Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1994 Jun;14(6):3707–3718. doi: 10.1128/mcb.14.6.3707

Two types of RAS mutants that dominantly interfere with activators of RAS.

V Jung 1, W Wei 1, R Ballester 1, J Camonis 1, S Mi 1, L Van Aelst 1, M Wigler 1, D Broek 1
PMCID: PMC358738  PMID: 8196614

Abstract

In the fission yeast Schizosaccharomyces pombe, ras1 regulates both sexual development (conjugation and sporulation) and cellular morphology. Two types of dominant interfering mutants were isolated in a genetic screen for ras1 mutants that blocked sexual development. The first type of mutation, at Ser-22, analogous to the H-rasAsn-17 mutant (L. A. Feig and G. M. Cooper, Mol. Cell. Biol. 8:3235-3243, 1988), blocked only conjugation, whereas a second type of mutation, at Asp-62, interfered with conjugation, sporulation, and cellular morphology. Analogous mutations at position 64 of Saccharomyces cerevisiae RAS2 or position 57 of human H-ras also resulted in dominant interfering mutants that interfered specifically and more profoundly than mutants of the first type with RAS-associated pathways in both S. pombe or S. cerevisiae. Genetic evidence indicating that both types of interfering mutants function upstream of RAS is provided. Biochemical evidence showing that the mutants are altered in their interaction with the CDC25 class of exchange factors is presented. We show that both H-rasAsn-17 and H-rasTyr-57, compared with wild-type H-ras, are defective in their guanine nucleotide-dependent release from human cdc25 and that this defect is more severe for the H-rasTyr-57 mutant. Such a defect would allow the interfering mutants to remain bound to, thereby sequestering RAS exchange factors. The more severe interference phenotype of this novel interfering mutant suggests that it functions by titrating out other positive regulators of RAS besides those encoded by ste6 and CDC25.

