Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1995 Nov 21;92(24):11009–11013. doi: 10.1073/pnas.92.24.11009

Differential activation of yeast adenylyl cyclase by Ras1 and Ras2 depends on the conserved N terminus.

N Hurwitz 1, M Segal 1, I Marbach 1, A Levitzki 1
PMCID: PMC40560  PMID: 7479926

Abstract

Although both Ras1 and Ras2 activate adenylyl cyclase in yeast, a number of differences can be observed regarding their function in the cAMP pathway. To explore the relative contribution of conserved and variable domains in determining these differences, chimeric RAS1-RAS2 or RAS2-RAS1 genes were constructed by swapping the sequences encoding the variable C-terminal domains. These constructs were expressed in a cdc25ts ras1 ras2 strain. Biochemical data show that the difference in efficacy of adenylyl cyclase activation between the two Ras proteins resides in the highly conserved N-terminal domain. This finding is supported by the observation that Ras2 delta, in which the C-terminal domain of Ras2 has been deleted, is a more potent activator of the yeast adenylyl cyclase than Ras1 delta, in which the C-terminal domain of Ras1 has been deleted. These observations suggest that amino acid residues other than the highly conserved residues of the effector domain within the N terminus may determine the efficiency of functional interaction with adenylyl cyclase. Similar levels of intracellular cAMP were found in Ras1, Ras1-Ras2, Ras1 delta, Ras2, and Ras2-Ras1 strains throughout the growth curve. This was found to result from the higher expression of Ras1 and Ras1-Ras2, which compensate for their lower efficacy in activating adenylyl cyclase. These results suggest that the difference between the Ras1 and the Ras2 phenotype is not due to their different efficacy in activating the cAMP pathway and that the divergent C-terminal domains are responsible for these differences, through interaction with other regulatory elements.

Full text

PDF
11009

Images in this article

11011Fig. 2
on p.11011
11012Fig. 5
on p.11012

Selected References

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

  1. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  2. Boguski M. S., McCormick F. Proteins regulating Ras and its relatives. Nature. 1993 Dec 16;366(6456):643–654. doi: 10.1038/366643a0. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Camonis J. H., Jacquet M. A new RAS mutation that suppresses the CDC25 gene requirement for growth of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jul;8(7):2980–2983. doi: 10.1128/mcb.8.7.2980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clarke S. Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu Rev Biochem. 1992;61:355–386. doi: 10.1146/annurev.bi.61.070192.002035. [DOI] [PubMed] [Google Scholar]
  7. DeFeo-Jones D., Scolnick E. M., Koller R., Dhar R. ras-Related gene sequences identified and isolated from Saccharomyces cerevisiae. Nature. 1983 Dec 15;306(5944):707–709. doi: 10.1038/306707a0. [DOI] [PubMed] [Google Scholar]
  8. Engelberg D., Klein C., Martinetto H., Struhl K., Karin M. The UV response involving the Ras signaling pathway and AP-1 transcription factors is conserved between yeast and mammals. Cell. 1994 May 6;77(3):381–390. doi: 10.1016/0092-8674(94)90153-8. [DOI] [PubMed] [Google Scholar]
  9. Engelberg D., Simchen G., Levitzki A. In vitro reconstitution of cdc25 regulated S. cerevisiae adenylyl cyclase and its kinetic properties. EMBO J. 1990 Mar;9(3):641–651. doi: 10.1002/j.1460-2075.1990.tb08156.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Goldberg D., Segal M., Levitzki A. Cdc25 is not the signal receiver for glucose induced cAMP response in S. cerevisiae. FEBS Lett. 1994 Dec 19;356(2-3):249–254. doi: 10.1016/0014-5793(94)01273-3. [DOI] [PubMed] [Google Scholar]
  12. Gross E., Marbach I., Engelberg D., Segal M., Simchen G., Levitzki A. Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein. Mol Cell Biol. 1992 Jun;12(6):2653–2661. doi: 10.1128/mcb.12.6.2653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Kataoka T., Powers S., McGill C., Fasano O., Strathern J., Broach J., Wigler M. Genetic analysis of yeast RAS1 and RAS2 genes. Cell. 1984 Jun;37(2):437–445. doi: 10.1016/0092-8674(84)90374-x. [DOI] [PubMed] [Google Scholar]
  15. Kikuchi A., Demo S. D., Ye Z. H., Chen Y. W., Williams L. T. ralGDS family members interact with the effector loop of ras p21. Mol Cell Biol. 1994 Nov;14(11):7483–7491. doi: 10.1128/mcb.14.11.7483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Lowy D. R., Willumsen B. M. Function and regulation of ras. Annu Rev Biochem. 1993;62:851–891. doi: 10.1146/annurev.bi.62.070193.004223. [DOI] [PubMed] [Google Scholar]
  19. Marshall M. S., Gibbs J. B., Scolnick E. M., Sigal I. S. Regulatory function of the Saccharomyces cerevisiae RAS C-terminus. Mol Cell Biol. 1987 Jul;7(7):2309–2315. doi: 10.1128/mcb.7.7.2309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Rodriguez-Viciana P., Warne P. H., Dhand R., Vanhaesebroeck B., Gout I., Fry M. J., Waterfield M. D., Downward J. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature. 1994 Aug 18;370(6490):527–532. doi: 10.1038/370527a0. [DOI] [PubMed] [Google Scholar]
  22. Segal M., Marbach I., Engelberg D., Simchen G., Levitzki A. Interaction between the Saccharomyces cerevisiae CDC25 gene product and mammalian ras. J Biol Chem. 1992 Nov 15;267(32):22747–22751. [PubMed] [Google Scholar]
  23. Segal M., Willumsen B. M., Levitzki A. Residues crucial for Ras interaction with GDP-GTP exchangers. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5564–5568. doi: 10.1073/pnas.90.12.5564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sun J., Kale S. P., Childress A. M., Pinswasdi C., Jazwinski S. M. Divergent roles of RAS1 and RAS2 in yeast longevity. J Biol Chem. 1994 Jul 15;269(28):18638–18645. [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. Tatchell K., Chaleff D. T., DeFeo-Jones D., Scolnick E. M. Requirement of either of a pair of ras-related genes of Saccharomyces cerevisiae for spore viability. Nature. 1984 Jun 7;309(5968):523–527. doi: 10.1038/309523a0. [DOI] [PubMed] [Google Scholar]
  30. Toda T., Cameron S., Sass P., Zoller M., Wigler M. Three different genes in S. cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase. Cell. 1987 Jul 17;50(2):277–287. doi: 10.1016/0092-8674(87)90223-6. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES