Critical amino acids of p21 protein are located within beta-turns: further evaluation

It has been shown that malignant activation of ras proto-oncogenes was mediated by point mutations which resulted in the single amino acid conversions at positions 12, 13 or 61 of the ras gene products (p21 proteins). By analyzing randomly mutated ras genes, it has been demonstrated that amino acid substitutions at residues 12, 13, 59 and 63 activated p21. Furthermore, it has been shown that residues 16, 116 and 119 in p21 played critical roles in the guanine nucleotide binding and, consequently, the ability of the protein to induce changes characteristic of cellular transformation. By using the protein conformational prediction method of Chou and Fasman, the present work predicts that these critical amino acids, except glutamic acid at position 63, are located within beta-turns. The major "hot spots" for ras activation are codons 12 and 61. The author has predicted in an earlier paper that the single amino acid conversions at positions 12 and 61 would occur at beta-turn conformation consisting of residues 10-13 and 58-61, respectively. In the present study, probabilities of beta-turn occurrence at residues 10-13 or 58-61 of the p21 proteins encoded by various ras genes are compared. The probability for the normal p21 containing glycine as residue 12 is greatest, and the cancer-associated variants show less probabilities. The single amino acid substitutions at position 61 do not cause so decreased probabilities of beta-turn potential at residues 58-61, except the replacement by histidine. Histidine at position 61 is not predicted as occurring within a beta-turn.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic and hydrodynamic studies reveal structural differences in normal and transforming H-ras gene products

We have recorded the circular dichroism spectra of the cellular and the viral H-ras gene products both in the absence and in the presence of guanine nucleotides and analyzed these spectra in terms of the secondary structure composition of these proteins. It is shown that the GTP complex of the ras proteins has a different secondary structure composition than the GDP complex and, furthermore, that there are differences in the secondary structure of the viral ras protein and the cellular ras protein. We have also recorded and analyzed the circular dichroism spectrum of the isolated guanine nucleotide binding domain of the Escherichia coli elongation factor Tu (EF-Tu), which has been considered as a model for the tertiary structure of the ras proteins [McCormick, F., Clark, B. F. C., LaCour, T. F. M., Kjeldgaard, M., Norskov-Lauritsen, L., & Nyborg, J. (1985) Science (Washington, D.C.) 230, 78-82]. Our data show that the guanine nucleotide binding domain of EF-Tu (30% alpha-helix and 16% beta-pleated sheet for the GDP complex) has quite a different secondary structure composition than the ras proteins (e.g., the cellular ras protein has 47% alpha-helix and 22% beta-pleated sheet for the GDP complex), indicating that the protein core comprising the guanine nucleotide binding site might be similar but that major structural differences must exist at the portion outside this core. Normal and transforming ras proteins also differ slightly in their hydrodynamic properties as shown by sedimentation velocity runs in the analytical ultracentrifuge.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.

Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.     

Crystal structures at 2.2 A resolution of the catalytic domains of normal ras protein and an oncogenic mutant complexed with GDP

The biological functions of ras proteins are controlled by the bound guanine nucleotide GDP or GTP. The GTP-bound conformation is biologically active, and is rapidly deactivated to the GDP-bound conformation through interaction with GAP (GTPase Activating Protein). Most transforming mutants of ras proteins have drastically reduced GTP hydrolysis rates even in the presence of GAP. The crystal structures of the GDP complexes of ras proteins at 2.2 A resolution reveal the detailed interaction between the ras proteins and the GDP molecule. All the currently known transforming mutation positions are clustered around the bound guanine nucleotide molecule. The presumed "effector" region and the GAP recognition region are both highly exposed. No significant structural differences were found between the GDP complexes of normal ras protein and the oncogenic mutant with valine at position 12, except the side-chain of the valine residue. However, comparison with GTP-analog complexes of ras proteins suggests that the valine side-chain may inhibit GTP hydrolysis in two possible ways: (1) interacting directly with the gamma-phosphate and altering its orientation or the conformation of protein residues around the phosphates; and/or (2) preventing either the departure of gamma-phosphate on GTP hydrolysis or the entrance of a nucleophilic group to attack the gamma-phosphate. The structural similarity between ras protein and the bacterial elongation factor Tu suggests that their common structural motif might be conserved for other guanine nucleotide binding proteins.     

