Identification of Effector Residues and a Neutralizing Epitope of Ha-ras-Encoded p21

• Published in: Proceedings of the National Academy of Sciences - PNAS 1986-07, Vol.83 (13), p.4725-4729
• Main Authors: Sigal, Irving S., Gibbs, Jackson B., D'Alonzo, Jill S., Scolnick, Edward M.
• Format: Article
• Language: English
• Subjects: 3T3 cells; Adenylyl Cyclases - metabolism; Amino Acid Sequence; Amino acid substitution; Amino acids; Antibodies; Antibodies, Monoclonal - immunology; Binding, Competitive; Biochemistry; Biological and medical sciences; Cell Membrane - metabolism; Cell physiology; Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes; Cloning, Molecular; Epitopes; Fundamental and applied biological sciences. Psychology; GTP-Binding Proteins - genetics; GTP-Binding Proteins - immunology; GTP-Binding Proteins - metabolism; Immunosorbent Techniques; Models, Molecular; Molecular and cellular biology; Monoclonal antibodies; Mutant proteins; Mutation; NIH 3T3 cells; Oligopeptides - immunology; Oncogene Proteins, Viral - genetics; Oncogene Proteins, Viral - immunology; Oncogene Proteins, Viral - metabolism; Peptide Elongation Factors - metabolism; Saccharomyces cerevisiae - enzymology; Sodium; Structure-Activity Relationship; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.83.13.4725

To identify the amino acid residues of the Harvey (Ha) ras-encoded protein that are involved in protein-protein interactions, we have created a series of mutant Ha-ras proteins. In particular, amino acid substitutions have been introduced within two regions, residues 32-42 and 61-80, that are conserved among ras proteins from different species. We observed that amino acid substitutions at positions 35, 36, 38, 40, and, to a lesser extent, 39 and 78 reduce the biological potency of Ha-ras protein in both mammalian and Saccharomyces cerevisiae cells, without noticeably affecting the known intrinsic biochemistry of these proteins. The reduction of in vivo activity for these mutant ras proteins correlates with their reduced ability to stimulate yeast adenylate cyclase. The ras-protein-neutralizing antibody Y13-259 binds to six residues: Glu-63, Ser-65, Ala-66, Met-67, Gln-70, and Arg-73. Single substitutions for these residues reduce Y13-259 antibody binding by at least a factor of 1000 but do not significantly affect biological activity. These data are discussed in terms of the model for Ha-ras protein based on the structure of the elongation factor EF-Tu-GDP complex.