Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1992 Jun;12(6):2606–2615. doi: 10.1128/mcb.12.6.2606

Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein.

A D Cox 1, M M Hisaka 1, J E Buss 1, C J Der 1
PMCID: PMC364454  PMID: 1375323

Abstract

While the Ras C-terminal CAAX sequence signals modification by a 15-carbon farnesyl isoprenoid, the majority of isoprenylated proteins in mammalian cells are modified instead by a 20-carbon geranylgeranyl moiety. To determine the structural and functional basis for modification of proteins by a specific isoprenoid group, we have generated chimeric Ras proteins containing C-terminal CAAX sequences (CVLL and CAIL) from geranylgeranyl-modified proteins and a chimeric Krev-1 protein containing the H-Ras C-terminal CAAX sequence (CVLS). Our results demonstrate that both oncogenic Ras transforming activity and Krev-1 antagonism of Ras transforming activity can be promoted by either farnesyl or geranylgeranyl modification. Similarly, geranylgeranyl-modified normal Ras [Ras(WT)CVLL], when overexpressed, exhibited the same level of transforming activity as the authentic farnesyl-modified normal Ras protein. Therefore, farnesyl and geranylgeranyl moieties are functionally interchangeable for these biological activities. In contrast, expression of moderate levels of geranylgeranyl-modified normal Ras inhibited the growth of untransformed NIH 3T3 cells. This growth inhibition was overcome by coexpression of the mutant protein with oncogenic Ras or Raf, but not with oncogenic Src or normal Ras. The similar growth-inhibiting activities of Ras(WT)CVLL and the previously described Ras(17N) dominant inhibitory mutant suggest that geranylgeranyl-modified normal Ras may exert its growth-inhibiting action by perturbing endogenous Ras function. These results suggest that normal Ras function may specifically require protein modification by a farnesyl, but not a geranylgeranyl, isoprenoid.

Full text

PDF
2606

Images in this article

2608Image
on p.2608
2608Image
on p.2608
2609Image
on p.2609
2610Image
on p.2610
2611Image
on p.2611
2612Image
on p.2612
2613Image
on p.2613

Selected References

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

  1. Buss J. E., Quilliam L. A., Kato K., Casey P. J., Solski P. A., Wong G., Clark R., McCormick F., Bokoch G. M., Der C. J. The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein. Mol Cell Biol. 1991 Mar;11(3):1523–1530. doi: 10.1128/mcb.11.3.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Buss J. E., Solski P. A., Schaeffer J. P., MacDonald M. J., Der C. J. Activation of the cellular proto-oncogene product p21Ras by addition of a myristylation signal. Science. 1989 Mar 24;243(4898):1600–1603. doi: 10.1126/science.2648572. [DOI] [PubMed] [Google Scholar]
  3. Casey P. J., Solski P. A., Der C. J., Buss J. E. p21ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8323–8327. doi: 10.1073/pnas.86.21.8323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Casey P. J., Thissen J. A., Moomaw J. F. Enzymatic modification of proteins with a geranylgeranyl isoprenoid. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8631–8635. doi: 10.1073/pnas.88.19.8631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Der C. J., Cox A. D. Isoprenoid modification and plasma membrane association: critical factors for ras oncogenicity. Cancer Cells. 1991 Sep;3(9):331–340. [PubMed] [Google Scholar]
  6. Der C. J., Pan B. T., Cooper G. M. rasH mutants deficient in GTP binding. Mol Cell Biol. 1986 Sep;6(9):3291–3294. doi: 10.1128/mcb.6.9.3291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Epstein W. W., Lever D. C., Rilling H. C. Prenylated proteins: synthesis of geranylgeranylcysteine and identification of this thioether amino acid as a component of proteins in CHO cells. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7352–7354. doi: 10.1073/pnas.87.19.7352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Farnsworth C. C., Gelb M. H., Glomset J. A. Identification of geranylgeranyl-modified proteins in HeLa cells. Science. 1990 Jan 19;247(4940):320–322. doi: 10.1126/science.2296721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Farnsworth C. C., Kawata M., Yoshida Y., Takai Y., Gelb M. H., Glomset J. A. C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated cysteine residues and a methyl ester. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6196–6200. doi: 10.1073/pnas.88.14.6196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Farnsworth C. C., Wolda S. L., Gelb M. H., Glomset J. A. Human lamin B contains a farnesylated cysteine residue. J Biol Chem. 1989 Dec 5;264(34):20422–20429. [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Finegold A. A., Johnson D. I., Farnsworth C. C., Gelb M. H., Judd S. R., Glomset J. A., Tamanoi F. Protein geranylgeranyltransferase of Saccharomyces cerevisiae is specific for Cys-Xaa-Xaa-Leu motif proteins and requires the CDC43 gene product but not the DPR1 gene product. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4448–4452. doi: 10.1073/pnas.88.10.4448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gibbs J. B., Schaber M. D., Schofield T. L., Scolnick E. M., Sigal I. S. Xenopus oocyte germinal-vesicle breakdown induced by [Val12]Ras is inhibited by a cytosol-localized Ras mutant. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6630–6634. doi: 10.1073/pnas.86.17.6630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Glomset J. A., Gelb M. H., Farnsworth C. C. Prenyl proteins in eukaryotic cells: a new type of membrane anchor. Trends Biochem Sci. 1990 Apr;15(4):139–142. doi: 10.1016/0968-0004(90)90213-u. [DOI] [PubMed] [Google Scholar]
  15. Goldstein J. L., Brown M. S. Regulation of the mevalonate pathway. Nature. 1990 Feb 1;343(6257):425–430. doi: 10.1038/343425a0. [DOI] [PubMed] [Google Scholar]
  16. Gutierrez L., Magee A. I., Marshall C. J., Hancock J. F. Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteolysis. EMBO J. 1989 Apr;8(4):1093–1098. doi: 10.1002/j.1460-2075.1989.tb03478.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hancock J. F., Cadwallader K., Paterson H., Marshall C. J. A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins. EMBO J. 1991 Dec;10(13):4033–4039. doi: 10.1002/j.1460-2075.1991.tb04979.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Haubruck H., McCormick F. Ras p21: effects and regulation. Biochim Biophys Acta. 1991 Dec 10;1072(2-3):215–229. doi: 10.1016/0304-419x(91)90015-d. [DOI] [PubMed] [Google Scholar]
  19. Khosravi-Far R., Lutz R. J., Cox A. D., Conroy L., Bourne J. R., Sinensky M., Balch W. E., Buss J. E., Der C. J. Isoprenoid modification of rab proteins terminating in CC or CXC motifs. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6264–6268. doi: 10.1073/pnas.88.14.6264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kinsella B. T., Maltese W. A. rab GTP-binding proteins implicated in vesicular transport are isoprenylated in vitro at cysteines within a novel carboxyl-terminal motif. J Biol Chem. 1991 May 5;266(13):8540–8544. [PubMed] [Google Scholar]
  21. 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]
  22. Kitayama H., Sugimoto Y., Matsuzaki T., Ikawa Y., Noda M. A ras-related gene with transformation suppressor activity. Cell. 1989 Jan 13;56(1):77–84. doi: 10.1016/0092-8674(89)90985-9. [DOI] [PubMed] [Google Scholar]