Cellular oncogenes and cancer

Human oncogenes have been identified either by the ability of normal or tumor DNAs to induce transformation of cells in culture or as the targets of chromosome translocations or DNA amplification in neoplasms. By the combination of these approaches, approximately 40 different genes have been implicated as potential contributors to the development of human neoplasms. The proteins which are encoded by these potential human oncogenes include plasma membrane proteins with tyrosine kinase activity, plasma membrane guanine nucleotide binding proteins, cytoplasmic proteins with serine/threonine kinase activity and nuclear proteins. In many tumors, more than 1 potential oncogene has been activated, suggesting that multiple genes may contribute to neoplasm pathogenesis. I will discuss the identification of these genes, their modes of activation, the diversity of their protein products and their potential roles in both neoplastic and normal cells.     

Expression and characterization of ras mRNAs from Saccharomyces cerevisiae.

The cellular homologs of the Harvey and Kirsten murine sarcoma virus oncogenes comprise a multigene family, ras, that displays striking evolutionary conservation. We recently reported [DeFeo-Jones et al., Nature (London) 306:707-709, 1983] the cloning of two ras homologs from the yeast Saccharomyces cerevisiae. The nucleotide sequences of these genes predict polypeptides that show remarkable homology to p21, the mammalian ras gene product. We have also found proteins in yeast lysates with serological cross-reactivity to p21 (Papageorge et al., Mol. Cell. Biol. 4:23-29, 1984). In this work, we explored the relationship between the immunoprecipitated proteins and the yeast ras genes. We show that both ras genes are expressed in the wild-type cell. Furthermore, we demonstrate by in vitro translation of hybrid-selected RASsc1 mRNA and immunoprecipitation of the translation products that the cloned RASsc1 gene encodes the proteins immunoprecipitated from yeast lysates by anti-p21 monoclonal antibody. Finally, we used anti-p21 monoclonal antibodies to detect a guanine nucleotide binding activity in yeast lysates. The structural and biochemical homologies between ras gene products of S. cerevisiae and mammalian cells suggest that information obtained by genetic analysis of ras function in a lower eucaryote should be applicable to higher organisms as well.     

Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins

The protein products of the mammalian ras genes, p21ras, are regulatory guanine nucleotide binding proteins that are involved in the control of cell proliferation, though the exact biochemical processes regulated are unknown. Recently a cytoplasmic protein has been identified that interacts with and increases the GTPase activity of p21ras. It has been shown that this GTPase-activating protein, or GAP, interacts with the effector domain of ras, leading us and others to propose that GAP may be the target for regulation by p21ras. It has become apparent that ras is part of a much larger family of proteins, and at least 15 ras-related genes have now been identified in the mammalian genome. Each encodes a small (about 21 kDa) guanine nucleotide binding protein, but the functions of none of these regulatory molecules are known. We report here that mammalian cytoplasmic extracts contain GAP-like activity toward the products of two other ras-related genes, R-ras and rho. It appears that p23R-ras interacts with the same 125-kDa GAP protein as p21ras whereas p21rho interacts with a distinct 29-kDa protein, rho GAP.     

Ras activation by insulin and epidermal growth factor through enhanced exchange of guanine nucleotides on p21ras.

A number of growth factors, including insulin and epidermal growth factor (EGF), induce accumulation of the GTP-bound form of p21ras. This accumulation could be caused either by an increase in guanine nucleotide exchange on p21ras or by a decrease in the GTPase activity of p21ras. To investigate whether insulin and EGF affect nucleotide exchange on p21ras, we measured binding of [alpha-32P]GTP to p21ras in cells permeabilized with streptolysin O. For this purpose, we used a cell line which expressed elevated levels of p21 H-ras and which was highly responsive to insulin and EGF. Stimulation with insulin or EGF resulted in an increase in the rate of nucleotide binding to p21ras. To determine whether this increased binding rate is due to the activation of a guanine nucleotide exchange factor, we made use of the inhibitory properties of a dominant negative mutant of p21ras, p21ras (Asn-17). Activation of p21ras by insulin and EGF in intact cells was abolished in cells infected with a recombinant vaccinia virus expressing p21ras (Asn-17). In addition, the enhanced nucleotide binding to p21ras in response to insulin and EGF in permeabilized cells was blocked upon expression of p21ras (Asn-17). From these data, we conclude that the activation of a guanine nucleotide exchange factor is involved in insulin- and EGF-induced activation of p21ras.     

The structure of Ras protein: a model for a universal molecular switch.

X-ray crystallography has revealed the molecular architecture of the cellular and oncogenic forms of p21Ha-ras, the protein encoded by the human Ha-ras gene, in both its active (GTP-bound) and in its inactive (GDP-bound) forms. From comparison of these two structures, a mechanism is suggested for the GTPase hydrolysis reaction that triggers the conformational change necessary for signal transduction. The structures have also allowed identification of the structural consequences of point mutations and the way in which they interfere with the intrinsic GTPase activity of p21ras. The p21ras structure is similar to that of the G-domain of elongation factor Tu (EF-Tu) from Escherichia coli, suggesting that p21ras can serve as a good model for other guanine nucleotide binding proteins.     

Biological and biochemical properties of human rasH genes mutated at codon 61

Using site-directed mutagenesis, we have introduced mutations encoding 17 different amino acids at codon 61 of the human rasH gene. Fifteen of these substitutions increased rasH transforming activity. The remaining two mutants, encoding proline and glutamic acid, displayed transforming activities similar to the normal gene. Overall, these mutants vary over 1000-fold in transforming potency. Increased levels of p21 expression were required for transformation by weakly transforming mutants. The mutant proteins were unaltered in guanine nucleotide binding properties. However, all 17 different mutant proteins displayed equivalently reduced rates of GTP hydrolysis, 8- to 10-fold lower than the normal protein. There was no quantitative correlation between reduction in GTPase activity and transformation, indicating that reduced GTP hydrolysis is not sufficient to activate ras transforming potential.     

Mutagenesis of bacterial elongation factor Tu at lysine 136. A conserved amino acid in GTP regulatory proteins

We have studied the effects of specific amino acid replacements in EF-Tu upon the protein's interactions with guanine nucleotides and elongation factor Ts (EFTs). We found that alterations at the lysine residue of the Asn-Lys-Cys-Asp sequence, the guanine ring-binding sequence, differentially affect the protein's ability to bind guanine nucleotides. Wild type EF-Tu (Lys-136) binds GDP and GTP much more tightly than do many of the altered proteins. Replacing lysine by arginine lowers the protein's affinity for GDP by about 20-fold relative to the change in its affinity for EF-Ts. Substitutions at residue 136 by glutamine (K136Q) and glutamic acid (K136E) further lower the protein relative affinity for GDP by factors of about 4 and 10, respectively. In contrast, replacement of the residue by isoleucine (K136I) eliminates guanine nucleotide binding as well as EF-Ts binding. Apparently, the distortion of this loop by substitution at residue 136 of a bulky hydrophobic residue can hamper the binding for both substrates or disrupt the folding of the protein. All altered proteins except EF-Tu(K136I) are able to bind tRNA(Phe); however, they require much higher concentrations of GTP than wild type EF-Tu. In minimal media, Escherichia coli cells harboring plasmids encoding EF-Tu(K136E) or EF-Tu(K136Q) suffer growth retardation relative to cells bearing the same plasmid encoding wild type EF-Tu. Co-transformation of these cells with a compatible plasmid bearing the EF-Ts gene reverses this growth problem. The growth retardation effect of some of the altered proteins can be explained by their sequestering EF-Ts. These results indicate that EF-Ts is essential to the growth of E. coli and suggest a technique for studying EF-Ts mutants as well as for identifying other guanine nucleotide exchange enzymes.     

