Structure and Function of ras p21: Studies BY Site-Directed Mutagenesis

  • Thomas Y. Shih
  • David J. Clanton
  • Pothana Saikumar
  • Linda S. Ulsh
  • Seisuke Hattori


Are studies by analogy fruitful to understanding protein structure and function? With protein sequences accumulating at an astonishing rate through gene cloning and DNA sequencing, a great deal of protein structure and function can be learned by analogy with members of the superfamily whose structures have been determined and whose molecular mechanisms of action are known. The best case in point, perhaps is in unraveling the elusive function of ras p21. The molecular model of p21 has been constructed by analogy with the crystal structure of the E. coli elongation factor, EF-Tu (McCormick et al., 1985; Jurnak, 1985). This p21 model is remarkably consistent with the actual three dimensional structure of p21 later determined by X-ray crystallography (De Vos et al., 1988). The recent identification of the GAP protein which stimulates GTPase activity of p21 (Trahey and McCormick, 1987), is a conceptual offspring of similar biochemical mechanism well understood for the function of EF-Tu in protein synthesis (Kaziro, 1978). Furthermore, analogy with the well characterized G-proteins, which regulate transmembrane cell signalling in the adenylate cyclase systems and light transduction in retina, forms the foundation for the current belief that ras p21 mediates transmission of growth signals to their intracellular effectors that control cell proliferation and differentiation (Bourne and Sullivan, 1986).


