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. 1992 Jun;11(6):2151–2157. doi: 10.1002/j.1460-2075.1992.tb05274.x

Cloning by functional complementation of a mouse cDNA encoding a homologue of CDC25, a Saccharomyces cerevisiae RAS activator.

E Martegani 1, M Vanoni 1, R Zippel 1, P Coccetti 1, R Brambilla 1, C Ferrari 1, E Sturani 1, L Alberghina 1
PMCID: PMC556682  PMID: 1376246

Abstract

In the yeast Saccharomyces cerevisiae genetic and biochemical evidence indicates that the product of the CDC25 gene activates the RAS/adenylyl cyclase/protein kinase A pathway by acting as a guanine nucleotide protein. Here we report the isolation of a mouse brain cDNA homologous to CDC25. The mouse cDNA, called CDC25Mm, complements specifically point mutations and deletion/disruptions of the CDC25 gene. In addition, it restores the cAMP levels and CDC25-dependent glucose-induced cAMP signalling in a yeast strain bearing a disruption of the CDC25 gene. The CDC25Mm-encoded protein is 34% identical with the catalytic carboxy terminal part of the CDC25 protein and shares significant homology with other proteins belonging to the same family. The protein encoded by CDC25Mm, prepared as a glutathione S-transferase fusion in Escherichia coli cells, activates adenylyl cyclase in yeast membranes in a RAS2-dependent manner. Northern blot analysis of mouse brain poly(A)+ RNA reveals two major transcripts of approximately 1700 and 5200 nucleotides. Transcripts were found also in mouse heart and at a lower level in liver and spleen.