G-protein linked polyphosphoinositide phospholipase C activity in cell membranes from clonally derived and ras-transformed Madin-Darby canine kidney cells

Membranes prepared from clone D1 of Madin-Darby canine kidney (MDCK) cells contain activity that can be attributed to Gp, a guanine nucleotide binding protein linked to phosphatidylinositol 4,5-bisphosphate dependent phospholipase C. Polyphosphoinositides are produced by addition of GTP, nonhydrolyzable GTP analogs, or fluoroaluminate. This production is inhibited by guanosine 5'-(beta-thiodiphosphate). While Ca2+ at 1 microM or more can generate high yields of inositol phosphates, guanine nucleotide activation of Gp can potentiate this Ca2(+)-dependent yield at resting levels of the cation. Membranes from cells expressing large amounts of ras-p21 exhibit small differences in guanine nucleotide induced polyphosphoinositide quantities. The greatest difference between normal and ras membranes was seen with AlF4- incubation. Of the three inositol phosphates measured, only the inositol bisphosphate yield was greatly increased in ras membranes compared with membranes from both parental and the D-1 clone of MDCK cells. From these data, we conclude that the presence of ras-p21 may affect production of polyphosphoinositides in MDCK cell membranes by some means other than direct participation in phospholipase C activation.     

Biochemical properties of the ras-related YPT protein in yeast: a mutational analysis.

Using site-directed mutagenesis, the ras-related and essential yeast YPT1 gene was changed to generate proteins with amino acid exchanges within conserved regions. Bacterially produced wild-type proteins were used for biochemical studies in vitro and were found to have properties very similar to mammalian ras proteins. Gene replacement allowed the study of physiological consequences of the mutations in yeast cells. Lys21----Met and Asn121----Ile substitutions rendered the protein incapable of binding GTP and caused lethality. Ser17----Gly and Ala65----Thr substitutions slightly changed the protein's apparent binding capacity for either GDP or GTP and altered its intrinsic GTPase activity. These mutations were without effect on cellular growth. The YPTgly17,thr65 mutant protein displayed a significantly altered relative capacity for guanine nucleotide binding but a GTPase activity comparable to the wild-type protein. In contrast to the Ala65----Thr substitution, the double mutant displayed a significantly reduced capacity for autophosphorylation and allowed cells to grow only poorly. Cellular growth was improved when this mutant protein was overproduced.     

The posttranslational processing of ras p21 is critical for its stimulation of yeast adenylate cyclase.

Mammalian ras genes substitute for the yeast RAS gene, and their products activate adenylate cyclase in yeast cells, although the direct target protein of mammalian ras p21s remains to be identified. ras p21s undergo posttranslational processing, including prenylation, proteolysis, methylation, and palmitoylation, at their C-terminal regions. We have previously reported that the posttranslational processing of Ki-ras p21 is essential for its interaction with one of its GDP/GTP exchange proteins named smg GDS. In this investigation, we have studied whether the posttranslational processing of Ki- and Ha-ras p21s is critical for their stimulation of yeast adenylate cyclase in a cell-free system. We show that the posttranslationally fully processed Ki- and Ha-ras p21s activate yeast adenylate cyclase far more effectively than do the unprocessed proteins. The previous and present results suggest that the posttranslational processing of ras p21s is important for their interaction not only with smg GDS but also with the target protein.     

Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu

Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu have been derived from the atomic coordinates of the trypsin-modified form of EF-Tu-GDP and by comparison with the ras p21 structures. The significance of the differences in the guanine nucleotide binding sites of EF-Tu and ras p21 are discussed. Crystallization of the EF-Tu-GMPPNP complex is reported.     

