Proc. Natl. Acad. Sci. USA Vol. 90, pp. 6706-6710, July 1993 Biochemistry Suppression of oncogenic Ras by mutant neurofibromatosis type 1 genes with single amino acid substitutions MASATO NAKAFUKU*t, MASARU NAGAMINE*, AKIHIRA OHTOSHI*, KAZUMA TANAKAO§, AKIo TOH-E*, AND YOSHITO KAZIRO*¶ *DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304-1104; and tDepartment of Biology, Faculty of Science, University of Tokyo, Tokyo 113, Japan Communicated by Paul Berg, April 1, 1993 ABSTRACT NFI was frit identified as the gene responsible growth through the regulation of adenylate cyclase (14, 15). for the pathogenesis of the human genetic disorder neurofibro- Thus, yeast cells carrying activated mutations in Ras (such as matosis type 1. cDNA cloning revealed that its putative protein [Val'9]Ras2 and [Leua]Ras2) are defective in responding to product has a domain showing signfifcant sequence homology environmental conditions and show a variety of phenotypes with the mammalian Ras GTPase activating protein and two including a heat-shock-sensitive phenotype (14, 15). yeast Saccharomyces cerevisiae proteins, Iral and Ira2. The Ras S. cerevisiae also possesses two NF1 homologs, Iral and GTPase activating protein-related domain of the NFI gene Ira2, and human NF1 is structurally closer to yeast Ira than product (NF1-GRD) stimulates GTPase activity of normal Ras to human GAP (1-4). NF1 and Ira are also functionally proteins but not of oncogenic mutant Ras from both mamma- related since NF1-GRD expressed in yeast cells can com- lian and yeast cells. Thus, in yeast, NF1-GRD can suppress the plement ira-deficient yeast. In ira- cells, the conversion of heat-shock-sensitive phenotype of ira- cells but not the same Ras'GTP to Ras-GDP is defective, and hence the cells show phenotype of activated RAS such as RAS2vEdl9 and RAS2Le"". a heat-shock-sensitive phenotype that is very similar to that We have screened a pool of mutagenized NF1 expression ofactivated Ras mutants (16, 17). NF1-GRD can suppress the plasmids and obtained two mutant NF1 cDNA clones that can heat-sensitive phenotype of ira-, but not of RAS2Val19 or suppress the heat-shock-sensitive phenotype ofRAS2Vdll9 cells. RAS2Leu68 (5-7). This is consistent with the fact that NFl- One clone (NF201) suppressed RAS2VLde6, RAS2Ser4l, and GRD stimulates GTPase activity of normal but not mutant RAS2Val'9, whereas another done (NF204) preferentially sup- Ras proteins. pressed RAS2vauI9. When expressed in mammalian cells, these In this study, we took advantage of the yeast Ras system mutant NFl-GRDs were able to induce the morphological to isolate NF1-GRD mutants that can act as antioncogenes reversion of v-ras-transformed NIH 3T3 cells. Both wild-type specific for oncogenic Ras. Suppressors of the heat-shock and mutant NFl-GRDs can stimulate the GTPase activity of phenotype of RAS2VaOI9 were selected from mutagenized normal but not transforming Ras. We suggest that mutant NF1-GRD plasmids in yeast cells, and two mutants with NFl-GRDs may bind tightly to transforming Ras, which stays single amino acid substitutions were obtained. We demon- in GTP-bound conformation, thus preventing the interaction strate that these mutant NF1-GRDs, when expressed in with the putative effector molecule. On the other hand, normal mammalian cells, are able to induce the morphological re- Ras cannot be sequestered since the bound GTP is rapidly version of v-ras-transformed NIH 3T3 cells. hydrolyzed upon interaction with mutant NFl-GRD to yield Ras-GDP, which is readily released from the NF1-GRD and recycled. MATERIALS AND METHODS Yeast Strains and Medium. The wild-type S. cerevisiae Recent progress in molecular biology of human genetic RAY-3A-D (MATa/MATa ura3/ura3 leu2/leu2 trpl/trpl disorders has identified a gene named NFl that is involved in his3/his3 ade8/ade8) and its isogenic strains, TK161-R2V-D neurofibromatosis type 1 disease (1-4). The NFI gene en- (RAS2VaIl9/RAS2val"9) and KT63-2B-D (ira2-/ira2-) (16, codes a protein of 2818 aa sharing significant sequence 17), were used in this study. Transformants of NF1-GRD homology with the members of Ras GTPase-activating pro- plasmids were grown in SDC medium, which contains 2% tein (GAP) family (1-4), and the GAP-related domain of the (wt/vol) dextrose, 0.67% yeast nitrogen base without amino NFl gene product (NF1-GRD) can stimulate the GTPase acids (Difco), 0.5% Casamino acids (Difco), 50 mg ofadenine activity of Ras proteins (5-7). sulfate per liter, and 50 mg of L-tryptophan per liter. Ras is a GTP-binding protein and serves as a molecular Mammalian Cell Lines. DT is a NIH 3T3-derived cell line switch in signal transduction controlling the proliferation and transformed by two copies of a v-Ki-ras gene that carries differentiation of cells (8-10). Somatic mutations that acti- both Ser-12 and Thr-59 mutations (18) and was obtained from