Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects.

Abstract

We report that residues Lys-16 and Asp-l19 play critical roles in the guanine nudeotide binding and, consequently, the biological function of the Ha-ras-encoded protein (Ha). Substitution of an asparagine residue for Lys-16 reduces the affinity of Ha for GDP and GTP by a factor of 100 but does not alter the specificity of nucleotide binding. The replacement of Asp-119 with an alanine residue reduces the affinity of Ha for GDP and GTP by a factor of 20 and reduces the relative affinity of Ha for GDP over IDP from 200-500 to 10. Based on these observations, a structural model for the GDP/GTP-binding site of Ha is proposed. By microinjecting purified proteins into NIH 3T3 cells, we observed that the ability of [Ala'19] Comparisons of amino acid sequences suggested two peptide regions of Ha-ras protein (Ha) p21 that might be involved in nucleotide interactions. Ha p21 sequence 5-20 is homologous to sequences present in a large series of nucleotide-binding proteins, with the sequence Gly-Lys that occurs at positions 15-16 being invariant (14). Structural studies of adenylate kinase have implicated the involvement of this region with binding to the phosphate groups of the nucleotide (15). The sequence Asn-Lys-Xaa-Asp that occurs at positions 116-119 is less universal but occurs in the known sequences for G proteins [e.g., EF-Tu, EF-G, and a-transducin (16, 17)].To examine the role of these residues in Ha-ras protein function, we investigated the biochemical properties of Ha proteins having amino acid substitutions at positions 16, 117, and 119. The biological properties of these proteins were determined in both mammalian and yeast cells. MATERIALS AND METHODSProtein Biochemistry. Oligonucleotide mutagenesis (11) of the genes for Ha p21 and [Val12,Thr59Ha was used to create ras variants, with all mutations confirmed by nucleotide sequencing. Mutant proteins were expressed in Escherichia coli strain HB101 and purified by chromatography on DEAESephacel and Sephadex G-75 in buffer A (50 mM sodium Hepes, pH 7.5/1 mM sodium EDTA/1 mM sodium EGTA/ 1 mM dithiothreitol/1 mM phenylmethylsulfonyl fluoride/0.01% n-octyl glucoside), as described (11). For [Ala'19]Ha, 5 mM MgCl2 and 10 ,M GDP were included in buffer A through the DEAE-Sephacel chromatography (4°C), and then purification was continued in buffer A by highperformance liquid chromatography (LKB) on a column of Superose 12 (Pharmacia) at 22°C, followed by immediate chilling. Protein determinations and other assays were as described (11).In trypsin-digestion experiments, pure Ha proteins (2-4 ,ug) were incubated at 4°C in 10 ,u of 50 mM sodium Hepes, pH 7.5/10 mM MgCl2/1 mM dithiothreitol with nucleotides (Sigma) for 15 min prior to the addition of 1 ug of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Sigma). Reactions were terminated by the addition of 3 ,ug of soybean trypsin inhibitor (Sigma).Equilibrium-dialysis measurements of nucleotide binding utilized 4-mm dialysis tubing with a 12-to 14-kDa cutoff (Spectra/Por 2, Spectrum Medic...

Signal transduction elements of TC21, an oncogenic member of the R-Ras subfamily of GTP-binding proteins

“…Since it is known that mutations that increase the nucleotide turn-over rates of Ras proteins are tumorigenic (Sigal et al, 1986), our results suggest that quiescent cells should have some inhibitory mechanism to downmodulate the high nucleotide exchange rate of TC21. One of these mechanisms could be the expression of TC21 only when it is required for speci®c biological Figure 8 Upper panels, activation of JNK by TC21.…”

Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence

“…The amino acids of this conserved region have been shown to be important for binding nucleotides in these proteins by nuclear magnetic resonance (17)(18)(19), X-ray crystallography (24,55,69), and genetic (23,49,60) studies. The highly conserved lysine is thought to function by interacting with the ␥-phosphate of ATP (17-19, 55, 69), and changes at this position greatly reduce nucleotide binding (5,23,46,48,52,60). To explore whether this nucleotide-binding site is important in virulence, the virB11 gene was modified by oligonucleotidedirected mutagenesis to inactivate the Walker nucleotide-binding site.…”

Systematic Structure-Function Analysis of the Small GTPase Arf1 in Yeast

“…Furthermore, although destabilization of the double mutant cannot be ruled out, the mutation arf1-117 alone clearly does not significantly decrease the stability of Arf1p because this mutation has a very mild phenotype on its own (Figure 2). Mutation arf1-118 is in the so-called guanine nucleotide specificity region, mutations of which have been shown by studies in multiple small GTPase family members to result in decreased affinity of protein for nucleotide primarily as a result of an increased nucleotide dissociation rate (Sigal et al, 1986;Walter et al, 1986). This, in turn, has been proposed to result in one or both of the following effects in cells: a shift toward the GTP-bound conformation and interaction with effector proteins or a shift toward nucleotide-free protein and subsequent sequestra-tion of GEFs (Feig et al, 1986;Ziman et al, 1991;Jones et al, 1995;Schmidt et al, 1996;Cool et al, 1999).…”