The product of ras is a GTPase and the T24 oncogenic mutant is deficient in this activity

Abstract

Ha-ras is a member of a multigene family in man which encode highly related proteins of 189 amino acids (p21)^1–6. In vitro, ras proteins bind GTP^7–9, and p21 mutants with threonine at position 59 autophosphorylate at that residue^10–12. Mutation (at amino acids 12 or 61)^6,13–6 and elevated expression¹⁷ of ras genes result in cell transformation in culture, and are also observed in many types of human tumours¹⁸. Normal and mutant transforming ras proteins show no differences in localization, lipidation or GTP binding⁹. However, mutations at position 12 in recombinant (Thr59) p21 molecules were observed to affect autophosphorylation¹². We have expressed the full-length normal and T24 transforming (Gly → Val at position 12) Ha-ras proteins in Escherichia coli and have purified them to homogeneity (ref. 19 and M.G. et al., in preparation); these proteins bound GTP with approximately molar stoichiometry and with an affinity comparable to partially purified mammalian proteins. Microinjection of the T24 protein into quiescent rodent fibroblasts resulted in a rapid alteration in cell morphology, stimulation of DNA synthesis and cell division¹⁹; in contrast, little response was observed with the normal protein. We now report that the normal ras protein has an intrinsic GTPase activity, yielding GDP and P_i. In contrast, the T24 transforming protein is reduced 10-fold in this activity. We suggest that this deficiency in GTPase is the probable cause for the transforming phenotype of the T24 protein.