vate Ras may contribute to tumorigenesis in up to 30% of M. Noda (Cancer Institute, Tokyo). COS-7 cells are simian human tumors (11, 12). Ras is active when it binds GTP, and cells expressing the simian virus 40 tumor antigen (19). Both the active RasGTP complex is converted to an inactive cell lines were cultured in Dulbecco's modified Eagle's Ras-GDP complex by intrinsic GTPase activity (9, 10). The medium containing high glucose (4.5 g/liter) supplemented members of the GAP family, including NF1, stimulate the with 10%o (vol/vol) fetal bovine serum (JRH Biosciences, conversion from Ras'GTP to Ras-GDP, and negatively reg- ulate the activity of Ras (13). Abbreviations: GAP, Ras GTPase activating protein; NF1-GRD, Ras is ubiquitously present among eukaryotic cells. Yeast GAP-related domain of the NFI gene product. Saccharomyces cerevisiae possesses two members of the tPresent address: National Institute of Neuroscience, Kodaira, Ras family (Rasl and Ras2) that play an important role in cell Tokyo 187, Japan. §Present address: Department of Biochemistry, Kobe University School of Medicine, Kobe 650, Japan. The publication costs of this article were defrayed in part by page charge lTo whom reprint requests should be addressed at: DNAX Research payment. This article must therefore be hereby marked "advertisement" Institute of Molecular and Cellular Biology, 901 California Avenue, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Palo Alto, CA 94304-1104. 6706 Downloaded by guest on September 30, 2021 Biochemistry: Nakafuku et al. Proc. Natl. Acad. Sci. USA 90 (1993) 6707 Lenexa, KS), 100 units of penicillin per ml, and 100 Ag of Table 1. Suppression of the heat-sensitive phenotypes of various streptomycin per ml. activated alleles of RAS2 by mutant NF1-GRD Plasmids. Construction of pKP11 was as described (5), in which wild-type NF1-GRD was cloned into the yeast expres- NF1-GRD Suppression of heat-sensitive phenotype sion vector pKT10 containing the glyceraldehyde-3-phos- type RAS2Val19 RAS2Leu68 RAS2Se14l RAS2wt phate dehydrogenase promoter (16). RAS2 plasmids, YCp- NF201 +++ +++ ++ +++ RAS2va"19, YCp-RAS2Ieu", and YCp-RAS2-er4l, were as NF204 +++ + - +++ described (20). For the expression of NF1-GRD in mamma- NF1 (wild-type) - + - + + + lian cells, 1.7-kb cDNA fragments of wild-type and two mutant NF1-GRDs, named NF201 and NF204 (see Results Wild-type S. cerevisiae RAY-3A-D, harboring a combination of RAS2 plasmids (YCp-RAS2v`al9, -RAS2Lu68, and -RAS2ser4l; ref. and Discussions), were cloned into pEF-BOS, which is a 20) and NF1-GRD plasmids, was subjected to heat-shock assay as mammalian expression vector containing human EFl-a pro- described in Fig. 1. The ability ofeach NF1-GRD plasmid to suppress moter (21), and named pEF-NF1, pEF-NF201, and pEF- the heat-sensitive phenotype was scored as follows: +++, strong NF204, respectively. A rat cDNA encoding full-length GAP suppression; + +, intermediate suppression; +, weak suppression; was also cloned into pEF-BOS to yield pEF-GAP (T. Satoh, -, no detectable suppression. The complementation activity in ira2 K. Saitoh, and Y.K., unpublished material). pKrev-1 carries cells (KT63-2B-D; ref. 17), which reflects the activity of these Krev-1 cDNA and the neomycin-resistance gene under the NFl-GRDs on wild-type RAS2 (RAS2Wt), was also scored and is control of the simian virus 40 early promoter (18). shown. Mutagenesis and Screening of Mutant NF1-GRD in Yeast Cells. pKP11 DNA (100 itg) was mutagenized by hydroxyl- expresses a domain of NF1 (aa 1063-1651; the numbers of amine in vitro as described (22) and transformed into the amino acid residues are from ref. 26) and a yeast strain RAS2Val19 strain, TK161-R2V-D. About 2 x 10W colonies carrying the RAS2VaII9 mutation. In a previous study, this were grown on selection plates, and the plates were incu- plasmid was shown to suppress ira2- but not RAS2Val"9 (5). bated at 57°C for 15 min. The resultant plates were incubated The plasmid was randomly mutagenized by treatment with at 30°C for 4 days, and growing colonies were picked. The hydroxylamine in vitro, and apool ofmutagenized DNAs was heat-shock sensitivity of these colonies was checked, and 12 transformed into RAS2Val19 cells. Subsequently, 2 x 105 clones were selected at this stage. Plasmid DNAs were colonies were screened for heat-shock resistance. Twelve recovered from these cells and retransformed into TK161- positive colonies were obtained in the initial screening. R2V-D. Phenotypic reversion was examined, and four plas- mids were found to be positive. Among them, two clones, Subsequently, two clones, NF201 and NF204, that had a NF201 and NF204, were characterized in this study. relatively strong suppression activity for RAS2Vall9 were Transfection of Plasmid DNAs into Mammalian Cells. NFl- selected and subjected to further analysis as described below.