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 resides in the C-terminal half, which activates mitogen-activated protein (MAP) kinase kinase, while it is the N-terminal half of c-Raf-1 (Raf257, 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-induced malignancy. In this paper we demonstrate for the first time that the 81-amino acid fragment (Raf81, residues 51-131), the minimal Ras-binding fragment of Raf, indeed can suppress v-Ha-Ras-induced malignant phenotype. A further deletion of the first 6 amino acids causes 65% reduction in the Ras binding of Raf81. The resultant 75 amino acid fragment (Raf75, residues 57-131) consists of a single alpha-helix, five anti-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-sheets/loops completely abolishes Ras binding. In addition we have found that the removal of the C-terminal 35 amino acids from a Ras-binding 91-amino acid fragment of NF1 (NF91, residues 1441-1531) does not abolish its ability to suppress the Ras-induced malignancy.
Fridman
M
M
Ludwig Institute for Cancer Research, P.O. Royal Melbourne Hospital, Victoria, Australia.
Tikoo
A
A
Varga
M
M
Murphy
A
A
Nur-E-Kamal
M S
MS
Maruta
H
H
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000502:physiology; D002471:Cell Transformation, Neoplastic; D006801:Humans; D007700:Kinetics; D008322:Mammals; D051379:Mice; D008969:Molecular Sequence Data; D025542:Neurofibromin 1; D015689:Oncogene Protein p21(ras) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D010641:Phenotype; D017354:Point Mutation; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D017433:Protein Structure, Secondary; D011506:Proteins / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D019908:Proto-Oncogene Proteins c-raf; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D025542:Neurofibromin 1; D010446:Peptide Fragments; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D015689:Oncogene Protein p21(ras)