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Characterization of Normal and Mutant Human Kirsten-ras(4B) p21 and of the Catalytic Domain of GAP

  • Peter N. Lowe
  • Susan Rhodes
  • Susan Bradley
  • Richard H. Skinner
Part of the NATO ASI Series book series (NSSA, volume 220)

Abstract

Point mutations in the Harvey-, N and Kirsten-ras genes are associated with 20–30% of human tumours1. Kirsten-ras is the gene most frequently activated in tumours. Certain tumour types contain high frequencies of K-ras mutation, for example colorectal (c. 50%) and pancreatic (>95%) tumours2–4. The high incidence of K-ras mutations in clinically important tumours makes the K-ras gene product of particular interest as a target for the discovery of potentially highly selective anti-tumour agents. Hitherto, biochemical studies on the ras proteins has focused on the products of the H- and N-ras genes. The product of the viral Kirsten-ras gene has been studied but it is the counterpart of a rare transcript of the cellular K-ras gene (transcript 2A or 4A). The commonly occuring form of K-ras (2B or 4B) expressed in human cells differs from the 4A transcript in having a lysine-rich C-terminus terminus, which is not modified by palmitoylation5.

Keywords

Purine Nucleoside Phosphorylase Fluorimetric Assay Immunoaffinity Purification ompT Protease Relative Binding Constant 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. 1.
    J.L. Bos, The ras gene family and human carcinogenesis, Mutat. Res., 195:255 (1988).PubMedGoogle Scholar
  2. 2.
    S. Kahn, F. Yamamoto, C. Almoguera, E. Winter, K. Forrester, J. Jordano, and M. Perucho, 1987, The c-K-ras gene and human cancer (review), Anticancer Res. 7:639 (1987)PubMedGoogle Scholar
  3. 3.
    C. Almoguera, D. Shibata, K. Forrester, J. Martin, N. Arnheim and M. Perucho, Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes, Cell, 53:549.Google Scholar
  4. 4.
    V.T.H.B.M. Smit, A.J.M. Boot, A.M.M. Smits, G.J. Fleuren, C.J. Cornelisse and J.L. Bos, Kras codon 12 mutations occur very frequently in pancreatic adenocarcinomas, Nucl. Acids Res. 16:7773 (1988).PubMedCrossRefGoogle Scholar
  5. 5.
    J.E. Hancock, A.I. Magee, J.E. Childs and C.J. Marshall, All ras proteins are polyisoprenylated but only some are palmitoylated, Cell, 57:1167(1989)PubMedCrossRefGoogle Scholar
  6. 6.
    P.N. Lowe, M.J. Page, S. Bradley, S. Rhodes, M. Sydenham, H. Paterson and R.H. Skinner, Characterization of recombinant human Kirstenras (4B) p21 produced at high levels in Escherichia coli and insect baculovirus expression systems, J. Biol. Chem. 266:1672(1991).PubMedGoogle Scholar
  7. 7.
    J. Grodberg and J.J. Dunn, ompT encodes the Escherichia coli outermembrane protease that cleaves T7 RNA polymerase during purification, J. Bacteriol. 170:1245 (1988).PubMedGoogle Scholar
  8. 8.
    J. Feuerstein, H.R. Kalbitzer, J. John, R.S. Goody and A. Wittinghofer, Characterisation of the metal-ion-GDP complex at the active sites of transforming and nontransforming p21 proteins by observation of the 170-Mn superhyperfine coupling and by kinetic methods, Eur. J. Biochem. 162:49(1987).PubMedCrossRefGoogle Scholar
  9. 9.
    J. John, M. Frech, and A. Wittinghofer, Biochemical properties of Ha-ras encoded p21 mutants and mechanism of the autophosphorylation reaction, J. Biol. Chem. 263:11792 (1988).PubMedGoogle Scholar
  10. 10.
    J. John, I. Schlichting, E. Schilz, P. Rosch, and A. Wittinghofer, C-terminal truncation of p21H preserves crucial kinetic and structural properties, J. Biol. Chem. 264:13086 (1989).PubMedGoogle Scholar
  11. 11.
    M.S. Marshall, W.S. Hill, A.S. Ng, U.S. Vogel, M.D. Schaber, E.M. Scolnick, R.A.F. Dixon, I.S. Sigal and J.B. Gibbs, A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity, EMBO J. 8:1105 (1989)PubMedGoogle Scholar
  12. 12.
    D.K. Stammers, M. Tisdale, S. Court, V. Parmar, C. Bradley, and C.K. Ross, Rapid purification and characterisation of HIV-1 reverse transcriptase & RNaseH engineered to incorporate a C-terminal tripeptide α-tubulin epitope, FEBS Lett. in press (1991).Google Scholar
  13. 13.
    J. Wehland, H.C. Schroder, and K. Weber, Amino acid sequence requirements in the epitope recognized by the α-tubulin-specific rat monoclonal antibody YL1/2, EMBO J. 3:1295 (1984)PubMedGoogle Scholar
  14. 14.
    J.V. Kilmartin, B. Wright, and C. Milstein, Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line, J.Cell Biol. 93:576 (1982)PubMedCrossRefGoogle Scholar
  15. 15.
    R. Halenbeck, W.J. Crosier, R. Clark, F. McCormick and K. Koth, Purification, characterization, and Western blot analysis of human GTPase-activating protein from native and recombinant sources, J. Biol. Chem. 265:21922 (1990).PubMedGoogle Scholar
  16. 16.
    U. Bank and S. Roy, A continuous fluorimetric assay for ATPase activity, Biochem. J. 266:611 (1990).Google Scholar

Copyright information

© Plenum Press, New York 1991

Authors and Affiliations

  • Peter N. Lowe
    • 1
  • Susan Rhodes
    • 1
  • Susan Bradley
    • 1
  • Richard H. Skinner
    • 1
  1. 1.Department of Cell Biology, Wellcome Research LaboratoriesLangley CourtBeckenham, KentUK

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