SMART MODE:

NORMAL GENOMIC

• Bar-Sagi D
• A Ras by any other name.
• Mol Cell Biol. 2001; 21: 1441-3

• Crul M, de Klerk GJ, Beijnen JH, Schellens JH
• Ras biochemistry and farnesyl transferase inhibitors: a literature survey.
• Anticancer Drugs. 2001; 12: 163-84
• Display abstract

• Over the last decades, knowledge on the genetic defects involved in tumor formation and growth has increased rapidly. This has launched the development of novel anticancer agents, interfering with the proteins encoded by the identified mutated genes. One gene of particular interest is ras, which is found mutated at high frequency in a number of malignancies. The Ras protein is involved in signal transduction: it passes on stimuli from extracellular factors to the cell nucleus, thereby changing the expression of a number of growth regulating genes. Mutated Ras proteins remain longer in their active form than normal Ras proteins, resulting in an overstimulation of the proliferative pathway. In order to function, Ras proteins must undergo a series of post-translational modifications, the most important of which is farnesylation. Inhibition of Ras can be accomplished through inhibition of farnesyl transferase, the enzyme responsible for this modification. With this aim, a number of agents, designated farnesyl transferase inhibitors (FTIs), have been developed that possess antineoplastic activity. Several of them have recently entered clinical trials. Even though clinical testing is still at an early stage, antitumor activity has been observed. At the same time, knowledge on the biochemical mechanisms through which these drugs exert their activity is expanding. Apart from Ras, they also target other cellular proteins that require farnesylation to become activated, e.g. RhoB. Inhibition of the farnesylation of RhoB results in growth blockade of the exposed tumor cells as well as an increase in the rate of apoptosis. In conclusion, FTIs present a promising class of anticancer agents, acting through biochemical modulation of the tumor cells.

• Kuhn K, Owen DJ, Bader B, Wittinghofer A, Kuhlmann J, Waldmann H
• Synthesis of functional Ras lipoproteins and fluorescent derivatives.
• J Am Chem Soc. 2001; 123: 1023-35
• Display abstract

• For the study of biological signal transduction, access to correctly lipidated proteins is of utmost importance. Furthermore, access to bioconjugates that embody the correct structure of the protein but that may additionally carry different lipid groups or labels (i.e., fluorescent tags) by which the protein can be traced in biological systems, could provide invaluable reagents. We report here of the development of techniques for the synthesis of a series of modified Ras proteins. These modified Ras proteins carry a number of different, natural and non-natural lipid residues, and the process was extended to also provide access to a number of fluorescently labeled derivatives. The maleimide group provided the key to link chemically synthesized lipopeptide molecules in a specific and efficient manner to a truncated form of the H-Ras protein. Furthermore, a preliminary study on the biological activity of the natural Ras protein derivative (containing the normal farnesyl and palmitoyl lipid residues) has shown full biological activity. This result highlights the usefulness of these compounds as invaluable tools for the study of Ras signal transduction processes and the plasma membrane localization of the Ras proteins.

• Crespo P, Leon J
• Ras proteins in the control of the cell cycle and cell differentiation.
• Cell Mol Life Sci. 2000; 57: 1613-36
• Display abstract

• The Ras family of small GTPases includes three closely related proteins: H-, K-, and N-Ras. Ras proteins are involved in the transduction of signals elicited by activated surface receptors, acting as key components by relaying signals downstream through diverse pathways. Mutant, constitutively activated forms of Ras proteins are frequently found in cancer. While constitutive Ras activation induces oncogenic-like transformation in immortalized fibroblasts, it causes growth arrest in primary vertebrate cells. Induction of p53 and cyclin-dependent kinase inhibitors such as p15INK4b, p16INK4a, p19ARF, and p21WAF1 accounts for this response. Interestingly, while ras has usually been regarded as a transforming oncogene, the analysis of Ras function in most of the cellular systems studied so far indicates that the promotion of differentiation is the most prominent effect of Ras. While in some cell types, particularly muscle, Ras inhibits differentiation, in others such as neuronal, adipocytic, or myeloid cells, Ras induces differentiation, in some cases accompanied by growth arrest. Several possible mechanisms for the pleiotropic effects' of Ras in animal cells are discussed.