Full text

PDF
3707

Images in this article

3711Image
on p.3711
3713Image
on p.3713
3714Image
on p.3714
3714Image
on p.3714

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. Ballester R., Michaeli T., Ferguson K., Xu H. P., McCormick F., Wigler M. Genetic analysis of mammalian GAP expressed in yeast. Cell. 1989 Nov 17;59(4):681–686. doi: 10.1016/0092-8674(89)90014-7. [DOI] [PubMed] [Google Scholar]
  3. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  4. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
  5. Broek D., Samiy N., Fasano O., Fujiyama A., Tamanoi F., Northup J., Wigler M. Differential activation of yeast adenylate cyclase by wild-type and mutant RAS proteins. Cell. 1985 Jul;41(3):763–769. doi: 10.1016/s0092-8674(85)80057-x. [DOI] [PubMed] [Google Scholar]
  6. Broek D., Toda T., Michaeli T., Levin L., Birchmeier C., Zoller M., Powers S., Wigler M. The S. cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway. Cell. 1987 Mar 13;48(5):789–799. doi: 10.1016/0092-8674(87)90076-6. [DOI] [PubMed] [Google Scholar]
  7. Cai H., Szeberényi J., Cooper G. M. Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells. Mol Cell Biol. 1990 Oct;10(10):5314–5323. doi: 10.1128/mcb.10.10.5314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Camonis J. H., Kalékine M., Gondré B., Garreau H., Boy-Marcotte E., Jacquet M. Characterization, cloning and sequence analysis of the CDC25 gene which controls the cyclic AMP level of Saccharomyces cerevisiae. EMBO J. 1986 Feb;5(2):375–380. doi: 10.1002/j.1460-2075.1986.tb04222.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cen H., Papageorge A. G., Zippel R., Lowy D. R., Zhang K. Isolation of multiple mouse cDNAs with coding homology to Saccharomyces cerevisiae CDC25: identification of a region related to Bcr, Vav, Dbl and CDC24. EMBO J. 1992 Nov;11(11):4007–4015. doi: 10.1002/j.1460-2075.1992.tb05494.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chardin P., Camonis J. H., Gale N. W., van Aelst L., Schlessinger J., Wigler M. H., Bar-Sagi D. Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2. Science. 1993 May 28;260(5112):1338–1343. doi: 10.1126/science.8493579. [DOI] [PubMed] [Google Scholar]
  11. Créchet J. B., Poullet P., Camonis J., Jacquet M., Parmeggiani A. Different kinetic properties of the two mutants, RAS2Ile152 and RAS2Val19, that suppress the CDC25 requirement in RAS/adenylate cyclase pathway in Saccharomyces cerevisiae. J Biol Chem. 1990 Jan 25;265(3):1563–1568. [PubMed] [Google Scholar]
  12. Deng W. P., Nickoloff J. A. Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem. 1992 Jan;200(1):81–88. doi: 10.1016/0003-2697(92)90280-k. [DOI] [PubMed] [Google Scholar]
  13. Downward J. Signal transduction. Exchange rate mechanisms. Nature. 1992 Jul 23;358(6384):282–283. doi: 10.1038/358282a0. [DOI] [PubMed] [Google Scholar]
  14. Farnsworth C. L., Feig L. A. Dominant inhibitory mutations in the Mg(2+)-binding site of RasH prevent its activation by GTP. Mol Cell Biol. 1991 Oct;11(10):4822–4829. doi: 10.1128/mcb.11.10.4822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Feig L. A., Cooper G. M. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol. 1988 Aug;8(8):3235–3243. doi: 10.1128/mcb.8.8.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Feig L. A., Pan B. T., Roberts T. M., Cooper G. M. Isolation of ras GTP-binding mutants using an in situ colony-binding assay. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4607–4611. doi: 10.1073/pnas.83.13.4607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Field J., Nikawa J., Broek D., MacDonald B., Rodgers L., Wilson I. A., Lerner R. A., Wigler M. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol. 1988 May;8(5):2159–2165. doi: 10.1128/mcb.8.5.2159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  19. Fukui Y., Kaziro Y. Molecular cloning and sequence analysis of a ras gene from Schizosaccharomyces pombe. EMBO J. 1985 Mar;4(3):687–691. doi: 10.1002/j.1460-2075.1985.tb03684.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fukui Y., Kozasa T., Kaziro Y., Takeda T., Yamamoto M. Role of a ras homolog in the life cycle of Schizosaccharomyces pombe. Cell. 1986 Jan 31;44(2):329–336. doi: 10.1016/0092-8674(86)90767-1. [DOI] [PubMed] [Google Scholar]
  21. Fukui Y., Miyake S., Satoh M., Yamamoto M. Characterization of the Schizosaccharomyces pombe ral2 gene implicated in activation of the ras1 gene product. Mol Cell Biol. 1989 Dec;9(12):5617–5622. doi: 10.1128/mcb.9.12.5617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Fukui Y., Yamamoto M. Isolation and characterization of Schizosaccharomyces pombe mutants phenotypically similar to ras1-. Mol Gen Genet. 1988 Dec;215(1):26–31. doi: 10.1007/BF00331298. [DOI] [PubMed] [Google Scholar]
