Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1985 Nov;4(11):2893–2896. doi: 10.1002/j.1460-2075.1985.tb04019.x

Transforming p21 ras protein: flexibility in the major variable region linking the catalytic and membrane-anchoring domains.

B M Willumsen, A G Papageorge, N Hubbert, E Bekesi, H F Kung, D R Lowy
PMCID: PMC554594  PMID: 2998761

Abstract

The mammalian p21 ras proteins contain a 20-amino acid region that is highly divergent, in contrast to the strong sequence conservation that is common to other regions of these proteins. This major variable region is located near the C terminus just upstream from a conserved cysteine residue that is required for post-translational processing, membrane localization and transforming activity of the proteins. We have now used the viral oncogene (v-rasH) of Harvey sarcoma virus to study the major variable region by deleting or duplicating parts of the gene. Reducing this region to five amino acids or increasing it to 50 amino acids has relatively little effect on the capacity of the gene to induce morphological transformation of NIH 3T3 cells. Assays of GTP binding, GTPase and autophosphorylating activities of such mutant v-rasH-encoded proteins synthesized in bacteria indicated that the sequences that encode these biochemical activities are located upstream from the major variable region. In the context of transformation, we propose that the region of sequence heterogeneity serves principally to connect the N-terminal catalytic domain with amino acids at the C terminus that are required to anchor the protein in the membrane.

