Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Aug;82(16):5280–5284. doi: 10.1073/pnas.82.16.5280

Antibodies specific for amino acid 12 of the ras oncogene product inhibit GTP binding.

R Clark, G Wong, N Arnheim, D Nitecki, F McCormick
PMCID: PMC390551  PMID: 3927300

Abstract

An antibody (anti-p21ser) was raised against a ras p21-related synthetic peptide and was able to recognize specifically the substitution of serine for glycine at amino acid 12 of p21. This substitution causes oncogenic activation of p21. Anti-p21ser was found to immunoprecipitate v-Ki-ras p21 and to strongly inhibit its ability to autophosphorylate and to bind GTP in an immunoabsorption assay. Furthermore, binding of the antibody to p21 was specifically inhibited by GTP or GDP, suggesting that amino acids around position 12 are part of the GTP/GDP binding site. These results, taken together with the observation that the microinjection of anti-p21ser into cells transformed by v-Ki-ras p21 causes a transient reversion of the cells to a normal phenotype [Feramisco, J. R., Clark, R., Wong, G., Arnheim, N., Milley, R. & McCormick, F. (1985) Nature (London) 314, 639-642], support the idea that interaction of p21 with guanine nucleotides is crucial to the transforming function of this protein.

Full text

PDF
5283

Images in this article

5281Image
on p.5281
5281Image
on p.5281
5281Image
on p.5281
5281Image
on p.5281
5281Image
on p.5281
5282Image
on p.5282
5282Image
on p.5282
5283Image
on p.5283

Selected References

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

  1. Alexander H., Johnson D. A., Rosen J., Jerabek L., Green N., Weissman I. L., Lerner R. A. Mimicking the alloantigenicity of proteins with chemically synthesized peptides differing in single amino acids. Nature. 1983 Dec 15;306(5944):697–699. doi: 10.1038/306697a0. [DOI] [PubMed] [Google Scholar]
  2. Bittner M., Kupferer P., Morris C. F. Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzyloxymethyl cellulose or nitrocellulose sheets. Anal Biochem. 1980 Mar 1;102(2):459–471. doi: 10.1016/0003-2697(80)90182-7. [DOI] [PubMed] [Google Scholar]
  3. Feramisco J. R., Clark R., Wong G., Arnheim N., Milley R., McCormick F. Transient reversion of ras oncogene-induced cell transformation by antibodies specific for amino acid 12 of ras protein. Nature. 1985 Apr 18;314(6012):639–642. doi: 10.1038/314639a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Gay N. J., Walker J. E. Homology between human bladder carcinoma oncogene product and mitochondrial ATP-synthase. Nature. 1983 Jan 20;301(5897):262–264. doi: 10.1038/301262a0. [DOI] [PubMed] [Google Scholar]
  6. Gibbs J. B., Ellis R. W., Scolnick E. M. Autophosphorylation of v-Ha-ras p21 is modulated by amino acid residue 12. Proc Natl Acad Sci U S A. 1984 May;81(9):2674–2678. doi: 10.1073/pnas.81.9.2674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Halliday K. R. Regional homology in GTP-binding proto-oncogene products and elongation factors. J Cyclic Nucleotide Protein Phosphor Res. 1983;9(6):435–448. [PubMed] [Google Scholar]
  8. Hurley J. B., Simon M. I., Teplow D. B., Robishaw J. D., Gilman A. G. Homologies between signal transducing G proteins and ras gene products. Science. 1984 Nov 16;226(4676):860–862. doi: 10.1126/science.6436980. [DOI] [PubMed] [Google Scholar]
  9. Leberman R., Egner U. Homologies in the primary structure of GTP-binding proteins: the nucleotide-binding site of EF-Tu and p21. EMBO J. 1984 Feb;3(2):339–341. doi: 10.1002/j.1460-2075.1984.tb01808.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lerner R. A. Tapping the immunological repertoire to produce antibodies of predetermined specificity. Nature. 1982 Oct 14;299(5884):593–596. doi: 10.1038/299592a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Merrifield R. B. Solid-phase peptide synthesis. Adv Enzymol Relat Areas Mol Biol. 1969;32:221–296. doi: 10.1002/9780470122778.ch6. [DOI] [PubMed] [Google Scholar]
  13. Nyari L. J., Tan Y. H., Erlich H. A. Monoclonal antibodies directed against human fibroblast interferon: characterization and functional studies. Hybridoma. 1983;2(1):79–86. doi: 10.1089/hyb.1983.2.79. [DOI] [PubMed] [Google Scholar]
  14. Rubin J. R., Morikawa K., Nyborg J., la Cour T. F., Clark B. F., Miller D. L. Structural features of the GDP binding site of elongation factor Tu from Escherichia coli as determined by x-ray diffraction. FEBS Lett. 1981 Jun 29;129(1):177–179. doi: 10.1016/0014-5793(81)80784-3. [DOI] [PubMed] [Google Scholar]
  15. Shih T. Y., Papageorge A. G., Stokes P. E., Weeks M. O., Scolnick E. M. Guanine nucleotide-binding and autophosphorylating activities associated with the p21src protein of Harvey murine sarcoma virus. Nature. 1980 Oct 23;287(5784):686–691. doi: 10.1038/287686a0. [DOI] [PubMed] [Google Scholar]
  16. Shih T. Y., Stokes P. E., Smythers G. W., Dhar R., Oroszlan S. Characterization of the phosphorylation sites and the surrounding amino acid sequences of the p21 transforming proteins coded for by the Harvey and Kirsten strains of murine sarcoma viruses. J Biol Chem. 1982 Oct 10;257(19):11767–11773. [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Thor A., Horan Hand P., Wunderlich D., Caruso A., Muraro R., Schlom J. Monoclonal antibodies define differential ras gene expression in malignant and benign colonic diseases. Nature. 1984 Oct 11;311(5986):562–565. doi: 10.1038/311562a0. [DOI] [PubMed] [Google Scholar]
  20. Walter G., Scheidtmann K. H., Carbone A., Laudano A. P., Doolittle R. F. Antibodies specific for the carboxy- and amino-terminal regions of simian virus 40 large tumor antigen. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5197–5200. doi: 10.1073/pnas.77.9.5197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wierenga R. K., Hol W. G. Predicted nucleotide-binding properties of p21 protein and its cancer-associated variant. Nature. 1983 Apr 28;302(5911):842–844. doi: 10.1038/302842a0. [DOI] [PubMed] [Google Scholar]
  22. Yuasa Y., Srivastava S. K., Dunn C. Y., Rhim J. S., Reddy E. P., Aaronson S. A. Acquisition of transforming properties by alternative point mutations within c-bas/has human proto-oncogene. Nature. 1983 Jun 30;303(5920):775–779. doi: 10.1038/303775a0. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES