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. 1997 Mar;17(3):1057–1064. doi: 10.1128/mcb.17.3.1057

Effect of association with adenylyl cyclase-associated protein on the interaction of yeast adenylyl cyclase with Ras protein.

F Shima 1, Y Yamawaki-Kataoka 1, C Yanagihara 1, M Tamada 1, T Okada 1, K Kariya 1, T Kataoka 1
PMCID: PMC231830  PMID: 9032232

Abstract

Posttranslational modification of Ras protein has been shown to be critical for interaction with its effector molecules, including Saccharomyces cerevisiae adenylyl cyclase. However, the mechanism of its action was unknown. In this study, we used a reconstituted system with purified adenylyl cyclase and Ras proteins carrying various degrees of the modification to show that the posttranslational modification, especially the farnesylation step, is responsible for 5- to 10-fold increase in Ras-dependent activation of adenylyl cyclase activity even though it has no significant effect on their binding affinity. The stimulatory effect of farnesylation is found to depend on the association of adenylyl cyclase with 70-kDa adenylyl cyclase-associated protein (CAP), which was known to be required for proper in vivo response of adenylyl cyclase to Ras protein, by comparing the levels of Ras-dependent activation of purified adenylyl cyclase with and without bound CAP. The region of CAP required for this effect is mapped to its N-terminal segment of 168 amino acid residues, which coincides with the region required for the in vivo effect. Furthermore, the stimulatory effect is successfully reconstituted by in vitro association of CAP with the purified adenylyl cyclase molecule lacking the bound CAP. These results indicate that the association of adenylyl cyclase with CAP is responsible for the stimulatory effect of posttranslational modification of Ras on its activity and that this may be the mechanism underlying its requirement for the proper in vivo cyclic AMP response.

