c-Raf-1 RBD associates with a subset of active v-H-Ras.
Mutational analysis of the cRaf-1 Ras binding domain (RBD) identified several point mutants with elevated Ras binding. Detailed examination of the binding kinetics of one mutant (A85K) suggests that it associates with a greater range of isomeric conformers of v-H-Ras than wt-RBD. At limiting v-H-Ras concentrations, saturation binding to A85K-RBD is higher than to wt-RBD. Notably, in assay systems where the RBD concentration is limiting, no difference exists between wt-RBD and A85K-RBD saturation levels in the presence of a sufficiently large molar excess of Ras. The inability of wt-RBD to saturate all bindable Ras/GTP (defined by its binding to A85K-RBD) suggests that Ras/GTP exists as several isoforms and that only a minority of these isoforms are capable of associating with wt-RBD. These findings provide the first experimental evidence in support of functionally distinct Ras/GTP isoforms. We also describe a novel analysis of such isoforms.
Fridman
M
M
Ludwig Institute for Cancer Research, P.O. Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia.
Walker
F
F
Catimel
B
B
Domagala
T
T
Nice
E
E
Burgess
A
A
D001665:Binding Sites / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins