Ha-ras and N-ras regulate MAPK activity by distinct mechanisms in vivo.
The Ras GTPases function as molecular switches, regulating a multiplicity of biological events. However the contribution, if any, of a specific c-Ras isoform (Ha-, N-, or Ki-ras A or B) in the regulation of a given biological or biochemical process, is unknown. Murine C3H1OT1/2 fibroblasts transformed with activated (G12V)Ha-ras or (Q61K)N-ras proliferate in serum-free media and have constitutive MAPK activity. The growth factor antagonist, suramin, inhibited the serum-independent proliferation of Ha-ras transformed fibroblasts, but not the serum-independent proliferation of N-ras transformed cells. The inhibition of cell proliferation was concomitant with the abrogation of the constitutive MAPK activity in the Ha-ras transformed fibroblasts. Analysis of the Ras-signalling complexes in immunoprecipitates from Ha-ras transformed cells revealed that Raf-1 co-immunoprecipitated with endogenous c-N-ras but not (G12V)Ha-ras. Pretreatment with suramin resulted in the loss of Raf-1 from c-N-ras immunoprecipitates. A c-N-ras antisense oligonucleotide, which down-regulated c-N-ras protein levels, abrogated the constitutive MAPK activity and serum-independent proliferation of (G12V)Ha-ras transformed cells. The data suggest that Raf-1 has a higher affinity for N-ras then Ha-ras in vivo, and c-N-ras function is required for the serum-independent proliferation of Ha-ras transformed cells.
Hamilton
M
M
Department of Cell Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Ohio 44195, USA.
Wolfman
A
A
D000818:Animals; D001769:Blood; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002461:Cell Line, Transformed; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D007106:Immune Sera; D051379:Mice; D008809:Mice, Inbred C3H; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology
D007106:Immune Sera; D016376:Oligonucleotides, Antisense; D013498:Suramin; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D015689:Oncogene Protein p21(ras)