• Nammi S, Lodagala DS
• Ras farnesyltransferase inhibition: a novel and safe approach for cancer chemotherapy.
• Acta Pharmacol Sin. 2000; 21: 396-404
• Display abstract

• The 21-kDa Ras proteins are well known for their regulatory role in oncogenic, mitogenic, and developmental signaling pathways. GTP activated Ras interacts directly with the Raf protein to recruit the MAP kinases and their subordinates. Attachment of Ras protein to the plasma membrane that requires farnesylation by farnesyl pyrophosphate at its C-terminus, is essential for its biological activity. Ras oncogenes are associated with a wide variety of solid tumors and leukemias for which existing chemotherapeutics have limited utility. A promising pharmacological approach of antagonizing oncogenic Ras activity is to develop inhibitors of farnesyl transferase. These inhibitors may be useful in blocking the action of Ras onco-proteins.

• Ayllon V, Rebollo A
• Ras-induced cellular events (review).
• Mol Membr Biol. 2000; 17: 65-73
• Display abstract

• Ras is a crucial regulator of cell growth in eukaryotic cells. Activated Ras can stimulate signal transduction cascades, leading to cell proliferation, differentiation or apoptosis. It is also one of the most commonly mutated genes in both solid tumours and haematologic neoplasias. In leukaemia and tumours, aberrant Ras signalling can be induced directly by Ras mutation or indirectly by altering genes that associate with Ras or its signalling pathways. A requisite for Ras function is localization to the plasma membrane, which is induced by the post-translational modification farnesylation. Molecules that interfere with this Ras modification have been used as antitumour agents. Ras is emerging as a dual regulator of cell functions, playing either positive or negative roles in the control of proliferation or apoptosis. The diversity of Ras-mediated effects may be related in part to the differential involvement of Ras homologues in distinct cellular processes or to the expanding array of Ras effectors.

• Ewen ME
• Relationship between Ras pathways and cell cycle control.
• Prog Cell Cycle Res. 2000; 4: 1-17
• Display abstract

• The ordered execution of the two main events of cellular reproduction, duplication of the genome and cell division, characterize progression through the cell cycle. Cultured cells can be switched between cycling and non-cycling states by alteration of extracellular conditions and the notion that a critical cellular control mechanism presides on this decision, whose temporal location is known as the restriction point, has become the focus for the study of how extracellular mitogenic signalling impinges upon the cell cycle to influence proliferation. This review attempts to cover the disparate pathways of Ras-mediated mitogenic signal transduction that impact upon restriction point control.

• Frame S, Balmain A
• Integration of positive and negative growth signals during ras pathway activation in vivo.
• Curr Opin Genet Dev. 2000; 10: 106-13
• Display abstract

• Expression of RAS proteins can have either positive or negative effects on cell growth, differentiation and death. New technologies are being developed for the generation of animal models to address the questions of where, when and how much Ras is expressed during tumorigenesis, and how these disparate signals are integrated during multistage carcinogenesis.

• Cook SJ, Balmanno K, Garner A, Millar T, Taverner C, Todd D
• Regulation of cell cycle re-entry by growth, survival and stress signalling pathways.
• Biochem Soc Trans. 2000; 28: 233-40
• Display abstract

• The mitogen-activated and stress-activated protein kinases transduce signals from plasma membrane signalling machinery into the nucleus to modulate gene expression. By regulating the genomic response to environmental cues (growth factors, stresses) these pathways determine whether a cell re-enters the cell cycle, undergoes cell cycle arrest, senescence or apoptosis. We are particularly interested in how these pathways integrate with each other, and interact with the cell cycle machinery to achieve these discrete biological responses.

