The C-terminal end of R-Ras contains a focal adhesion targeting signal.
R-Ras promotes cell adhesion and activation of integrins through a process that is yet unknown. We show here that active R-Ras (38V) promotes the formation of focal adhesions and a spread cell shape. By contrast, the dominant-negative mutant of R-Ras (43N) reduces the number of focal adhesions, leading to the formation of refractile cells. In adherent cells wild-type R-Ras, activated (38V) R-Ras and endogeous R-Ras were preferentially targeted to focal adhesions, whereas the dominant-negative mutant (43N) of R-Ras was excluded from these structures. Activated mutants of H-Ras and K-Ras were not found in focal adhesions. We dissected R-Ras to find out the determinants that are important for the targeting process. The outermost region in the N-terminus of R-Ras, as well as the intact proline-rich sequence in the C-terminus of RRas that mediates binding to Nck, were not essential. Mutating the potential palmitoylation site (C213A) of RRas results in depalmitoylation and accumulation of R-Ras in the Golgi. Using H-Ras/R-Ras, R-Ras/H-Ras and RRas/K-Ras hybrid molecules we showed that the C-termini (175-218 amino acids) of R-Ras contains the signal for focal adhesions targeting. Exchanging the hypervariable region of H-Ras to R-Ras inhibited the targeting of R-Ras to focal adhesions, whereas H-Ras obtained the ability to localize to focal adhesions after receiving the hypervariable region of R-Ras. This indicates that R-Ras targeting is mediated both by the nucleotide binding status as well as through a specific region in the C-terminus of R-Ras. These results indicate that targeting and activation of R-Ras are linked processes in the formation of focal adhesions.
Furuhjelm
Johanna
J
Institute of Biotechnology, Program in Cellular Biotechnology, PO Box 56 (Viikinkaari 9), FIN-00014 University of Helsinki, Finland.
Peränen
Johan
J
D048868:Adaptor Proteins, Signal Transducing; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D022001:Focal Adhesions / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D011905:Genes, ras / Q000502:physiology; D006056:Golgi Apparatus / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D015513:Oncogene Proteins / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D018631:ras Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C078332:Nck protein; D015513:Oncogene Proteins; D011993:Recombinant Fusion Proteins; D006160:Guanosine Triphosphate; D002784:Cholesterol; D020558:GTP Phosphohydrolases; C085929:RRAS protein, human; D018631:ras Proteins