GTPase Activity Adenylate Cyclase System Murine Sarcoma Virus Tryptic Peptide Mapping Autokinase Activity 
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  1. Adari, H., Lowy, D. R., Willumsen, B. M., Der, C. J. and McCormick, F., 1988, Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain, Science, 240:518–521.PubMedCrossRefGoogle Scholar
  2. Barbacid, M., 1987, ras genes, Annu. Rev. Biochem., 56:779–827.PubMedCrossRefGoogle Scholar
  3. Bourne, H. R. and Sullivan, K. S., 1986, Mammalian G proteins: model for ras proteins in transmembrane signalling? Cancer Surveys, 5:257–274.PubMedGoogle Scholar
  4. Cales, C., Hancock, J. F., Marshall, C. J. and Hall, A., 1988, The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product, Nature, 332:548–551.PubMedCrossRefGoogle Scholar
  5. Clanton, D. J., Hattori, S. and Shih, T. Y., 1986, Mutations of the ras gene product p21 that abolish guanine nucleotide binding, Proc. Nat. Acad. Sci., USA, 83:5076–5080.CrossRefGoogle Scholar
  6. Clanton, D. J., Lu, Y., Blair, D. G. and Shih, T. Y., 1987, Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis, Mol. Cell. Biol., 7:3092–3097.PubMedGoogle Scholar
  7. Dever, T. E., Glynias, M. J. and Merrick, W. C., 1987, GTP-binding domain: three consensus sequence elements with distinct spacing, Proc. Nat. Acad. Sci., USA, 84:1814–1818.CrossRefGoogle Scholar
  8. De Vos, A. M., Tong, L., Milburn, M. V., Matias, P. M., Jancarik, J., Noguchi, S., Nishimura, S., Miura, K., Ohtsuka, E. and Kim, S. H., 1988, Three-dimensional structure of an oncogenic protein: catalytic domain of human c-H-ras p21, Science, 239:888–893.PubMedCrossRefGoogle Scholar
  9. Feig, L. A., Pan, B. T., Roberts, T. M. and Cooper, G. M., 1986, Isolation of ras GTP-binding mutants using an in situ colony-binding assay, Proc. Nat. Acad. Sci., USA, 83:4607–4611.CrossRefGoogle Scholar
  10. Gibbs, J. B., Schaber, M. D., Marshall, M. S., Scolnick, E. M. and Sigal, I.S., 1987, Identification of guanine nucleotides bound to ras-encoded proteins in growing yeast cells, J, Biol1. Chem., 262:10426–10429.Google Scholar
  11. Gibbs, J. B., Sigal, I. S., Poe, M. and Scolnick, E. M., 1984, Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules, Proc. Nat. Acad. Sci., USA, 81:5704–5708.CrossRefGoogle Scholar
  12. Hall, A. and Self, A. J., 1986, The effect of Mg2+ on the guanine nucleo tide exchange rate of p2lN-ras, J. Biol. Chem., 261:10963–10965.PubMedGoogle Scholar
  13. Halliday, K. R., 1984, Regional homology in GTP-binding proto-oncogene products and elongation factors, J. Cyclic Nucleotide and Protein Phosphorylation Research, 9:435–448.Google Scholar
  14. Hattori, S., Ulsh, L. S., Halliday, K. and Shih, T. Y., 1985, Biochemical properties of a highly purified v-ras H p21 protein overproduced in E. coli and inhibition of its activities by a monoclonal antibody, Mol. Cell. Biol., 5:1449–1455.PubMedGoogle Scholar
  15. Hattori, S., Yamashita, T., Copeland, T. D., Oroszlan, S. and Shih, T. Y., 1986, Reactivity of a sulfhydryl group of the ras oncogene product p21 modulated by GTP binding, J. Biol. Chem., 751: 14582–14586.Google Scholar
  16. Hoshino, M., Clanton, D. J., Shih, T. Y., Kawakita, M. and Hattori, S., 1987, Interaction of ras oncogene product p21 with guanine nucleo tides, J. Biochem. (Tokyo), 102:503–511.Google Scholar
  17. Jurnak, F., 1985, Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins, Science, 230: 32–36.PubMedCrossRefGoogle Scholar
  18. Kaziro, Y., 1978, The role of guanosine 5’-triphosphate in polypeptide chain elongation, Biochim. Biophys. Acta, 505:95–127.PubMedCrossRefGoogle Scholar
  19. Lautenberger, J. A., Ulsh, L., Shih, T. Y. and Papas, T. S., 1983, High level expression in E. coli of enzymatically active Harvey murine sarcoma virus p21 ras protein, Science, 221:858–860.PubMedCrossRefGoogle Scholar
  20. Leberman, R. and Egner, U., 1984, Homologies in the primary structure of GTP-binding proteins: the nucleotide-binding site of EF-Tu and p21, The EMBO J., 3:339–341.Google Scholar
  21. Manne, V., Bekesi, E. and Kung, H. F., 1985, Ha-ras proteins exhibit GTPase activity: point mutations that activate Ha-ras products result in decreased GTPase activity, Proc. Nat. Acad. Sci., USA, 82:376–380.CrossRefGoogle Scholar
  22. McCormick, F., Clark, B. F., La Cour, T. F. M., Kjeldgaard, M., Norskov- Lauritsen, L. and Nyborg, J., 1985, A model for the tertiary struc ture of p21, the product of the ras oncogene, Science, 230: 78–82.PubMedCrossRefGoogle Scholar
  23. McGrath, J. P., Capon, D. V., Goeddel, D. V. and Levinson, A. D., 1984, Comparative biochemical properties of normal and activated human ras p21 proteins, Nature, 310:644–649.PubMedCrossRefGoogle Scholar
  24. Moeller, W. and Amons, R., 1985, Phosphate-binding sequences in nucleotide- binding proteins, FEBS Letters, 186:1–7.CrossRefGoogle Scholar
  25. Rubin, J. R., Morikawa, K., Nyborg, J., La Cour, T. F. M., Clark, B. F. C. and Miller, D. L., 1981, Structural features of the GDP binding site of elongation factor Tu from E. coli as determined by X-ray diffrac tion, FEBS Letters, 129:177–179.PubMedCrossRefGoogle Scholar
  26. Saikumar, P., Ulsh, L. S., Clanton, D. J., Huang, K. P. and Shih, T. Y., 1988, Novel phosphorylation of c-ras p21 by protein kinases, Oncogene Research, in press.Google Scholar
  27. Satoh, T., Nakamura, S. and Kaziro, Y., 1987, Induction of neurite formation in PC12 cells by microinjection of proto-oncogenic Ha-ras protein preincubated with guanosine-5’-0-(3-thiotriphosphate), Mol. Cell.Biol., 7:4553–4556.PubMedGoogle Scholar
  28. Scolnick, E. M., Papageorge, A. and Shih, T. Y., 1979, Guanine nucleotide- binding activity as an assay for sre protein of rat-derived murine sarcoma viruses, Proc. Nat. Acad. Sci., USA, 76:5355–5359.CrossRefGoogle Scholar
  29. Shih, T. Y., Papageorge, A. G., Stokes, P. E., Weeks, M. O. and Scolnick, E. M., 1980, Guanine nucleotide-binding and autophosphorylating activities associated with the p21src protein of Harvey murine sarcoma virus, Nature, 287:686–691.PubMedCrossRefGoogle Scholar
  30. Shih, T. Y., Weeks, M. O., Young, H. A. and Scolnick, E. M., 1979a, Identification of a sarcoma virus coded phosphoprotein in nonproducer cells transformed by Kirsten or Harvey murine sarcoma virus. Virology, 96:64–79.PubMedCrossRefGoogle Scholar
  31. Shih, T. Y., Weeks, M. O., Young, H. A. and Scolnick, E. M., 1979b, p21 of Kirsten murine sarcoma virus is thermolabile in a viral mutant temperature-sensitive for the maintenance of transformation, J. Virol., 31:546–556.PubMedGoogle Scholar
  32. Sigal, I. S., 1988, A structure and some function, Nature, 332:485–486.PubMedCrossRefGoogle Scholar
  33. Sigal, I. S., Gibbs, J. B., D’Alonzo, J. S., Temeles, G. L., Wolanski, B. S., Socher, S. and Scolnick, E. S., 1986, Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects, Proc. Nat. Acad. Sci., USA, 83:952–956.CrossRefGoogle Scholar
  34. Sweet, R. W., Yokoyama, S., Kamata, T., Feramisco, J. R., Rosenberg, M. and Gross, M., 1984, The product of ras is a GTPase and T24 oncogenic mutant is deficient in this activity, Nature, 311:273–275.PubMedCrossRefGoogle Scholar
  35. Trahey, M. and McCormick, F., 1987, A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants, Science, 238:542–545.PubMedCrossRefGoogle Scholar
  36. Walter, M., Clark, S. G. and Levinson, A. D., 1986, The oncogenic activation of human p2iras by a novel mechanism, Science, 233:649–652.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1989

Authors and Affiliations

  • Thomas Y. Shih
    • 1
  • David J. Clanton
    • 1
  • Pothana Saikumar
    • 1
  • Linda S. Ulsh
    • 1
  • Seisuke Hattori
    • 1
  1. 1.Laboratory of Molecular OncologyNational Cancer Institute-Frederick Cancer Research FacilityFrederickUSA

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