We used linker insertion-deletion mutagenesis to study the catalytic domain of the Harvey murine sarcoma virus v-rasH transforming protein, which is closely related to the cellular rasH protein. The mutants displayed a wide range of in vitro biological activity, from those that induced focal transformation of NIH 3T3 cells with approximately the same efficiency as the wild-type v-rasH gene to those that failed to induce any detectable morphologic changes. Correlation of transforming activity with the location of the mutations enabled us to identify three nonoverlapping segments within the catalytic domain that were dispensable for transformation and six other segments that were required for transformation. Segments that were necessary for guanosine nucleotide (GDP) binding corresponded to three of the segments that were essential for transformation; two of the three segments share strong sequence homology with other purine nucleotide-binding proteins. Loss of GDP binding was associated with apparent instability of the protein. Lesions in two of the three other required regions significantly reduced GDP binding, while small lesions in the last required region did not impair GDP binding or membrane localization. We speculate that this latter region interacts with the putative cellular target of ras. The results suggest that transforming ras proteins require membrane localization, guanosine nucleotide binding, and an additional undefined function that may represent interaction with their target.
Several ras genes have been expressed at high levels in Escherichia coli and the resultant ras proteins were shown to be functional with respect to their well-known specific, high-affinity, GDP/GTP binding. We were able to detect a weak GTPase activity associated with the purified proteins. The normal cellular ras protein (p21N) exhibits -40 times higher GTPase activity than the "activated" proteins. Even though the turnover rate of the reaction is very low (0.02 mol of GTP hydrolyzed per mol of p21N protein per minute), the reaction appears to be catalytic; one molecule of p21N hydrolyzes more than one molecule of GTP. The GTPase and the GDP binding activities both have been recovered from a Mr 23,000 protein eluted following NaDodSO4/polyacrylamide gel electrophoresis, suggesting that these two activities are associated with the same protein. Mg2e ions and dithiotheitol are required for GTPase activity and the optimal pH is between 7 and 8. Guanidine HCl, which is required for solubilizing bacterially expressed ras protein, is strongly inhibitory to GTPase activity at concentrations higher than 0.5 M.Members of the ras gene family encode closely related proteins of "189 amino acid residues and Mr 21,000 termed p21 ras (1, 2). The ras genes were first identified as the oncogenic sequences of certain strains of acute transforming retroviruses (3). Their normal counterparts (cellular ras genes) are highly conserved in eukaryotic cells (4-10). Cellular ras genes acquire transforming properties by single point mutations within their coding sequences (11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and the "activated" ras genes are detected in a significant fraction of human cancers and in experimentally obtained animal tumors (21)(22)(23). The observed conservation of cellular ras genes and analysis of their expression (24, 25) have led to speculations that p21 proteins are essential components of normal cells involved in cell division and differentiation (26). It has also been observed that elevated expression of the normal cellular p21 gene can induce transformation (27). More recently, Stacey and Kung (28) observed that both mutated and normal p21 proteins can induce transformation of NIH 3T3 cells, when introduced by microinjection, but that higher concentrations of the normal protein were required. These observations strongly suggest that high levels of cellular p21 protein, as well as low amounts of activated p21 protein have the same effect on NIH 3T3 cells and this might be due to altered or lack of regulation of cellular p21 activity consequent to its "activation" by mutation. However, various studies aimed at understanding the biochemical basis for the transforming activity of ras gene products gave no major differences in their subcellular localization, post-translational modification, and in vitro guanine nucleotide binding properties of normal and activated ras proteins (29,30).We have shown recently that bacterially produced p21 protein binds guanine nucleotides with high affinity using a simple nitro...
The mammalian p21 ras proteins contain a 20‐amino acid region that is highly divergent, in contrast to the strong sequence conservation that is common to other regions of these proteins. This major variable region is located near the C terminus just upstream from a conserved cysteine residue that is required for post‐translational processing, membrane localization and transforming activity of the proteins. We have now used the viral oncogene (v‐rasH) of Harvey sarcoma virus to study the major variable region by deleting or duplicating parts of the gene. Reducing this region to five amino acids or increasing it to 50 amino acids has relatively little effect on the capacity of the gene to induce morphological transformation of NIH 3T3 cells. Assays of GTP binding, GTPase and autophosphorylating activities of such mutant v‐rasH‐encoded proteins synthesized in bacteria indicated that the sequences that encode these biochemical activities are located upstream from the major variable region. In the context of transformation, we propose that the region of sequence heterogeneity serves principally to connect the N‐terminal catalytic domain with amino acids at the C terminus that are required to anchor the protein in the membrane.
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