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. 1995 Feb 28;92(5):1272–1276. doi: 10.1073/pnas.92.5.1272

Biological and structural characterization of a Ras transforming mutation at the phenylalanine-156 residue, which is conserved in all members of the Ras superfamily.

L A Quilliam 1, S Zhong 1, K M Rabun 1, J W Carpenter 1, T L South 1, C J Der 1, S Campbell-Burk 1
PMCID: PMC42501  PMID: 7877967

Abstract

Although Ras residue phenylalanine-156 (F156) is strictly conserved in all members of the Ras superfamily of proteins, it is located outside of the consensus GDP/GTP-binding pocket. Its location within the hydrophobic core of Ras suggests that its strict conservation reflects a crucial role in structural stability. However, mutation of the equivalent residue (F157L) in the Drosophila Ras-related protein Rap results in a gain-of-function phenotype, suggesting an alternative role for this residue. Therefore, we have introduced an F156L mutation into Ras to evaluate the role of this residue in Ras structure and function. Whereas introduction of this mutation activated the transforming potential of wild-type Ras, it did not impair that of oncogenic Ras. Further, Ras (156L) exhibited an extremely rapid off rate for bound GDP/GTP in vitro and showed increased levels of Ras.GTP in vivo. To determine the structural basis for these altered properties, we used high-resolution nuclear magnetic resonance spectroscopy. The F156L mutation caused loss of contact with residues 6, 23, 55, and 79, resulting in disruption of secondary structure in alpha-helix 1 and in beta-sheets 1-5. These major structural changes contrast with the isolated alterations induced by oncogenic mutation (residues 12 or 61) that perturb GTPase activity, and instead, weaken Ras contacts with Mg2+ and its guanine nucleotide substrate and result in increased rates of GDP/GTP dissociation. Altogether, these observations demonstrate the essential role of this conserved residue in Ras structure and its function as a regulated GDP/GTP switch.

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Selected References

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

  1. 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]
  2. Cepko C. L., Roberts B. E., Mulligan R. C. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell. 1984 Jul;37(3):1053–1062. doi: 10.1016/0092-8674(84)90440-9. [DOI] [PubMed] [Google Scholar]
  3. DeLoskey R. J., Van Dyk D. E., Van Aken T. E., Campbell-Burk S. Isolation and refolding of H-ras from inclusion bodies of Escherichia coli: refold procedure and comparison of refolded and soluble H-ras. Arch Biochem Biophys. 1994 May 15;311(1):72–78. doi: 10.1006/abbi.1994.1210. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Hariharan I. K., Carthew R. W., Rubin G. M. The Drosophila roughened mutation: activation of a rap homolog disrupts eye development and interferes with cell determination. Cell. 1991 Nov 15;67(4):717–722. doi: 10.1016/0092-8674(91)90066-8. [DOI] [PubMed] [Google Scholar]
  6. John J., Rensland H., Schlichting I., Vetter I., Borasio G. D., Goody R. S., Wittinghofer A. Kinetic and structural analysis of the Mg(2+)-binding site of the guanine nucleotide-binding protein p21H-ras. J Biol Chem. 1993 Jan 15;268(2):923–929. [PubMed] [Google Scholar]
  7. John J., Schlichting I., Schiltz E., Rösch P., Wittinghofer A. C-terminal truncation of p21H preserves crucial kinetic and structural properties. J Biol Chem. 1989 Aug 5;264(22):13086–13092. [PubMed] [Google Scholar]
  8. Khosravi-Far R., Der C. J. The Ras signal transduction pathway. Cancer Metastasis Rev. 1994 Mar;13(1):67–89. doi: 10.1007/BF00690419. [DOI] [PubMed] [Google Scholar]
  9. Kraulis P. J., Domaille P. J., Campbell-Burk S. L., Van Aken T., Laue E. D. Solution structure and dynamics of ras p21.GDP determined by heteronuclear three- and four-dimensional NMR spectroscopy. Biochemistry. 1994 Mar 29;33(12):3515–3531. doi: 10.1021/bi00178a008. [DOI] [PubMed] [Google Scholar]
  10. Krengel U., Schlichting I., Scherer A., Schumann R., Frech M., John J., Kabsch W., Pai E. F., Wittinghofer A. Three-dimensional structures of H-ras p21 mutants: molecular basis for their inability to function as signal switch molecules. Cell. 1990 Aug 10;62(3):539–548. doi: 10.1016/0092-8674(90)90018-a. [DOI] [PubMed] [Google Scholar]
  11. Privé G. G., Milburn M. V., Tong L., de Vos A. M., Yamaizumi Z., Nishimura S., Kim S. H. X-ray crystal structures of transforming p21 ras mutants suggest a transition-state stabilization mechanism for GTP hydrolysis. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3649–3653. doi: 10.1073/pnas.89.8.3649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Quilliam L. A., Kato K., Rabun K. M., Hisaka M. M., Huff S. Y., Campbell-Burk S., Der C. J. Identification of residues critical for Ras(17N) growth-inhibitory phenotype and for Ras interaction with guanine nucleotide exchange factors. Mol Cell Biol. 1994 Feb;14(2):1113–1121. doi: 10.1128/mcb.14.2.1113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Reinstein J., Schlichting I., Frech M., Goody R. S., Wittinghofer A. p21 with a phenylalanine 28----leucine mutation reacts normally with the GTPase activating protein GAP but nevertheless has transforming properties. J Biol Chem. 1991 Sep 15;266(26):17700–17706. [PubMed] [Google Scholar]
  15. Valencia A., Chardin P., Wittinghofer A., Sander C. The ras protein family: evolutionary tree and role of conserved amino acids. Biochemistry. 1991 May 14;30(19):4637–4648. doi: 10.1021/bi00233a001. [DOI] [PubMed] [Google Scholar]

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