Post-translational modification of H-Ras is required for activation of, but not for association with, B-Raf.

B-Raf is regulated by Ras protein and acts as a mitogen-activated protein (MAP) kinase kinase kinase in PC12 cells and brain. Ras protein undergoes a series of post-translational modifications on its C-terminal CAAX motif, and the modifications are critical for its function. To elucidate the role of the post-translational modifications ...
in interaction with, and activation of, B-Raf, we have analyzed a direct association between H-Ras and B-Raf, and constructed an in vitro system for B-Raf activation by H-Ras. By using methods based on inhibition of yeast adenylyl cyclase or RasGAP activity and by in vitro binding assays, we have shown that the segment of B-Raf corresponding to amino acid 1-326 binds directly to H-Ras with a dissociation constant (Kd) comparable to that of Raf-1 and that the binding is not significantly affected by the post-translational modifications. However, when the activity of B-Raf to stimulate MAP kinase was measured by using a cell-free system derived from rat brain cytosol, we observed that the unmodified form of H-Ras possesses an almost negligible activity to activate B-Raf in vitro compared to the fully modified form. H-RasSer-181,184 mutant, which was farnesylated but not palmitoylated, was equally active as the fully modified form. These results indicate that the post-translational modifications, especially farnesylation, are required for H-Ras to activate B-Raf even though they have no apparent effect on the binding properties of H-Ras to B-Raf.
Mesh Terms:
Adenylyl Cyclases, Amino Acid Sequence, Animals, Binding Sites, Brain, Calcium-Calmodulin-Dependent Protein Kinases, Cell Line, Cell-Free System, Cytosol, Enzyme Activation, GTPase-Activating Proteins, Humans, Kinetics, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Binding, Protein Processing, Post-Translational, Protein-Serine-Threonine Kinases, Proteins, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-raf, Proto-Oncogene Proteins p21(ras), Recombinant Proteins, Saccharomyces cerevisiae, Spodoptera, Transfection, ras GTPase-Activating Proteins
J. Biol. Chem.
Date: Mar. 01, 1996
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