• Scheidig AJ, Burmester C, Goody RS
• The pre-hydrolysis state of p21(ras) in complex with GTP: new insights into the role of water molecules in the GTP hydrolysis reaction of ras-like proteins.
• Structure Fold Des. 1999; 7: 1311-24
• Display abstract

• BACKGROUND: In numerous biological events the hydrolysis of guanine triphosphate (GTP) is a trigger to switch from the active to the inactive protein form. In spite of the availability of several high-resolution crystal structures, the details of the mechanism of nucleotide hydrolysis by GTPases are still unclear. This is partly because the structures of the proteins in their active states had to be determined in the presence of non-hydrolyzable GTP analogues (e.g. GppNHp). Knowledge of the structure of the true Michaelis complex might provide additional insights into the intrinsic protein hydrolysis mechanism of GTP and related nucleotides. RESULTS: The structure of the complex formed between p21(ras) and GTP has been determined by X-ray diffraction at 1.6 A using a combination of photolysis of an inactive GTP precursor (caged GTP) and rapid freezing (100K). The structure of this complex differs from that of p21(ras)-GppNHp (determined at 277K) with respect to the degree of order and conformation of the catalytic loop (loop 4 of the switch II region) and the positioning of water molecules around the gamma-phosphate group. The changes in the arrangement of water molecules were induced by the cryo-temperature technique. CONCLUSIONS: The results shed light on the function of Gln61 in the intrinsic GTP hydrolysis reaction. Furthermore, the possibility of a proton shuffling mechanism between two attacking water molecules and an oxygen of the gamma-phosphate group can be proposed for the basal GTPase mechanism, but arguments are presented that render this protonation mechanism unlikely for the GTPase activating protein (GAP)-activated GTPase.

• Futatsugi N, Hata M, Hoshino T, Tsuda M
• Ab initio study of the role of lysine 16 for the molecular switching mechanism of Ras protein p21.
• Biophys J. 1999; 77: 3287-92
• Display abstract

• Quantum chemical computations using the ab initio molecular orbital (MO) method have been performed to investigate the molecular switching mechanism of Ras protein p21, which has an important role in intracellular signal cascades. Lys(16) was demonstrated to be crucial to the function of Ras p21, and the hydrolysis of GTP to GDP was found to be an one-step reaction. The potential energy barrier of this hydrolysis reaction from GTP to (GDP + P) was calculated to be approximately 42 kcal/mol. The role of GAP (GTPase-activating protein) was also discussed in terms of the delivery of the water molecules required for the hydrolysis.

• Sternberg PW, Schmid SL
• Caveolin, cholesterol and Ras signalling.
• Nat Cell Biol. 1999; 1: 357-357

• Rebollo A, Martinez-A C
• Ras proteins: recent advances and new functions.
• Blood. 1999; 94: 2971-80

• de Gunzburg J
• Proteins of the Ras pathway as novel potential anticancer therapeutic targets.
• Cell Biol Toxicol. 1999; 15: 345-58
• Display abstract

• Ras proteins are molecular switches that constitute a pivotal element in the control of cellular responses to many incoming signals, and in particular mitogenic stimulations. They act through multiple effector pathways that carry out the biological functions of Ras in cells. Since mutations that constitutively activate Ras proteins have been found in a high proportion of human malignancies and participate in oncogenesis, a number of therapeutic anticancer strategies aimed against the activity or action of Ras proteins have been developed. This paper reviews the principal aspects of the Ras signaling pathway and describes some of the attempts to develop antitumor drugs based on this concept.

• Aharonson Z, Gana-Weisz M, Varsano T, Haklai R, Marciano D, Kloog Y
• Stringent structural requirements for anti-Ras activity of S-prenyl analogues.
• Biochim Biophys Acta. 1998; 1406: 40-50
• Display abstract

