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Proc Natl Acad Sci U S A 1994 Jun 21;9113:5982-6.
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A single amino acid change in Raf-1 inhibits Ras binding and alters Raf-1 function.

Fabian JR , Vojtek AB , Cooper JA , Morrison DK .

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.

PubMed ID: 8016101
PMC ID: PMC44121
Article link: Proc Natl Acad Sci U S A
Grant support: [+]

Species referenced: Xenopus laevis
Genes referenced: raf1

References [+] :
Birchmeier, ras proteins can induce meiosis in Xenopus oocytes. 1985, Pubmed, Xenbase