  23. Lacal P. M., Pennington C. Y., Lacal J. C. Transforming activity of ras proteins translocated to the plasma membrane by a myristoylation sequence from the src gene product. Oncogene. 1988 Jun;2(6):533–537. [PubMed] [Google Scholar]
  24. Lai R. K., Perez-Sala D., Cañada F. J., Rando R. R. The gamma subunit of transducin is farnesylated. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7673–7677. doi: 10.1073/pnas.87.19.7673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Maltese W. A. Posttranslational modification of proteins by isoprenoids in mammalian cells. FASEB J. 1990 Dec;4(15):3319–3328. doi: 10.1096/fasebj.4.15.2123808. [DOI] [PubMed] [Google Scholar]
  26. Marcus S., Caldwell G. A., Miller D., Xue C. B., Naider F., Becker J. M. Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone. Mol Cell Biol. 1991 Jul;11(7):3603–3612. doi: 10.1128/mcb.11.7.3603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Marshall M. S., Davis L. J., Keys R. D., Mosser S. D., Hill W. S., Scolnick E. M., Gibbs J. B. Identification of amino acid residues required for Ras p21 target activation. Mol Cell Biol. 1991 Aug;11(8):3997–4004. doi: 10.1128/mcb.11.8.3997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Maruta H., Holden J., Sizeland A., D'Abaco G. The residues of Ras and Rap proteins that determine their GAP specificities. J Biol Chem. 1991 Jun 25;266(18):11661–11668. [PubMed] [Google Scholar]
  29. Michaeli T., Field J., Ballester R., O'Neill K., Wigler M. Mutants of H-ras that interfere with RAS effector function in Saccharomyces cerevisiae. EMBO J. 1989 Oct;8(10):3039–3044. doi: 10.1002/j.1460-2075.1989.tb08454.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mulcahy L. S., Smith M. R., Stacey D. W. Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells. Nature. 1985 Jan 17;313(5999):241–243. doi: 10.1038/313241a0. [DOI] [PubMed] [Google Scholar]
  31. Patel G., MacDonald M. J., Khosravi-Far R., Hisaka M. M., Der C. J. Alternate mechanisms of ras activation are complementary and favor and formation of ras-GTP. Oncogene. 1992 Feb;7(2):283–288. [PubMed] [Google Scholar]
  32. Plutner H., Cox A. D., Pind S., Khosravi-Far R., Bourne J. R., Schwaninger R., Der C. J., Balch W. E. Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments. J Cell Biol. 1991 Oct;115(1):31–43. doi: 10.1083/jcb.115.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Quilliam L. A., Der C. J., Clark R., O'Rourke E. C., Zhang K., McCormick F., Bokoch G. M. Biochemical characterization of baculovirus-expressed rap1A/Krev-1 and its regulation by GTPase-activating proteins. Mol Cell Biol. 1990 Jun;10(6):2901–2908. doi: 10.1128/mcb.10.6.2901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Quinn M. T., Parkos C. A., Walker L., Orkin S. H., Dinauer M. C., Jesaitis A. J. Association of a Ras-related protein with cytochrome b of human neutrophils. Nature. 1989 Nov 9;342(6246):198–200. doi: 10.1038/342198a0. [DOI] [PubMed] [Google Scholar]
  36. Reiss Y., Goldstein J. L., Seabra M. C., Casey P. J., Brown M. S. Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides. Cell. 1990 Jul 13;62(1):81–88. doi: 10.1016/0092-8674(90)90242-7. [DOI] [PubMed] [Google Scholar]
  37. Ricketts M. H., Levinson A. D. High-level expression of c-H-ras1 fails to fully transform rat-1 cells. Mol Cell Biol. 1988 Apr;8(4):1460–1468. doi: 10.1128/mcb.8.4.1460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rine J., Kim S. H. A role for isoprenoid lipids in the localization and function of an oncoprotein. New Biol. 1990 Mar;2(3):219–226. [PubMed] [Google Scholar]
  39. 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]
  40. Seabra M. C., Reiss Y., Casey P. J., Brown M. S., Goldstein J. L. Protein farnesyltransferase and geranylgeranyltransferase share a common alpha subunit. Cell. 1991 May 3;65(3):429–434. doi: 10.1016/0092-8674(91)90460-g. [DOI] [PubMed] [Google Scholar]
  41. Shalloway D., Zelenetz A. D., Cooper G. M. Molecular cloning and characterization of the chicken gene homologous to the transforming gene of Rous sarcoma virus. Cell. 1981 May;24(2):531–541. doi: 10.1016/0092-8674(81)90344-5. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Smith M. R., DeGudicibus S. J., Stacey D. W. Requirement for c-ras proteins during viral oncogene transformation. Nature. 1986 Apr 10;320(6062):540–543. doi: 10.1038/320540a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stacey D. W., Feig L. A., Gibbs J. B. Dominant inhibitory Ras mutants selectively inhibit the activity of either cellular or oncogenic Ras. Mol Cell Biol. 1991 Aug;11(8):4053–4064. doi: 10.1128/mcb.11.8.4053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stanton V. P., Jr, Nichols D. W., Laudano A. P., Cooper G. M. Definition of the human raf amino-terminal regulatory region by deletion mutagenesis. Mol Cell Biol. 1989 Feb;9(2):639–647. doi: 10.1128/mcb.9.2.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zhang K., Noda M., Vass W. C., Papageorge A. G., Lowy D. R. Identification of small clusters of divergent amino acids that mediate the opposing effects of ras and Krev-1. Science. 1990 Jul 13;249(4965):162–165. doi: 10.1126/science.2115210. [DOI] [PubMed] [Google Scholar]

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

RESOURCES