• The carboxy terminal S-farnesylcysteine of Ras oncoproteins is required for their membrane anchorage and transforming activities. We showed previously that S-farnesylthiosalicylic acid (FTS) affects the membrane anchorage of activated H-Ras in EJ cells and inhibits their growth. We report here on structural elements in S-prenyl derivatives that specifically inhibit the growth of EJ cells, but not of untransformed Rat-1 cells. Inhibition of the Ras-dependent extracellular signal-regulated protein kinase (ERK), of DNA synthesis and of EJ cell growth were apparent after treatment with FTS or its 5-fluoro, 5-chloro and 4-fluoro derivatives or with the C20 S-geranylgeranyl derivative of thiosalicylic acid. The 4-Cl-FTS analogue was a weak inhibitor of EJ cell growth. The 3-Cl-FTS analogue and the FTS carboxyl methyl ester were inactive, as were the C10 S-geranyl derivative of thiosalicylic acid, farnesoic acid, N-acetyl-S-farnesyl-L-cysteine and S-farne-sylthiopropionic acid. The structural requirements for anti-Ras activity of S-prenyl analogues thus appear to be rather stringent. With regard to chain length, the C15 farnesyl group linked to a rigid backbone seems to be necessary and sufficient. A free carboxyl group in an appropriately rigid orientation, as in thiosalicylic acid, is also required. Halogenic substitutents on the benzene ring of the thiosalicylic acid are tolerated only at position 5 or 4. This information may facilitate the design of potent Ras antagonists and deepen our understanding of the mode of association of Ras with the plasma membrane.

• Malumbres M, Pellicer A
• RAS pathways to cell cycle control and cell transformation.
• Front Biosci. 1998; 3: 887912-887912
• Display abstract

• Ras genes are among the most frequently activated oncogenes in cancer. The corresponding protooncogenes are proteins expressed in the majority of tissues in mammals and have a signal transduction activity. Ras proteins interact with a wide spectrum of regulators and downstream effectors producing different cellular responses, including proliferation, differentiation or apoptosis. This review deals with the most recent advances on the role of Ras in the signal transduction pathway from external signals to the cell cycle and gene expression control. We specially address the new developments on the effect of Ras activation in the regulation of different molecules driving the cell cycle progression. Both positive and negative regulators of the cyclin-dependent kinases (CDK), cyclins and CDK inhibitors, are targets of Ras, giving rise to different effects in the cell cycle progression. These Ras-mediated interactions are an extraordinary example of the complexity of the signal transduction networks and the diversity of pathways used by Ras to propagate molecular signals. FBS Ras, Tumorigenesis, Signal Transduction, Cell Cycle, Cyclin-Dependent Kinases, CDK-inhibitors.

• Kinoshita H, Shimizu K
• Prediction of the GTPase activities by using the semiempirical molecular orbital theory.
• Bioorg Med Chem Lett. 1998; 8: 1083-8
• Display abstract

• Assuming that substrate-assisted catalysis is the mechanism of GTP hydrolysis for ras p21 and other GTP-binding proteins, we used the PM3 semiempirical molecular orbital method to predict from the calculated reaction profiles of GTP hydrolysis reactions the changes in GTPase activities caused by mutations. We succeeded in making qualitative predictions for mutants.

• Rommel C, Hafen E
• Ras--a versatile cellular switch.
• Curr Opin Genet Dev. 1998; 8: 412-8
• Display abstract

• With the number of known roles played by Ras proteins increasing rapidly, finding answers to how the diverse cellular responses are triggered is becoming increasingly pertinent. Although our understanding of the control of specificity of signal transduction is still small, the combination of biochemical, structural and genetic analyses is starting to reveal how the cell-specific responses to Ras activation are controlled.

• Haklai R et al.
• Dislodgment and accelerated degradation of Ras.
• Biochemistry. 1998; 37: 1306-14
• Display abstract

