Abstract
We have used linker insertion-deletion mutagenesis to study the Harvey murine sarcoma virus v-ras H transforming protein. The mutants were characterized with respect to their ability to induce morphological transformation of NIH 3T3 cells and the capacity of their proteins to bind guanosine nucleotides, undergo post-translational processing, and localize to the plasma membrane. We have identified four non-overlapping segments that are dispensable for morphological transformation of NIH 3T3 cells, as well as several segments that are required for transformation and stability in mammalian cells and guanosine nucleotide binding. One essential segment that does not affect guanine nucleotide binding or stability, which appears to lie on the exterior of the protein and therefore may interact with the putative ras protein target, has been identified (the effector domain, Willumsen et al., 1986, Sigal et al., 1986). A selected group of these mutations, which leave the v-ras H protein stable, processed and correctly localized, have been transferred to the c-ras H allele; the proteins were expressed in E. coli and assayed for the susceptibility to acceleration of their intrinsic GTPase activities by the protein GAP (GTPase Activating Protein, Trahey and McCormick, 1987). The results show that only mutations in the effector domain destroy GAP susceptibility (Adari et al., 1988); however, not all mutations affect both activities coordinately. These results suggest that GAP as well as the effector mediating p21 transformation interact through the same region on p21. The identification of mutations that destroy transformation when present in the v-ras H allele which do not destroy the GAP susceptibility of p21 protein from the c-ras H allele raises the possibility that the two factors may not be the same.
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Willumsen, B.M. et al. (1989). A Mutational Analysis of ras Function. In: Bosch, L., Kraal, B., Parmeggiani, A. (eds) The Guanine — Nucleotide Binding Proteins. Springer, Boston, MA. https://doi.org/10.1007/978-1-4757-2037-2_16
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DOI: https://doi.org/10.1007/978-1-4757-2037-2_16
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