THE MINIMAL FRAGMENTS OF C-RAF-1 AND NF1 THAT CAN SUPPRESS V-HA-RAS-INDUCED MALIGNANT PHENOTYPE

v-Ha-Ras, an oncogenic Ras mutant, causes malignant transformation of mammalian cells by recruiting c-Raf-1, a cytosolic Ser/Thr kinase, to the plasma membranes/cytoskeleton, The kinase activity of c-Raf-1 resi des in the C-terminal half, which activates mitogen-activated protein (MAP) kinase kinase, while it is the N-terminal half of c-Raf-1 (Raf25 7, residues 1-257) that binds the Ras-GTP complex and can compete Ras GTPase-activating proteins such as NF1 for binding to Ras. However, it still remains to be clarified whether overexpression of Raf257 or its minimal Ras-binding fragment alone is sufficient to suppress Ras-indu ced malignancy. In this paper we demonstrate for the first time that t he 81-amino acid fragment (Raf81, residues 51-131), the minimal Ras-bi nding fragment of Raf, indeed can suppress v-Ha-Ras-induced malignant phenotype. A further deletion of the first 6 amino acids causes 65% re duction in the Ras binding of Raf81. The resultant 75 amino acid fragm ent (Raf75, residues 57-131) consists of a single alpha-helix, five an ti-paralleled beta-sheets and five loops. We have found that a further deletion of either the first beta-sheet/loop or the last two beta-she ets/loops completely abolishes Ras binding. In addition we have found that the removal of the C-terminal 35 amino acids from a Ras-binding 9 1-amino acid fragment of NF1 (NF91, residues 1441-1531) does not aboli sh its ability to suppress the Ras-induced malignancy.