity of cotransfected, activated, sis, ras and v-raf oncogenes in NIH 3T3 cells. In this report, we show that C3G also interferes with dbl and R-Ras focus-forming activity and demonstrate that the transformation suppressor ability of C3G maps to its Crk-binding region (SH3-b domain). Using full-length C3G and C3GDeltaCat mutant, lacking catalytic domain, we showed here that overexpression of cotransfected C3G or C3GDeltaCat inhibited oncogenic Hraslys12-mediated phosphorylation of ERK, without altering Ras and Raf-1 kinase activation. We also showed that, overexpressed C3G and C3GdeltaCat inhibited the viability of oncogenic Ras-induced colonies in soft agar, indicating that C3G interferes with the anchorage-independent growth of Ras-transformed cells in a Rap1-independent manner. Consistent with both observations, overexpression of exogenous C3G and C3GDeltaCat also caused downregulation of Ras-induced cyclin A expression. Altogether, our results indicate that C3G interferes with at least two separate aspects of oncogenic transformation - cell cycle progression and loss of contact inhibition - and that these inhibitory effects probably account for its transformation suppressor activity.
Guerrero
Carmen
C
Centro de Investigación del Cáncer, IBMCC, Universidad de Salamanca-CSIC, 37007-Salamanca, Spain.
Martín-Encabo
Susana
S
Fernández-Medarde
Alberto
A
Santos
Eugenio
E
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002448:Cell Adhesion; D002455:Cell Division; D003001:Cloning, Molecular; D019925:Cyclin A / Q000235:genetics*; D017931:DNA Primers; D005347:Fibroblasts / Q000502:physiology; D011905:Genes, ras; D020571:Genes, sis; D020735:Guanine Nucleotide-Releasing Factor 2 / Q000235:genetics*; D006801:Humans; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D027384:Oncogene Proteins v-raf / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D017384:Sequence Deletion
D019925:Cyclin A; D017931:DNA Primers; D020735:Guanine Nucleotide-Releasing Factor 2; D027384:Oncogene Proteins v-raf; D020928:Mitogen-Activated Protein Kinases