A single amino acid change in Raf-1 inhibits Ras binding and alters Raf-1 function.
Ras and Raf-1 are key proteins involved in the transmission of developmental and proliferative signals generated by receptor and nonreceptor tyrosine kinases. Genetic and biochemical studies demonstrate that Raf-1 functions downstream of Ras in many signaling pathways. Although Raf-1 directly associates with GTP-bound Ras, an effect of this interaction on Raf-1 activity in vivo has not been established. To examine the biological consequence of the Ras/Raf-1 interaction in vivo, we set out to identify key residues of Raf-1 required for Ras binding. In this report, we show that a single amino acid mutation in Raf-1 (Arg89 to Leu) disrupted the interaction with Ras in vitro and in the yeast two-hybrid system. This mutation prevented Ras-mediated but not tyrosine kinase-mediated enzymatic activation of Raf-1 in the baculovirus/Sf9 expression system. Furthermore, kinase-defective Raf-1 proteins containing the Arg89-->Leu mutation were no longer dominant-inhibitory or capable of blocking Ras-mediated signal transduction in Xenopus laevis oocytes. These results demonstrate that the association of Raf-1 and Ras modulates both the kinase activity and the biological function of Raf-1 and identify Arg89 as a critical residue involved in this interaction. In addition, the finding that tyrosine kinases can stimulate the enzymatic activity of Raf-1 proteins containing a mutation at the Ras-interaction site suggests that Raf-1 can be activated by Ras-independent pathways.
Molecular Mechanisms of Carcinogenesis Laboratory, National Cancer Institute-Frederick Cancer Research and Development Center, MD 21702.
J R
JR
Fabian
A B
AB
Vojtek
J A
JA
Cooper
D K
DK
Morrison
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D002460:Cell Line; D005260:Female; D005982:Glutathione Transferase / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007930:Leucine; D009036:Moths; D009865:Oocytes / Q000502:physiology; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / 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; D015533:Transcriptional Activation; D014162:Transfection; D014982:Xenopus laevis
D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D001120:Arginine; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras); D007930:Leucine