Heterologous expression and characterization of the human R-ras gene product.

Abstract

We directly expressed human R-ras 23,000-dalton protein (p23) cDNA in Escherichia coli under the control of the tip promoter. GTP-dependent phosphorylation of a p23 threonine 85 substitution mutant was observed. This result is in direct analogy to the autokinase activity of H-ras and K-ras threonine 59 substitution mutants. Normal p23 protein was detected in the human fibrosarcoma cell line HT1080 by immunoprecipitation with rabbit antibodies raised against an E. coli-expressed R-ras fusion protein. The R-ras p23 protein was found to be 3H labeled in the presence of [9,10(n)-3H]palmitic acid and is associated with the P100 membrane fraction of HT1080 cells. These data suggest that human R-ras p23 has biochemical properties very similar to those of the p21 products of the H-, K-, and N-ras proto-oncogenes. We constructed an R-ras minigene and engineered the expression of normal and mutant alleles from the simian virus 40 early region promoter. Normal and mutant R-ras gene products were authenticated by transient expression in COS-7 cells and immunoprecipitation. The valine 38-substituted R-ras p23 displayed reduced electrophoretic mobility. R-ras p21-like proteins, made by eliminating the first 26 R-ras codons, displayed evident mobility differences between the pro form and mature form, along with a valine 12 substitution-dependent change in electrophoretic mobility. Rat-l fibroblasts were transfected with normal and mutant R-ras alleles and normal and activated H-ras alleles. Unlike the human T24 bladder oncogene-encoded p21, mutant R-ras alleles do not cause monolayer focus formation or growth in soft agar of rat fibroblasts.The human genome contains three closely related protooncogenes, H-rasl, K-ras2, and N-ras, which encode closely related 21,000-dalton proteins (p21s) of 188 to 189 amino acids (5,6,38,57,64). These genes were first identified through their mutant alleles, found as the transforming genes in transducing retroviruses and in high-molecular-weight tumor DNA (67). To understand the dominant phenotype of mutant ras genes in malignant transformation, considerable attention has been paid to the biochemical properties of ras p21 proteins (70). H-ras p21 is synthesized on free ribosomes as pro-21 (56), whereupon it is modified to its mature form by the addition of palmitic acid to cysteine 186 (8, 72, 73) and is localized to the inner surface of the plasma membrane (71). The H-, K-, and N-ras p2ls are GTP-binding proteins with intrinsic GTPase activity (17,37,51,62). The unique regulatory roles of GTP-binding proteins (27) and the biochemical similarities between ras p2ls and the adenylyl cyclase G proteins (18, 27) suggest that ras proteins play a role in cell surface signal transduction.By virtue of nucleic acid sequence homology to viral H-ras, we recently cloned another member of the mammalian ras gene family, R-ras (33). The predicted human R-ras gene product of 218 amino acids and 23,400 daltons (p23) has an amino-terminal extension of 26 amino acids relative to the H-, K-, and N-ras p2ls. T...

The C-terminal end of R-Ras contains a focal adhesion targeting signal

“…R-Ras is palmitoylated and contains a potential palmitoylation site (C213) upstream of the CAAX box (Lowe and Goeddel, 1987;Schmittberger and Waldmann, 1999). We showed that EGFP-R-Ras38V is labelled with palmitate, whereas EGFP-R-Ras38V/C213A, which contains a mutation in the potential palmitoylation site did not incorporate palmitate, suggesting that cysteine 213 is the target for palmitoylation in R-Ras (Fig.…”

Intracellular localization of the P21rho proteins.

“…COS-I ceils were transfected with 10 ~g plasmid DNA per 10-cm 2 petri dish using a DEAE-dextran method as described previously (Lowe and Goeddel, 1987). After transfection, cells were grown for 48 h after which they were harvested and fractionated as previously described (Cales et al, 1988) except that membranes were pelleted at 120,000 g for 30 min to produce P100 pellets and S100 supernatants.…”

Characterization of four novel ras-like genes expressed in a human teratocarcinoma cell line.

“…GTPase-activating protein, a possible ras effector protein, has recently been shown to stimulate the GTPase activity of R-ras but not rho proteins (15). The recently discovered transformation suppressor activity of rapl (20), and the lack of transforming activity of R-ras mutants containing in vitro-constructed mutations corresponding to those that activate ras proto-oncogenes (22), has led to the suggestion that members of this distinct ras family group (R-ras, rap genes, and TC21) might transduce growth inhibitory signals across the cell membrane, exerting their effects through an effector shared with the ras proteins but in an antagonistic fashion.…”