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. 1993 Jul 15;90(14):6706–6710. doi: 10.1073/pnas.90.14.6706

Suppression of oncogenic Ras by mutant neurofibromatosis type 1 genes with single amino acid substitutions.

M Nakafuku 1, M Nagamine 1, A Ohtoshi 1, K Tanaka 1, A Toh-e 1, Y Kaziro 1
PMCID: PMC47001  PMID: 8341688

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ballester R., Marchuk D., Boguski M., Saulino A., Letcher R., Wigler M., Collins F. The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins. Cell. 1990 Nov 16;63(4):851–859. doi: 10.1016/0092-8674(90)90151-4. [DOI] [PubMed] [Google Scholar]
  2. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  3. Bollag G., McCormick F. Differential regulation of rasGAP and neurofibromatosis gene product activities. Nature. 1991 Jun 13;351(6327):576–579. doi: 10.1038/351576a0. [DOI] [PubMed] [Google Scholar]
  4. Bollag G., McCormick F. Regulators and effectors of ras proteins. Annu Rev Cell Biol. 1991;7:601–632. doi: 10.1146/annurev.cb.07.110191.003125. [DOI] [PubMed] [Google Scholar]
  5. Bos J. L. ras oncogenes in human cancer: a review. Cancer Res. 1989 Sep 1;49(17):4682–4689. [PubMed] [Google Scholar]
  6. Broach J. R., Deschenes R. J. The function of ras genes in Saccharomyces cerevisiae. Adv Cancer Res. 1990;54:79–139. doi: 10.1016/s0065-230x(08)60809-x. [DOI] [PubMed] [Google Scholar]
  7. Buchberg A. M., Cleveland L. S., Jenkins N. A., Copeland N. G. Sequence homology shared by neurofibromatosis type-1 gene and IRA-1 and IRA-2 negative regulators of the RAS cyclic AMP pathway. Nature. 1990 Sep 20;347(6290):291–294. doi: 10.1038/347291a0. [DOI] [PubMed] [Google Scholar]
  8. Gibbs J. B., Marshall M. S. The ras oncogene--an important regulatory element in lower eucaryotic organisms. Microbiol Rev. 1989 Jun;53(2):171–185. doi: 10.1128/mr.53.2.171-185.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  10. Gutmann D. H., Boguski M., Marchuk D., Wigler M., Collins F. S., Ballester R. Analysis of the neurofibromatosis type 1 (NF1) GAP-related domain by site-directed mutagenesis. Oncogene. 1993 Mar;8(3):761–769. [PubMed] [Google Scholar]
  11. Gutmann D. H., Collins F. S. Recent progress toward understanding the molecular biology of von Recklinghausen neurofibromatosis. Ann Neurol. 1992 May;31(5):555–561. doi: 10.1002/ana.410310515. [DOI] [PubMed] [Google Scholar]
  12. Hattori S., Maekawa M., Nakamura S. Identification of neurofibromatosis type I gene product as an insoluble GTPase-activating protein toward ras p21. Oncogene. 1992 Mar;7(3):481–485. [PubMed] [Google Scholar]
  13. Imai Y., Miyake S., Hughes D. A., Yamamoto M. Identification of a GTPase-activating protein homolog in Schizosaccharomyces pombe. Mol Cell Biol. 1991 Jun;11(6):3088–3094. doi: 10.1128/mcb.11.6.3088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kaziro Y., Itoh H., Kozasa T., Nakafuku M., Satoh T. Structure and function of signal-transducing GTP-binding proteins. Annu Rev Biochem. 1991;60:349–400. doi: 10.1146/annurev.bi.60.070191.002025. [DOI] [PubMed] [Google Scholar]
  15. Kitamura T., Sato N., Arai K., Miyajima A. Expression cloning of the human IL-3 receptor cDNA reveals a shared beta subunit for the human IL-3 and GM-CSF receptors. Cell. 1991 Sep 20;66(6):1165–1174. doi: 10.1016/0092-8674(91)90039-2. [DOI] [PubMed] [Google Scholar]
  16. Kitayama H., Matsuzaki T., Ikawa Y., Noda M. Genetic analysis of the Kirsten-ras-revertant 1 gene: potentiation of its tumor suppressor activity by specific point mutations. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4284–4288. doi: 10.1073/pnas.87.11.4284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Li Y., Bollag G., Clark R., Stevens J., Conroy L., Fults D., Ward K., Friedman E., Samowitz W., Robertson M. Somatic mutations in the neurofibromatosis 1 gene in human tumors. Cell. 1992 Apr 17;69(2):275–281. doi: 10.1016/0092-8674(92)90408-5. [DOI] [PubMed] [Google Scholar]