Evidence for multiple, ras-like, guanine nucleotide-binding proteins in Swiss 3T3 plasma membranes. Stimulation of GTPase activity by cytosolic factors

A novel method for the analysis of putative G-proteins has been developed that reveals the existence of a large family of GTP/GDP-binding proteins with similar characteristics to those of p21ras in 3T3 cell plasma membranes. In the presence of Mg2+, exchange of [alpha-32P]GDP with prebound ligand was very slow, but, as with p21ras, exchange was dramatically accelerated by excess EDTA. In the presence of Mg2+, three classes of binding sites were distinguishable. However, no p21ras was detected in the membranes with the pan-reactive anti-ras antibody, Y13-259. Gel filtration analysis resolved two peaks of binding activity centering at 60 and 21 kilodaltons. High resolution anion exchange chromatography separated at least 11 unique GDP-binding proteins from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate-solubilized membranes, none of which cross-reacted with a pan-G alpha antiserum. Analogous to p21ras, the binding activities of 9 of the 11 species were sensitive to the thiol reagent N-ethylmaleimide, and six peaks possessed detectable GTPase activity in the absence of extrinsic factors. Addition of cytosol activated the GTPase activity of four of the peaks. We infer that the 11 peaks represent novel, small molecular weight guanine nucleotide-binding proteins, similar to those recently described in brain membranes.     

Hydrogen bond interactions of G proteins with the guanine ring moiety of guanine nucleotides.

We have utilized Raman difference spectroscopy to investigate hydrogen bonding interactions of the guanine moiety in guanine nucleotides with the binding site of two G proteins, EF-Tu (elongation factor Tu from Escherichia coli) and the c-Harvey ras protein, p21 (the gene product of the human c-H-ras proto-oncogene). Raman spectra of proteins complexed with GDP (guanosine 5' diphosphate), IDP (inosine 5' diphosphate), 6-thio-GDP, and 6-18O-GDP were measured, and the various difference spectra were determined. These were compared to the difference spectra obtained in solution, revealing vibrational features of the nucleotide that are altered upon binding. Specifically, we observed significant frequency shifts in the vibrational modes associated with the 6-keto and 2-amino positions of the guanine group of GDP and IDP that result from hydrogen bonding interactions between these groups and the two proteins. These shifts are interpreted as being proportional to the local energy of interaction (delta H) between the two groups and protein residues at the nucleotide binding site. Consistent with the tight binding between the nucleotides and the two proteins, the shifts indicate that the enthalpic interactions are stronger between these two polar groups and protein than with water. In general, the spectral shifts provide a rationale for the stronger binding of GDP and IDP with p21 compared to EF-Tu. Despite the structural similarity of the binding sites of EF-Tu and p21, the strengths of the observed hydrogen bonds at the 6-keto and 2-amino positions vary substantially, by up to a factor of 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in ascorbic acid content and ascorbate peroxidase activity during the development of Acetabularia mediterranea

We cloned ras-related sequences from goldfish genomic libraries constructed as recombinants using the lambda phage. Restriction enzyme mapping of the clones obtained revealed three kinds of ras-related sequences among approximately 350,000 genomic clones. One of these clones was partially sequenced. Comparison with the nucleotide sequences of mammalian ras genes showed that the determined sequences covered the predicted amino acid coding regions and parts of the intervening regions. The predicted amino acid sequences of the cloned ras-related goldfish gene suggested that the coding region is localized separately in DNA, and that its exon-intron boundaries are exactly the same as those of corresponding mammalian genes. The nucleotide and amino acid sequences of the goldfish ras-related gene may have extensive homologies to mammalian p 21 protein. Among the three mammalian ras proteins, the predicted amino acid sequence of the sequenced ras-related goldfish clone is most closely homologous (96%) to the Kirsten ras protein. Differences in the predicted amino acid sequence were greatest in the sequence predicted from the fourth exon; fewer differences were found in the sequence from the third exon, and only slight or no differences were found in the sequence predicted for the first and second exons. The 12th and 61st amino acids from the N-terminal of the protein, which are thought to be critical positions for GTP binding and catalysis, are both conserved in the goldfish protein.(ABSTRACT TRUNCATED AT 250 WORDS)