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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., 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]
  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. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  4. Boutelet F., Petitjean A., Hilger F. Yeast cdc35 mutants are defective in adenylate cyclase and are allelic with cyr1 mutants while CAS1, a new gene, is involved in the regulation of adenylate cyclase. EMBO J. 1985 Oct;4(10):2635–2641. doi: 10.1002/j.1460-2075.1985.tb03981.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  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. 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]
  8. Chant J., Corrado K., Pringle J. R., Herskowitz I. Yeast BUD5, encoding a putative GDP-GTP exchange factor, is necessary for bud site selection and interacts with bud formation gene BEM1. Cell. 1991 Jun 28;65(7):1213–1224. doi: 10.1016/0092-8674(91)90016-r. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Créchet J. B., Poullet P., Mistou M. Y., Parmeggiani A., Camonis J., Boy-Marcotte E., Damak F., Jacquet M. Enhancement of the GDP-GTP exchange of RAS proteins by the carboxyl-terminal domain of SCD25. Science. 1990 May 18;248(4957):866–868. doi: 10.1126/science.2188363. [DOI] [PubMed] [Google Scholar]
  11. Damak F., Boy-Marcotte E., Le-Roscouet D., Guilbaud R., Jacquet M. SDC25, a CDC25-like gene which contains a RAS-activating domain and is a dispensable gene of Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jan;11(1):202–212. doi: 10.1128/mcb.11.1.202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. De Vendittis E., Vitelli A., Zahn R., Fasano O. Suppression of defective RAS1 and RAS2 functions in yeast by an adenylate cyclase activated by a single amino acid change. EMBO J. 1986 Dec 20;5(13):3657–3663. doi: 10.1002/j.1460-2075.1986.tb04696.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. DeFeo-Jones D., Tatchell K., Robinson L. C., Sigal I. S., Vass W. C., Lowy D. R., Scolnick E. M. Mammalian and yeast ras gene products: biological function in their heterologous systems. Science. 1985 Apr 12;228(4696):179–184. doi: 10.1126/science.3883495. [DOI] [PubMed] [Google Scholar]
  14. Feig L. A., Cooper G. M. Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated ras proteins. Mol Cell Biol. 1988 Jun;8(6):2472–2478. doi: 10.1128/mcb.8.6.2472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Frascotti G., Coccetti P., Vanoni M. A., Alberghina L., Martegani E. The overexpression of the 3' terminal region of the CDC25 gene of Saccharomyces cerevisiae causes growth inhibition and alteration of purine nucleotides pools. Biochim Biophys Acta. 1991 Jun 13;1089(2):206–212. doi: 10.1016/0167-4781(91)90009-b. [DOI] [PubMed] [Google Scholar]
  16. Garreau H., Camonis J. H., Guitton C., Jacquet M. The Saccharomyces cerevisiae CDC25 gene product is a 180 kDa polypeptide and is associated with a membrane fraction. FEBS Lett. 1990 Aug 20;269(1):53–59. doi: 10.1016/0014-5793(90)81117-7. [DOI] [PubMed] [Google Scholar]
  17. Gibbs J. B., Schaber M. D., Marshall M. S., Scolnick E. M., Sigal I. S. Identification of guanine nucleotides bound to ras-encoded proteins in growing yeast cells. J Biol Chem. 1987 Aug 5;262(22):10426–10429. [PubMed] [Google Scholar]
  18. 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]
  19. Gribskov M., Burgess R. R. Sigma factors from E. coli, B. subtilis, phage SP01, and phage T4 are homologous proteins. Nucleic Acids Res. 1986 Aug 26;14(16):6745–6763. doi: 10.1093/nar/14.16.6745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  21. Huang Y. K., Kung H. F., Kamata T. Purification of a factor capable of stimulating the guanine nucleotide exchange reaction of ras proteins and its effect on ras-related small molecular mass G proteins. Proc Natl Acad Sci U S A. 1990 Oct;87(20):8008–8012. doi: 10.1073/pnas.87.20.8008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Jones S., Vignais M. L., Broach J. R. The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras. Mol Cell Biol. 1991 May;11(5):2641–2646. doi: 10.1128/mcb.11.5.2641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kaspar P., Fábry M., Sedlácek J., Zadrazil S. Expression and properties of prochymosin derivatives containing extensions of various length in the pro-part. Gene. 1988 Jul 15;67(1):131–136. doi: 10.1016/0378-1119(88)90016-9. [DOI] [PubMed] [Google Scholar]
  26. Kataoka T., Powers S., Cameron S., Fasano O., Goldfarb M., Broach J., Wigler M. Functional homology of mammalian and yeast RAS genes. Cell. 1985 Jan;40(1):19–26. doi: 10.1016/0092-8674(85)90304-6. [DOI] [PubMed] [Google Scholar]
  27. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  28. Levitzki A. Transmembrane signalling to adenylate cyclase in mammalian cells and in Saccharomyces cerevisiae. Trends Biochem Sci. 1988 Aug;13(8):298–301. doi: 10.1016/0968-0004(88)90122-3. [DOI] [PubMed] [Google Scholar]
  29. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  30. Martegani E., Baroni M., Wanoni M. Interaction of cAMP with the CDC25-mediated step in the cell cycle of budding yeast. Exp Cell Res. 1986 Feb;162(2):544–548. doi: 10.1016/0014-4827(86)90358-7. [DOI] [PubMed] [Google Scholar]