  18. Marchuk D. A., Saulino A. M., Tavakkol R., Swaroop M., Wallace M. R., Andersen L. B., Mitchell A. L., Gutmann D. H., Boguski M., Collins F. S. cDNA cloning of the type 1 neurofibromatosis gene: complete sequence of the NF1 gene product. Genomics. 1991 Dec;11(4):931–940. doi: 10.1016/0888-7543(91)90017-9. [DOI] [PubMed] [Google Scholar]
  19. Martin G. A., Viskochil D., Bollag G., McCabe P. C., Crosier W. J., Haubruck H., Conroy L., Clark R., O'Connell P., Cawthon R. M. The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell. 1990 Nov 16;63(4):843–849. doi: 10.1016/0092-8674(90)90150-d. [DOI] [PubMed] [Google Scholar]
  20. Mizushima S., Nagata S. pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 1990 Sep 11;18(17):5322–5322. doi: 10.1093/nar/18.17.5322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nishi T., Saya H. Neurofibromatosis type 1 (NF1) gene: implication in neuroectodermal differentiation and genesis of brain tumors. Cancer Metastasis Rev. 1991 Dec;10(4):301–310. doi: 10.1007/BF00554792. [DOI] [PubMed] [Google Scholar]
  22. Rodenhuis S. ras and human tumors. Semin Cancer Biol. 1992 Aug;3(4):241–247. [PubMed] [Google Scholar]
  23. Rose M. D., Fink G. R. KAR1, a gene required for function of both intranuclear and extranuclear microtubules in yeast. Cell. 1987 Mar 27;48(6):1047–1060. doi: 10.1016/0092-8674(87)90712-4. [DOI] [PubMed] [Google Scholar]
  24. Satoh T., Nakafuku M., Kaziro Y. Function of Ras as a molecular switch in signal transduction. J Biol Chem. 1992 Dec 5;267(34):24149–24152. [PubMed] [Google Scholar]
  25. Satoh T., Nakamura S., Nakafuku M., Kaziro Y. Studies on ras proteins. Catalytic properties of normal and activated ras proteins purified in the absence of protein denaturants. Biochim Biophys Acta. 1988 Jan 25;949(1):97–109. doi: 10.1016/0167-4781(88)90059-0. [DOI] [PubMed] [Google Scholar]
  26. Schweighoffer F., Barlat I., Chevallier-Multon M. C., Tocque B. Implication of GAP in Ras-dependent transactivation of a polyoma enhancer sequence. Science. 1992 May 8;256(5058):825–827. doi: 10.1126/science.1317056. [DOI] [PubMed] [Google Scholar]
  27. Tanaka K., Matsumoto K., Toh-E A. IRA1, an inhibitory regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Feb;9(2):757–768. doi: 10.1128/mcb.9.2.757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tanaka K., Nakafuku M., Tamanoi F., Kaziro Y., Matsumoto K., Toh-e A. IRA2, a second gene of Saccharomyces cerevisiae that encodes a protein with a domain homologous to mammalian ras GTPase-activating protein. Mol Cell Biol. 1990 Aug;10(8):4303–4313. doi: 10.1128/mcb.10.8.4303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tanaka K., Wood D. R., Lin B. K., Khalil M., Tamanoi F., Cannon J. F. A dominant activating mutation in the effector region of RAS abolishes IRA2 sensitivity. Mol Cell Biol. 1992 Feb;12(2):631–637. doi: 10.1128/mcb.12.2.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Trahey M., Wong G., Halenbeck R., Rubinfeld B., Martin G. A., Ladner M., Long C. M., Crosier W. J., Watt K., Koths K. Molecular cloning of two types of GAP complementary DNA from human placenta. Science. 1988 Dec 23;242(4886):1697–1700. doi: 10.1126/science.3201259. [DOI] [PubMed] [Google Scholar]
  31. White R., Viskochil D., O'Connell P. Identification and characterization of the gene for neurofibromatosis type 1. Curr Opin Neurobiol. 1991 Oct;1(3):462–467. doi: 10.1016/0959-4388(91)90070-n. [DOI] [PubMed] [Google Scholar]
  32. Wiesmüller L., Wittinghofer A. Expression of the GTPase activating domain of the neurofibromatosis type 1 (NF1) gene in Escherichia coli and role of the conserved lysine residue. J Biol Chem. 1992 May 25;267(15):10207–10210. [PubMed] [Google Scholar]
  33. Xu G. F., Lin B., Tanaka K., Dunn D., Wood D., Gesteland R., White R., Weiss R., Tamanoi F. The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae. Cell. 1990 Nov 16;63(4):835–841. doi: 10.1016/0092-8674(90)90149-9. [DOI] [PubMed] [Google Scholar]
  34. Zhang K., DeClue J. E., Vass W. C., Papageorge A. G., McCormick F., Lowy D. R. Suppression of c-ras transformation by GTPase-activating protein. Nature. 1990 Aug 23;346(6286):754–756. doi: 10.1038/346754a0. [DOI] [PubMed] [Google Scholar]

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