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Selected References

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  1. 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]
  2. 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]
  3. Casey P. J. Lipid modifications of G proteins. Curr Opin Cell Biol. 1994 Apr;6(2):219–225. doi: 10.1016/0955-0674(94)90139-2. [DOI] [PubMed] [Google Scholar]
  4. Colicelli J., Field J., Ballester R., Chester N., Young D., Wigler M. Mutational mapping of RAS-responsive domains of the Saccharomyces cerevisiae adenylyl cyclase. Mol Cell Biol. 1990 Jun;10(6):2539–2543. doi: 10.1128/mcb.10.6.2539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fedor-Chaiken M., Deschenes R. J., Broach J. R. SRV2, a gene required for RAS activation of adenylate cyclase in yeast. Cell. 1990 Apr 20;61(2):329–340. doi: 10.1016/0092-8674(90)90813-t. [DOI] [PubMed] [Google Scholar]
  6. Field J., Vojtek A., Ballester R., Bolger G., Colicelli J., Ferguson K., Gerst J., Kataoka T., Michaeli T., Powers S. Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein. Cell. 1990 Apr 20;61(2):319–327. doi: 10.1016/0092-8674(90)90812-s. [DOI] [PubMed] [Google Scholar]
  7. Freeman N. L., Chen Z., Horenstein J., Weber A., Field J. An actin monomer binding activity localizes to the carboxyl-terminal half of the Saccharomyces cerevisiae cyclase-associated protein. J Biol Chem. 1995 Mar 10;270(10):5680–5685. doi: 10.1074/jbc.270.10.5680. [DOI] [PubMed] [Google Scholar]
  8. Freeman N. L., Lila T., Mintzer K. A., Chen Z., Pahk A. J., Ren R., Drubin D. G., Field J. A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization. Mol Cell Biol. 1996 Feb;16(2):548–556. doi: 10.1128/mcb.16.2.548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fukada Y., Takao T., Ohguro H., Yoshizawa T., Akino T., Shimonishi Y. Farnesylated gamma-subunit of photoreceptor G protein indispensable for GTP-binding. Nature. 1990 Aug 16;346(6285):658–660. doi: 10.1038/346658a0. [DOI] [PubMed] [Google Scholar]
  10. Gerst J. E., Ferguson K., Vojtek A., Wigler M., Field J. CAP is a bifunctional component of the Saccharomyces cerevisiae adenylyl cyclase complex. Mol Cell Biol. 1991 Mar;11(3):1248–1257. doi: 10.1128/mcb.11.3.1248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gerst J. E., Rodgers L., Riggs M., Wigler M. SNC1, a yeast homolog of the synaptic vesicle-associated membrane protein/synaptobrevin gene family: genetic interactions with the RAS and CAP genes. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4338–4342. doi: 10.1073/pnas.89.10.4338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Gieselmann R., Mann K. ASP-56, a new actin sequestering protein from pig platelets with homology to CAP, an adenylate cyclase-associated protein from yeast. FEBS Lett. 1992 Feb 24;298(2-3):149–153. doi: 10.1016/0014-5793(92)80043-g. [DOI] [PubMed] [Google Scholar]
  14. Hancock J. F., Paterson H., Marshall C. J. A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell. 1990 Oct 5;63(1):133–139. doi: 10.1016/0092-8674(90)90294-o. [DOI] [PubMed] [Google Scholar]
  15. Hiroyoshi M., Kaibuchi K., Kawamura S., Hata Y., Takai Y. Role of the C-terminal region of smg p21, a ras p21-like small GTP-binding protein, in membrane and smg p21 GDP/GTP exchange protein interactions. J Biol Chem. 1991 Feb 15;266(5):2962–2969. [PubMed] [Google Scholar]
  16. Horiuchi H., Kaibuchi K., Kawamura M., Matsuura Y., Suzuki N., Kuroda Y., Kataoka T., Takai Y. The posttranslational processing of ras p21 is critical for its stimulation of yeast adenylate cyclase. Mol Cell Biol. 1992 Oct;12(10):4515–4520. doi: 10.1128/mcb.12.10.4515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hu C. D., Kariya K., Tamada M., Akasaka K., Shirouzu M., Yokoyama S., Kataoka T. Cysteine-rich region of Raf-1 interacts with activator domain of post-translationally modified Ha-Ras. J Biol Chem. 1995 Dec 22;270(51):30274–30277. doi: 10.1074/jbc.270.51.30274. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Kataoka T., Broek D., Wigler M. DNA sequence and characterization of the S. cerevisiae gene encoding adenylate cyclase. Cell. 1985 Dec;43(2 Pt 1):493–505. doi: 10.1016/0092-8674(85)90179-5. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Kawamukai M., Gerst J., Field J., Riggs M., Rodgers L., Wigler M., Young D. Genetic and biochemical analysis of the adenylyl cyclase-associated protein, cap, in Schizosaccharomyces pombe. Mol Biol Cell. 1992 Feb;3(2):167–180. doi: 10.1091/mbc.3.2.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kikuchi A., Williams L. T. The post-translational modification of ras p21 is important for Raf-1 activation. J Biol Chem. 1994 Aug 5;269(31):20054–20059. [PubMed] [Google Scholar]