  23. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  24. Gulbins E., Coggeshall K. M., Baier G., Katzav S., Burn P., Altman A. Tyrosine kinase-stimulated guanine nucleotide exchange activity of Vav in T cell activation. Science. 1993 May 7;260(5109):822–825. doi: 10.1126/science.8484124. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Hughes D. A., Fukui Y., Yamamoto M. Homologous activators of ras in fission and budding yeast. Nature. 1990 Mar 22;344(6264):355–357. doi: 10.1038/344355a0. [DOI] [PubMed] [Google Scholar]
  27. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. John J., Rensland H., Schlichting I., Vetter I., Borasio G. D., Goody R. S., Wittinghofer A. Kinetic and structural analysis of the Mg(2+)-binding site of the guanine nucleotide-binding protein p21H-ras. J Biol Chem. 1993 Jan 15;268(2):923–929. [PubMed] [Google Scholar]
  29. Kawamukai M., Ferguson K., Wigler M., Young D. Genetic and biochemical analysis of the adenylyl cyclase of Schizosaccharomyces pombe. Cell Regul. 1991 Feb;2(2):155–164. doi: 10.1091/mbc.2.2.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. LEUPOLD U. Methodisches zur Genetik von Schizosaccharomyces pombe. Schweiz Z Pathol Bakteriol. 1955;18(5):1141–1146. [PubMed] [Google Scholar]
  31. Lacal J. C., Aaronson S. A. Activation of ras p21 transforming properties associated with an increase in the release rate of bound guanine nucleotide. Mol Cell Biol. 1986 Dec;6(12):4214–4220. doi: 10.1128/mcb.6.12.4214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lai C. C., Boguski M., Broek D., Powers S. Influence of guanine nucleotides on complex formation between Ras and CDC25 proteins. Mol Cell Biol. 1993 Mar;13(3):1345–1352. doi: 10.1128/mcb.13.3.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Martegani E., Vanoni M., Zippel R., Coccetti P., Brambilla R., Ferrari C., Sturani E., Alberghina L. Cloning by functional complementation of a mouse cDNA encoding a homologue of CDC25, a Saccharomyces cerevisiae RAS activator. EMBO J. 1992 Jun;11(6):2151–2157. doi: 10.1002/j.1460-2075.1992.tb05274.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. McLeod M., Stein M., Beach D. The product of the mei3+ gene, expressed under control of the mating-type locus, induces meiosis and sporulation in fission yeast. EMBO J. 1987 Mar;6(3):729–736. doi: 10.1002/j.1460-2075.1987.tb04814.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Milburn M. V., Tong L., deVos A. M., Brünger A., Yamaizumi Z., Nishimura S., Kim S. H. Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science. 1990 Feb 23;247(4945):939–945. doi: 10.1126/science.2406906. [DOI] [PubMed] [Google Scholar]
  36. Mosteller R. D., Han J., Broek D. Identification of residues of the H-ras protein critical for functional interaction with guanine nucleotide exchange factors. Mol Cell Biol. 1994 Feb;14(2):1104–1112. doi: 10.1128/mcb.14.2.1104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Munder T., Fürst P. The Saccharomyces cerevisiae CDC25 gene product binds specifically to catalytically inactive ras proteins in vivo. Mol Cell Biol. 1992 May;12(5):2091–2099. doi: 10.1128/mcb.12.5.2091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nadin-Davis S. A., Nasim A. A gene which encodes a predicted protein kinase can restore some functions of the ras gene in fission yeast. EMBO J. 1988 Apr;7(4):985–993. doi: 10.1002/j.1460-2075.1988.tb02905.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nadin-Davis S. A., Nasim A., Beach D. Involvement of ras in sexual differentiation but not in growth control in fission yeast. EMBO J. 1986 Nov;5(11):2963–2971. doi: 10.1002/j.1460-2075.1986.tb04593.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nadin-Davis S. A., Yang R. C., Narang S. A., Nasim A. The cloning and characterization of a RAS gene from Schizosaccharomyces pombe. J Mol Evol. 1986;23(1):41–51. doi: 10.1007/BF02100997. [DOI] [PubMed] [Google Scholar]
  41. Pai E. F., Kabsch W., Krengel U., Holmes K. C., John J., Wittinghofer A. Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation. Nature. 1989 Sep 21;341(6239):209–214. doi: 10.1038/341209a0. [DOI] [PubMed] [Google Scholar]
  42. Pai E. F., Krengel U., Petsko G. A., Goody R. S., Kabsch W., Wittinghofer A. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 1990 Aug;9(8):2351–2359. doi: 10.1002/j.1460-2075.1990.tb07409.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Powers S., Gonzales E., Christensen T., Cubert J., Broek D. Functional cloning of BUD5, a CDC25-related gene from S. cerevisiae that can suppress a dominant-negative RAS2 mutant. Cell. 1991 Jun 28;65(7):1225–1231. doi: 10.1016/0092-8674(91)90017-s. [DOI] [PubMed] [Google Scholar]
