To understand the molecular principles involved in the cellular transformation by ras oncogenes has been the major focus of this project. The understanding of the structure-function relationship of ras protein is important in this regard. As a necessary prelude, we have changed v-H-ras DNA at two positions with point mutations by oligonucleotide-directed mutagenesis. The v-ras protein differs from the cellular proto-oncogene product at the amino acid positions 12 and 59. We have obtained mutants 12R/59T (equivalent to v-ras), 12R/59A, 12R/59S, 12G/59T, 12G/59S and 12G/59A (equivalent to c-ras) We compared some of the biochemical properties of these proteins, especially guanine nucleotide binding, nucleotide exchange and GTPase activities. Our preliminary results indicate that single point mutations, either at positions 12 or 59, produce oncogenic activation reflected in their GTPase activity (lowered). Position 59 is important in the nucleotide exchange of ras proteins. Threonine or serine are required for higher rate of exchange. Alanine at position 59 renders a poor exchange of nucleotides. Thus, altered amino acids at positions 12 and 59 sustain the v-ras protein in an activated state. Our studies are continuing in the direction of understanding the signal-transducing role of p21.