  23. Kobe B., Deisenhofer J. The leucine-rich repeat: a versatile binding motif. Trends Biochem Sci. 1994 Oct;19(10):415–421. doi: 10.1016/0968-0004(94)90090-6. [DOI] [PubMed] [Google Scholar]
  24. Kuroda Y., Suzuki N., Kataoka T. The effect of posttranslational modifications on the interaction of Ras2 with adenylyl cyclase. Science. 1993 Jan 29;259(5095):683–686. doi: 10.1126/science.8430318. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Lambright D. G., Sondek J., Bohm A., Skiba N. P., Hamm H. E., Sigler P. B. The 2.0 A crystal structure of a heterotrimeric G protein. Nature. 1996 Jan 25;379(6563):311–319. doi: 10.1038/379311a0. [DOI] [PubMed] [Google Scholar]
  27. Matsumoto K., Uno I., Oshima Y., Ishikawa T. Isolation and characterization of yeast mutants deficient in adenylate cyclase and cAMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2355–2359. doi: 10.1073/pnas.79.7.2355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Minato T., Wang J., Akasaka K., Okada T., Suzuki N., Kataoka T. Quantitative analysis of mutually competitive binding of human Raf-1 and yeast adenylyl cyclase to Ras proteins. J Biol Chem. 1994 Aug 19;269(33):20845–20851. [PubMed] [Google Scholar]
  29. Okada T., Masuda T., Shinkai M., Kariya K., Kataoka T. Post-translational modification of H-Ras is required for activation of, but not for association with, B-Raf. J Biol Chem. 1996 Mar 1;271(9):4671–4678. doi: 10.1074/jbc.271.9.4671. [DOI] [PubMed] [Google Scholar]
  30. Qiu M. S., Pitts A. F., Winters T. R., Green S. H. ras isoprenylation is required for ras-induced but not for NGF-induced neuronal differentiation of PC12 cells. J Cell Biol. 1991 Nov;115(3):795–808. doi: 10.1083/jcb.115.3.795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  32. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  33. Schafer W. R., Kim R., Sterne R., Thorner J., Kim S. H., Rine J. Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans. Science. 1989 Jul 28;245(4916):379–385. doi: 10.1126/science.2569235. [DOI] [PubMed] [Google Scholar]
  34. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  35. Suzuki N., Choe H. R., Nishida Y., Yamawaki-Kataoka Y., Ohnishi S., Tamaoki T., Kataoka T. Leucine-rich repeats and carboxyl terminus are required for interaction of yeast adenylate cyclase with RAS proteins. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8711–8715. doi: 10.1073/pnas.87.22.8711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Vojtek A., Haarer B., Field J., Gerst J., Pollard T. D., Brown S., Wigler M. Evidence for a functional link between profilin and CAP in the yeast S. cerevisiae. Cell. 1991 Aug 9;66(3):497–505. doi: 10.1016/0092-8674(81)90013-1. [DOI] [PubMed] [Google Scholar]
  40. Wall M. A., Coleman D. E., Lee E., Iñiguez-Lluhi J. A., Posner B. A., Gilman A. G., Sprang S. R. The structure of the G protein heterotrimer Gi alpha 1 beta 1 gamma 2. Cell. 1995 Dec 15;83(6):1047–1058. doi: 10.1016/0092-8674(95)90220-1. [DOI] [PubMed] [Google Scholar]
  41. Wang J., Suzuki N., Kataoka T. The 70-kilodalton adenylyl cyclase-associated protein is not essential for interaction of Saccharomyces cerevisiae adenylyl cyclase with RAS proteins. Mol Cell Biol. 1992 Nov;12(11):4937–4945. doi: 10.1128/mcb.12.11.4937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wang J., Suzuki N., Nishida Y., Kataoka T. Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding. Mol Cell Biol. 1993 Jul;13(7):4087–4097. doi: 10.1128/mcb.13.7.4087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Yamamori B., Kuroda S., Shimizu K., Fukui K., Ohtsuka T., Takai Y. Purification of a Ras-dependent mitogen-activated protein kinase kinase kinase from bovine brain cytosol and its identification as a complex of B-Raf and 14-3-3 proteins. J Biol Chem. 1995 May 19;270(20):11723–11726. doi: 10.1074/jbc.270.20.11723. [DOI] [PubMed] [Google Scholar]
  45. Yamawaki-Kataoka Y., Tamaoki T., Choe H. R., Tanaka H., Kataoka T. Adenylate cyclases in yeast: a comparison of the genes from Schizosaccharomyces pombe and Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5693–5697. doi: 10.1073/pnas.86.15.5693. [DOI] [PMC free article] [PubMed] [Google Scholar]

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