Antibodies specific for amino acid 12 of the ras oncogene product inhibit GTP binding.

Abstract

An antibody (anti-p2lser) was raised against a ras p21-related synthetic peptide and was able to recognize specifically the substitution of serine for glycine at amino acid 12 of p21. This substitution causes oncogenic activation of p21. Anti-p2lser was found to immunoprecipitate v-Ki-ras p21 and to strongly inhibit its ability to autophosphorylate and to bind GTP in an immunoabsorption assay. Furthermore, binding of the antibody to p21 was specifically inhibited by GTP or GDP, suggesting that amino acids around position 12 are part of the GTP/GDP binding site. These results, taken together with the observation that the microinjection of anti-p2lser into cells transformed by v-Ki-ras p21 causes a transient reversion of the cells to a normal phenotype [Feramisco, J. R., Clark, R., Wong, G., Arnheim, N., Milley, R. & McCormick, F. (1985) Nature (London) 314; 639-642], support the idea that interaction of p21 with guanine nucleotides is crucial to the transforming function of this protein.The p21 proteins, encoded by members of the ras protooncogene family, cause the neoplastic transformation of certain cells in culture when activated through somatic mutation and have been implicated in the generation of a variety of human cancers. The most frequently observed mutation leading to the oncogenic activation of p21 results in the alteration of the glycine residue at position 12 of the protein to any of several other residues (1). p21 is known to specifically bind to GTP and hydrolyze it to GDP and inorganic phosphate (2-5). Substitution of valine for glycine at position 12 reduces the rate of GTP hydrolysis by a factor of about 10 (3-5). In forms of p21 that contain a threonine residue at position 59, phosphate is transferred to this residue by an autophosphorylation reaction (2, 6). The precise role of these activities in the transforming potential of activated p21 is not known. To learn more about the mechanism of p21 activation, we have used synthetic peptides (7-9) to produce an antibody (anti-p2lser) that specifically recognizes the substitution of serine for glycine at position 12 in an activated p21 (v-Ki-ras) and have used this antibody as a specific probe to study the structure and biochemical functions of p21. MATERIALS AND METHODSSynthetic Peptides. The peptide shown in Fig. 1 was synthesized with an automated SAM Peptide Synthesizer (Biosearch, San Rafael, CA) using standard solid-phase chemistry (11). Homogeneity of the peptide was confirmed by reversed-phase HPLC and amino acid analysis. Conjugation of the peptide to the carrier was achieved by a crosslinking reagent, a 6-(N-maleimido)aminocaproic acid ester of the sodium salt of 4-hydroxy-3-nitro-benzenesulfonic acid. This reagent couples via its active ester to amino groups of carrier proteins; the coupling can be quantitated by monitoring the release of 4-hydroxy-3-nitrobenzenesulfonate. Coupling of peptide to carrier results from the reaction of the maleimido group with the sulfhydryl group of the cysteine on the peptide. A complete descri...

Monoclonal antibody Y13-259 recognizes an epitope of the p21 ras molecule not directly involved in the GTP-binding activity of the protein.

“…While the results of some investigations have been consistent with our conclusions concerning the lack of effect of Y13-259 on GTP binding by p21 (4), there have been contradictory reports as well. Furth et al (9) found that Y13-259 inhibited GTP binding in studies utilizing crude cell extracts, results that were not reproducible with more purified eucaryotic p21 preparations and highly purified antibody (Furth, personal …”

Mutational analysis of a ras catalytic domain.

“…Certain sequences outside these nonessential segments appear to be required for the in vitro GDP-binding activity of the protein, and this activity appears to be essential for efficient NIH 3T3 transformation. The importance of guanosine nucleotide binding for the transforming activity of ras has also been suggested by the observation that microinjection of ras-transformed cells with a ras antibody that prevents GDP binding in vitro can induce morphologic reversion (5).…”

“…This linker results in the addition of three novel amino acids within the protein at the site of the deletion, as shown in Table 1 Table 1; data for mutants in D, 7, and 8, which are included for comparative purposes, are from reference 42. The two transformation-competent mutants with undetectable GDP-binding activities whose deleted sequences border essential region 5 are pBW1244 and pBW1220. The hydropathic index has been plotted by the method of Kyte and Doolittle (19); hydrophobic regions are above the axis of the midpoint line, hydrophilic regions are below.…”

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

“…Antigen-antibody complexes were absorbed to 20 pA of 50% (wt/vol) PAS for 10 min at 4°C. The PAS was pelleted by centrifugation and washed four times in 50 mM Tris (pH 8.0)-0.5% Nonidet P-40-0.12 M NaCl-1 M LiCl (9). Proteins were eluted from the PAS by boiling in sample mix (29) at 250 puCi/ml was in Dulbecco MEM plus 5% dialyzed calf serum for 16 h. Immunoprecipitation of R-ras p23 from labeled HT1080 cells with fusion-protein antiserum was performed exactly as described by Platt et al (47).…”

“…R-ras codon 27 Ser (AGC) was mutated to Met (ATG) by using the mutant oligonucleotide 5'-GTGTGTCTCCATGGGCGGGG and the protection primer 5'-GGGATGCCATCCACA CTG, which hybridizes 119 bp 5' of the mutagenesis primer. The R-ras expression vectors utilizing the SV40 early promoter consisted of the BamHI-EcoRI vector fragment of pML-1 (34) with the EcoRI site fused to the HinclI site of the HinclI-HindIll 600-bp fragment of SV40 (13) For cotransformation and selection, the plasmid pNeo DHFR was constructed using the BamiHI-SalI fragment of pMTE4E (48), which contains, in order of 5' to 3', an SV40 early promoter linked to the aminoglycosyl phosphotransferase II gene (59), followed by the hepatitis B surface antigen gene 3' untranslated region (10) and an SV40 early promoter linked to dihydrofolate reductase coding sequence (58) (9). Cells from 20-ml cultures were suspended in 600 ,ul of 50 mM glucose-25 mM Tris hydrochloride (pH 8.0)-10 mM EDTA-5 mg of lysozyme per ml-0.01% aprotinin-1 mM phenylmethylsulfonyl fluoride and incubated at 4°C for 30 min.…”