  44. Powers S., Kataoka T., Fasano O., Goldfarb M., Strathern J., Broach J., Wigler M. Genes in S. cerevisiae encoding proteins with domains homologous to the mammalian ras proteins. Cell. 1984 Mar;36(3):607–612. doi: 10.1016/0092-8674(84)90340-4. [DOI] [PubMed] [Google Scholar]
  45. Powers S., Michaelis S., Broek D., Santa Anna S., Field J., Herskowitz I., Wigler M. RAM, a gene of yeast required for a functional modification of RAS proteins and for production of mating pheromone a-factor. Cell. 1986 Nov 7;47(3):413–422. doi: 10.1016/0092-8674(86)90598-2. [DOI] [PubMed] [Google Scholar]
  46. Powers S., O'Neill K., Wigler M. Dominant yeast and mammalian RAS mutants that interfere with the CDC25-dependent activation of wild-type RAS in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Feb;9(2):390–395. doi: 10.1128/mcb.9.2.390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sass P., Field J., Nikawa J., Toda T., Wigler M. Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9303–9307. doi: 10.1073/pnas.83.24.9303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Shou C., Farnsworth C. L., Neel B. G., Feig L. A. Molecular cloning of cDNAs encoding a guanine-nucleotide-releasing factor for Ras p21. Nature. 1992 Jul 23;358(6384):351–354. doi: 10.1038/358351a0. [DOI] [PubMed] [Google Scholar]
  50. Sigal I. S., Gibbs J. B., D'Alonzo J. S., Scolnick E. M. Identification of effector residues and a neutralizing epitope of Ha-ras-encoded p21. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4725–4729. doi: 10.1073/pnas.83.13.4725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Stotz A., Linder P. The ADE2 gene from Saccharomyces cerevisiae: sequence and new vectors. Gene. 1990 Oct 30;95(1):91–98. doi: 10.1016/0378-1119(90)90418-q. [DOI] [PubMed] [Google Scholar]
  52. Szeberényi J., Cai H., Cooper G. M. Effect of a dominant inhibitory Ha-ras mutation on neuronal differentiation of PC12 cells. Mol Cell Biol. 1990 Oct;10(10):5324–5332. doi: 10.1128/mcb.10.10.5324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Toda T., Uno I., Ishikawa T., Powers S., Kataoka T., Broek D., Cameron S., Broach J., Matsumoto K., Wigler M. In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell. 1985 Jan;40(1):27–36. doi: 10.1016/0092-8674(85)90305-8. [DOI] [PubMed] [Google Scholar]
  54. Van Aelst L., Barr M., Marcus S., Polverino A., Wigler M. Complex formation between RAS and RAF and other protein kinases. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6213–6217. doi: 10.1073/pnas.90.13.6213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Vojtek A. B., Hollenberg S. M., Cooper J. A. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 1993 Jul 16;74(1):205–214. doi: 10.1016/0092-8674(93)90307-c. [DOI] [PubMed] [Google Scholar]
  56. Wang Y., Xu H. P., Riggs M., Rodgers L., Wigler M. byr2, a Schizosaccharomyces pombe gene encoding a protein kinase capable of partial suppression of the ras1 mutant phenotype. Mol Cell Biol. 1991 Jul;11(7):3554–3563. doi: 10.1128/mcb.11.7.3554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wei W., Mosteller R. D., Sanyal P., Gonzales E., McKinney D., Dasgupta C., Li P., Liu B. X., Broek D. Identification of a mammalian gene structurally and functionally related to the CDC25 gene of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7100–7104. doi: 10.1073/pnas.89.15.7100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Willumsen B. M., Christensen A., Hubbert N. L., Papageorge A. G., Lowy D. R. The p21 ras C-terminus is required for transformation and membrane association. Nature. 1984 Aug 16;310(5978):583–586. doi: 10.1038/310583a0. [DOI] [PubMed] [Google Scholar]
  59. Willumsen B. M., Norris K., Papageorge A. G., Hubbert N. L., Lowy D. R. Harvey murine sarcoma virus p21 ras protein: biological and biochemical significance of the cysteine nearest the carboxy terminus. EMBO J. 1984 Nov;3(11):2581–2585. doi: 10.1002/j.1460-2075.1984.tb02177.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Willumsen B. M., Papageorge A. G., Kung H. F., Bekesi E., Robins T., Johnsen M., Vass W. C., Lowy D. R. Mutational analysis of a ras catalytic domain. Mol Cell Biol. 1986 Jul;6(7):2646–2654. doi: 10.1128/mcb.6.7.2646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wolfman A., Macara I. G. A cytosolic protein catalyzes the release of GDP from p21ras. Science. 1990 Apr 6;248(4951):67–69. doi: 10.1126/science.2181667. [DOI] [PubMed] [Google Scholar]
  62. Zhou Y. H., Zhang X. P., Ebright R. H. Random mutagenesis of gene-sized DNA molecules by use of PCR with Taq DNA polymerase. Nucleic Acids Res. 1991 Nov 11;19(21):6052–6052. doi: 10.1093/nar/19.21.6052. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES