Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro.
The Ras proteins are key regulators of the growth of eukaryotic cells, but their direct target enzymes, or 'effectors', are unknown. The protein encoded by the c-raf-1 proto-oncogene is thought to function downstream of p21ras because disruption of Raf blocks signalling by Ras in a number of systems. Here we report that the amino-terminal cysteine-rich regulatory region of p74c-raf-1 expressed as a glutathione-S-transferase (GST) fusion protein binds directly to Ras with relatively high affinity (50 nM). The binding is strictly dependent on the Ras protein being in the active GTP-bound conformation rather than the inactive GDP-bound state. Raf-GST interacts with wild-type and oncogenic Ras (Val 12) but fails to interact with a biologically inert effector mutant of Ras (Ala 38) and a dominant negative mutant (Asn 17). A peptide based on the effector region of Ras inhibits the interaction. Raf-GST acts as a potent competitive inhibitor of the GTPase-activating proteins p120GAP and neurofibromin. In addition, Raf itself displays weak GTPase-stimulating activity towards Ras. It is therefore likely that Raf is a direct effector of Ras.
Warne
P H
PH
Imperial Cancer Research Fund, London, UK.
Viciana
P R
PR
Downward
J
J
D005982:Glutathione Transferase / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D015032:Zinc / Q000502:physiology
D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras); D015032:Zinc