  31. Martegani Enzo, Baroni Maurizio D., Frascotti Gianni, Alberghina Lilia. Molecular cloning and transcriptional analysis of the start gene CDC25 of Saccharomyces cerevisiae. EMBO J. 1986 Sep;5(9):2363–2369. doi: 10.1002/j.1460-2075.1986.tb04505.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Matsumoto K., Uno I., Ishikawa T. Control of cell division in Saccharomyces cerevisiae mutants defective in adenylate cyclase and cAMP-dependent protein kinase. Exp Cell Res. 1983 Jun;146(1):151–161. doi: 10.1016/0014-4827(83)90333-6. [DOI] [PubMed] [Google Scholar]
  33. Mbonyi K., Beullens M., Detremerie K., Geerts L., Thevelein J. M. Requirement of one functional RAS gene and inability of an oncogenic ras variant to mediate the glucose-induced cyclic AMP signal in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1988 Aug;8(8):3051–3057. doi: 10.1128/mcb.8.8.3051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mbonyi K., van Aelst L., Argüelles J. C., Jans A. W., Thevelein J. M. Glucose-induced hyperaccumulation of cyclic AMP and defective glucose repression in yeast strains with reduced activity of cyclic AMP-dependent protein kinase. Mol Cell Biol. 1990 Sep;10(9):4518–4523. doi: 10.1128/mcb.10.9.4518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Robinson L. C., Gibbs J. B., Marshall M. S., Sigal I. S., Tatchell K. CDC25: a component of the RAS-adenylate cyclase pathway in Saccharomyces cerevisiae. Science. 1987 Mar 6;235(4793):1218–1221. doi: 10.1126/science.3547648. [DOI] [PubMed] [Google Scholar]
  39. Russell P., Nurse P. Schizosaccharomyces pombe and Saccharomyces cerevisiae: a look at yeasts divided. Cell. 1986 Jun 20;45(6):781–782. doi: 10.1016/0092-8674(86)90550-7. [DOI] [PubMed] [Google Scholar]
  40. Schomerus C., Munder T., Küntzel H. Site-directed mutagenesis of the Saccharomyces cerevisiae CDC25 gene: effects on mitotic growth and cAMP signalling. Mol Gen Genet. 1990 Sep;223(3):426–432. doi: 10.1007/BF00264449. [DOI] [PubMed] [Google Scholar]
  41. Sigal I. S., Gibbs J. B., D'Alonzo J. S., Temeles G. L., Wolanski B. S., Socher S. H., Scolnick E. M. Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects. Proc Natl Acad Sci U S A. 1986 Feb;83(4):952–956. doi: 10.1073/pnas.83.4.952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Simon M. A., Bowtell D. D., Dodson G. S., Laverty T. R., Rubin G. M. Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase. Cell. 1991 Nov 15;67(4):701–716. doi: 10.1016/0092-8674(91)90065-7. [DOI] [PubMed] [Google Scholar]
  43. Tanaka K., Nakafuku M., Satoh T., Marshall M. S., Gibbs J. B., Matsumoto K., Kaziro Y., Toh-e A. S. cerevisiae genes IRA1 and IRA2 encode proteins that may be functionally equivalent to mammalian ras GTPase activating protein. Cell. 1990 Mar 9;60(5):803–807. doi: 10.1016/0092-8674(90)90094-u. [DOI] [PubMed] [Google Scholar]
  44. Tatchell K., Robinson L. C., Breitenbach M. RAS2 of Saccharomyces cerevisiae is required for gluconeogenic growth and proper response to nutrient limitation. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3785–3789. doi: 10.1073/pnas.82.11.3785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Thevelein J. M. Fermentable sugars and intracellular acidification as specific activators of the RAS-adenylate cyclase signalling pathway in yeast: the relationship to nutrient-induced cell cycle control. Mol Microbiol. 1991 Jun;5(6):1301–1307. doi: 10.1111/j.1365-2958.1991.tb00776.x. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Van Aelst L., Boy-Marcotte E., Camonis J. H., Thevelein J. M., Jacquet M. The C-terminal part of the CDC25 gene product plays a key role in signal transduction in the glucose-induced modulation of cAMP level in Saccharomyces cerevisiae. Eur J Biochem. 1990 Nov 13;193(3):675–680. doi: 10.1111/j.1432-1033.1990.tb19386.x. [DOI] [PubMed] [Google Scholar]
  48. Vanoni M., Vavassori M., Frascotti G., Martegani E., Alberghina L. Overexpression of the CDC25 gene, an upstream element of the RAS/adenylyl cyclase pathway in Saccharomyces cerevisiae, allows immunological identification and characterization of its gene product. Biochem Biophys Res Commun. 1990 Oct 15;172(1):61–69. doi: 10.1016/s0006-291x(05)80173-1. [DOI] [PubMed] [Google Scholar]
  49. Walter M., Clark S. G., Levinson A. D. The oncogenic activation of human p21ras by a novel mechanism. Science. 1986 Aug 8;233(4764):649–652. doi: 10.1126/science.3487832. [DOI] [PubMed] [Google Scholar]
  50. Wickner R. B., Koh T. J., Crowley J. C., O'Neil J., Kaback D. B. Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperatures. Yeast. 1987 Mar;3(1):51–57. doi: 10.1002/yea.320030108. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. van Aelst L., Jans A. W., Thevelein J. M. Involvement of the CDC25 gene product in the signal transmission pathway of the glucose-induced RAS-mediated cAMP signal in the yeast Saccharomyces cerevisiae. J Gen Microbiol. 1991 Feb;137(2):341–349. doi: 10.1099/00221287-137-2-341. [DOI] [PubMed] [Google Scholar]

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