Full text

PDF
2893

Images in this article

2895Fig. 1.
on p.2895

Selected References

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

  1. Bolanowski M. A., Earles B. J., Lennarz W. J. Fatty acylation of proteins during development of sea urchin embryos. J Biol Chem. 1984 Apr 25;259(8):4934–4940. [PubMed] [Google Scholar]
  2. Buss J. E., Sefton B. M. Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog. J Virol. 1985 Jan;53(1):7–12. doi: 10.1128/jvi.53.1.7-12.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Capon D. J., Seeburg P. H., McGrath J. P., Hayflick J. S., Edman U., Levinson A. D., Goeddel D. V. Activation of Ki-ras2 gene in human colon and lung carcinomas by two different point mutations. Nature. 1983 Aug 11;304(5926):507–513. doi: 10.1038/304507a0. [DOI] [PubMed] [Google Scholar]
  4. Chang E. H., Furth M. E., Scolnick E. M., Lowy D. R. Tumorigenic transformation of mammalian cells induced by a normal human gene homologous to the oncogene of Harvey murine sarcoma virus. Nature. 1982 Jun 10;297(5866):479–483. doi: 10.1038/297479a0. [DOI] [PubMed] [Google Scholar]
  5. Courtneidge S. A., Levinson A. D., Bishop J. M. The protein encoded by the transforming gene of avian sarcoma virus (pp60src) and a homologous protein in normal cells (pp60proto-src) are associated with the plasma membrane. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3783–3787. doi: 10.1073/pnas.77.7.3783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Dhar R., Ellis R. W., Shih T. Y., Oroszlan S., Shapiro B., Maizel J., Lowy D., Scolnick E. Nucleotide sequence of the p21 transforming protein of Harvey murine sarcoma virus. Science. 1982 Sep 3;217(4563):934–936. doi: 10.1126/science.6287572. [DOI] [PubMed] [Google Scholar]
  8. Dhar R., Nieto A., Koller R., DeFeo-Jones D., Scolnick E. M. Nucleotide sequence of two rasH related-genes isolated from the yeast Saccharomyces cerevisiae. Nucleic Acids Res. 1984 Apr 25;12(8):3611–3618. doi: 10.1093/nar/12.8.3611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fasano O., Aldrich T., Tamanoi F., Taparowsky E., Furth M., Wigler M. Analysis of the transforming potential of the human H-ras gene by random mutagenesis. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4008–4012. doi: 10.1073/pnas.81.13.4008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Furth M. E., Davis L. J., Fleurdelys B., Scolnick E. M. Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family. J Virol. 1982 Jul;43(1):294–304. doi: 10.1128/jvi.43.1.294-304.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gibbs J. B., Sigal I. S., Poe M., Scolnick E. M. Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5704–5708. doi: 10.1073/pnas.81.18.5704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  13. Gross M., Sweet R. W., Sathe G., Yokoyama S., Fasano O., Goldfarb M., Wigler M., Rosenberg M. Purification and characterization of human H-ras proteins expressed in Escherichia coli. Mol Cell Biol. 1985 May;5(5):1015–1024. doi: 10.1128/mcb.5.5.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Land H., Parada L. F., Weinberg R. A. Cellular oncogenes and multistep carcinogenesis. Science. 1983 Nov 18;222(4625):771–778. doi: 10.1126/science.6356358. [DOI] [PubMed] [Google Scholar]
  16. Manne V., Bekesi E., Kung H. F. Ha-ras proteins exhibit GTPase activity: point mutations that activate Ha-ras gene products result in decreased GTPase activity. Proc Natl Acad Sci U S A. 1985 Jan;82(2):376–380. doi: 10.1073/pnas.82.2.376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Manne V., Yamazaki S., Kung H. F. Guanosine nucleotide binding by highly purified Ha-ras-encoded p21 protein produced in Escherichia coli. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6953–6957. doi: 10.1073/pnas.81.22.6953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McGrath J. P., Capon D. J., Goeddel D. V., Levinson A. D. Comparative biochemical properties of normal and activated human ras p21 protein. Nature. 1984 Aug 23;310(5979):644–649. doi: 10.1038/310644a0. [DOI] [PubMed] [Google Scholar]
  19. Neuman-Silberberg F. S., Schejter E., Hoffmann F. M., Shilo B. Z. The Drosophila ras oncogenes: structure and nucleotide sequence. Cell. 1984 Jul;37(3):1027–1033. doi: 10.1016/0092-8674(84)90437-9. [DOI] [PubMed] [Google Scholar]
  20. Papageorge A., Lowy D., Scolnick E. M. Comparative biochemical properties of p21 ras molecules coded for by viral and cellular ras genes. J Virol. 1982 Nov;44(2):509–519. doi: 10.1128/jvi.44.2.509-519.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Reymond C. D., Gomer R. H., Mehdy M. C., Firtel R. A. Developmental regulation of a Dictyostelium gene encoding a protein homologous to mammalian ras protein. Cell. 1984 Nov;39(1):141–148. doi: 10.1016/0092-8674(84)90199-5. [DOI] [PubMed] [Google Scholar]
  23. Schejter E. D., Shilo B. Z. Characterization of functional domains of p21 ras by use of chimeric genes. EMBO J. 1985 Feb;4(2):407–412. doi: 10.1002/j.1460-2075.1985.tb03643.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schultz A. M., Henderson L. E., Oroszlan S., Garber E. A., Hanafusa H. Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein. Science. 1985 Jan 25;227(4685):427–429. doi: 10.1126/science.3917576. [DOI] [PubMed] [Google Scholar]
  25. Sefton B. M., Trowbridge I. S., Cooper J. A., Scolnick E. M. The transforming proteins of Rous sarcoma virus, Harvey sarcoma virus and Abelson virus contain tightly bound lipid. Cell. 1982 Dec;31(2 Pt 1):465–474. doi: 10.1016/0092-8674(82)90139-8. [DOI] [PubMed] [Google Scholar]
  26. Shih T. Y., Weeks M. O., Gruss P., Dhar R., Oroszlan S., Scolnick E. M. Identification of a precursor in the biosynthesis of the p21 transforming protein of harvey murine sarcoma virus. J Virol. 1982 Apr;42(1):253–261. doi: 10.1128/jvi.42.1.253-261.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shih T. Y., Weeks M. O. Oncogenes and cancer: the p21 ras genes. Cancer Invest. 1984;2(2):109–123. doi: 10.3109/07357908409020294. [DOI] [PubMed] [Google Scholar]
  28. Shimizu K., Birnbaum D., Ruley M. A., Fasano O., Suard Y., Edlund L., Taparowsky E., Goldfarb M., Wigler M. Structure of the Ki-ras gene of the human lung carcinoma cell line Calu-1. Nature. 1983 Aug 11;304(5926):497–500. doi: 10.1038/304497a0. [DOI] [PubMed] [Google Scholar]
  29. Sukumar S., Notario V., Martin-Zanca D., Barbacid M. Induction of mammary carcinomas in rats by nitroso-methylurea involves malignant activation of H-ras-1 locus by single point mutations. Nature. 1983 Dec 15;306(5944):658–661. doi: 10.1038/306658a0. [DOI] [PubMed] [Google Scholar]
  30. Sweet R. W., Yokoyama S., Kamata T., Feramisco J. R., Rosenberg M., Gross M. The product of ras is a GTPase and the T24 oncogenic mutant is deficient in this activity. Nature. 1984 Sep 20;311(5983):273–275. doi: 10.1038/311273a0. [DOI] [PubMed] [Google Scholar]
  31. Tabin C. J., Bradley S. M., Bargmann C. I., Weinberg R. A., Papageorge A. G., Scolnick E. M., Dhar R., Lowy D. R., Chang E. H. Mechanism of activation of a human oncogene. Nature. 1982 Nov 11;300(5888):143–149. doi: 10.1038/300143a0. [DOI] [PubMed] [Google Scholar]
  32. Taparowsky E., Shimizu K., Goldfarb M., Wigler M. Structure and activation of the human N-ras gene. Cell. 1983 Sep;34(2):581–586. doi: 10.1016/0092-8674(83)90390-2. [DOI] [PubMed] [Google Scholar]
  33. Temeles G. L., Gibbs J. B., D'Alonzo J. S., Sigal I. S., Scolnick E. M. Yeast and mammalian ras proteins have conserved biochemical properties. Nature. 1985 Feb 21;313(6004):700–703. doi: 10.1038/313700a0. [DOI] [PubMed] [Google Scholar]
  34. Tsuchida N., Ryder T., Ohtsubo E. Nucleotide sequence of the oncogene encoding the p21 transforming protein of Kirsten murine sarcoma virus. Science. 1982 Sep 3;217(4563):937–939. doi: 10.1126/science.6287573. [DOI] [PubMed] [Google Scholar]
  35. Willingham M. C., Pastan I., Shih T. Y., Scolnick E. M. Localization of the src gene product of the Harvey strain of MSV to plasma membrane of transformed cells by electron microscopic immunocytochemistry. Cell. 1980 Apr;19(4):1005–1014. doi: 10.1016/0092-8674(80)90091-4. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Yuasa Y., Gol R. A., Chang A., Chiu I. M., Reddy E. P., Tronick S. R., Aaronson S. A. Mechanism of activation of an N-ras oncogene of SW-1271 human lung carcinoma cells. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3670–3674. doi: 10.1073/pnas.81.12.3670. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES