Suppression of oncogenic Ras by mutant neurofibromatosis type 1 genes with single amino acid substitutions.
NF1 was first identified as the gene responsible for the pathogenesis of the human genetic disorder neurofibromatosis type 1. cDNA cloning revealed that its putative protein product has a domain showing significant sequence homology with the mammalian Ras GTPase activating protein and two yeast Saccharomyces cerevisiae proteins, Ira1 and Ira2. The Ras GTPase activating protein-related domain of the NF1 gene product (NF1-GRD) stimulates GTPase activity of normal Ras proteins but not of oncogenic mutant Ras from both mammalian and yeast cells. Thus, in yeast, NF1-GRD can suppress the heat-shock-sensitive phenotype of ira- cells but not the same phenotype of activated RAS such as RAS2Val19 and RAS2Leu68. We have screened a pool of mutagenized NF1 expression plasmids and obtained two mutant NF1 cDNA clones that can suppress the heat-shock-sensitive phenotype of RAS2Val19 cells. One clone (NF201) suppressed RAS2Leu68, RAS2Ser41, and RAS2Val19, whereas another clone (NF204) preferentially suppressed RAS2Val19. When expressed in mammalian cells, these mutant NF1-GRDs were able to induce the morphological reversion of v-ras-transformed NIH 3T3 cells. Both wild-type and mutant NF1-GRDs can stimulate the GTPase activity of normal but not transforming Ras. We suggest that mutant NF1-GRDs may bind tightly to transforming Ras, which stays in GTP-bound conformation, thus preventing the interaction with the putative effector molecule. On the other hand, normal Ras cannot be sequestered since the bound GTP is rapidly hydrolyzed upon interaction with mutant NF1-GRD to yield Ras-GDP, which is readily released from the NF1-GRD and recycled.
Nakafuku
M
M
DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304-1104.
Nagamine
M
M
Ohtoshi
A
A
Tanaka
K
K
Toh-e
A
A
Kaziro
Y
Y
D016475:3T3 Cells / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism; D016514:Genes, Neurofibromatosis 1 / Q000235:genetics*; D016147:Genes, Tumor Suppressor / Q000235:genetics*; D011905:Genes, ras / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D025542:Neurofibromin 1; D010641:Phenotype; D011506:Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D018631:ras Proteins
D005656:Fungal Proteins; D006360:Heat-Shock Proteins; D025542:Neurofibromin 1; D011506:Proteins; D020558:GTP Phosphohydrolases; D018631:ras Proteins