• Membrane anchorage of Ras oncoproteins, required for transforming activity, depends on their carboxy-terminal farnesylcysteine. We previously showed that S-trans,trans-farnesylthiosalicylic acid (FTS), a synthetic farnesylcysteine mimetic, inhibits growth of ErbB2- and Ras-transformed cells, but not of v-Raf-transformed cells, suggesting that FTS interferes specifically with Ras functions. Here we demonstrate that FTS dislodges Ras from membranes of H-Ras-transformed (EJ) cells, facilitating its degradation and decreasing total cellular Ras. The dislodged Ras that was transiently present in the cytosol was degraded relatively rapidly, causing a decrease of up to 80% in total cellular Ras. The half-life of Ras was 10 +/- 4 h in FTS-treated EJ cells and 27 +/- 4 h in controls. The dislodgment of membrane Ras and decrease in total cellular Ras were dose-dependent: 50% of the effects occurred at 10-15 microM, comparable to concentrations (7-10 microM) required for 50% growth inhibition in EJ cells. Higher concentrations of FTS (25-50 microM) were required to dislodge Ras from Rat-1 cell membranes expressing normal Ras, suggesting some selectivity of FTS toward oncogenic Ras. Membrane localization of the prenylated G beta gamma of heterotrimeric G proteins was not affected by FTS in EJ cells. An FTS-related compound, N-acetyl-S-farnesyl-L-cysteine, which does not inhibit EJ cell growth, did not affect Ras. FTS did not inhibit growth of Rat-1 cells transformed by N-myristylated H-Ras and did not reduce the total amount of this Ras isoform. The results suggest that FTS affects docking of Ras in the cell membrane in a rather specific manner, rendering the protein susceptible to proteolytic degradation.

• Rowell CA, Kowalczyk JJ, Lewis MD, Garcia AM
• Direct demonstration of geranylgeranylation and farnesylation of Ki-Ras in vivo.
• J Biol Chem. 1997; 272: 14093-7
• Display abstract

• It has recently been reported that Ki-Ras protein can be modified in vitro by farnesylation or geranylgeranylation. However, a previous analysis of Ki-Ras prenylation in vivo found only farnesylated Ki-Ras. In this report it is shown that under normal conditions, Ki-Ras is farnesylated in vivo and when cells are treated with the farnesyl transferase inhibitors B956 or B957, farnesylation is inhibited and Ki-Ras becomes geranylgeranylated in a dose dependent manner. These results have strong implications in the design of anticancer drugs based on inhibition of prenylation.

• Muegge I, Schweins T, Langen R, Warshel A
• Electrostatic control of GTP and GDP binding in the oncoprotein p21ras.
• Structure. 1996; 4: 475-89
• Display abstract

• BACKGROUND: p21ras is one of the GTP-binding proteins that act as intercellular molecular switches. The GTP-bound form of p21ras sends a growth-promoting signal that is terminated once the protein is cycled back into its GDP-bound form. The interaction of guanine-nucleotide-exchange factors (GEFs) with p21ras leads to activation of the protein by promoting GDP --> GTP exchange. Oncogenic mutations of p21ras trap the protein in its biological active GTP-bound form. Other mutations interfere with the activity of GEF. Thus, it is important to explore the structural basis for the action of different mutations. RESULTS: The crystal structures of p21ras are correlated with the binding affinities of GTP and GDP by calculating the relevant electrostatic energies. It is demonstrated that such calculations can provide a road map to the location of 'hot' residues whose mutations are likely to change functional properties of the protein. Furthermore, calculations of the effect of specific mutations on GTP and GDP binding are consistent with those observed. This helps to analyze and locate functionally important parts of the protein. CONCLUSIONS: Our calculations indicate that the protein main chain provides a major contribution to the binding energies of nucleotides and probably plays a key role in relaying the effect of GEF action. Analysis of p21ras mutations in residues that are important for the proper function of GEFs suggests that the region comprising residues 62-67 in p21ras is the major GEF-binding site. This analysis and our computer simulations indicate that the effect of GEF is probably propagated to the P-loop (residues 10-17) through interaction between Gly60 and Gly12. This then reduces the interaction between the main-chain dipoles of the P-loop and the nucleotide. Finally, the results also suggest a possible relationship between the GTP --> GDP structural transition and the catalytic effect of the GTPase-activating protein.

• Downward J
• KSR: a novel player in the RAS pathway.
• Cell. 1995; 83: 831-4

• Bar-Sagi D
• Mammalian cell microinjection assay.
• Methods Enzymol. 1995; 255: 436-42

• Volker C, Stock JB
• Carboxyl methylation of Ras-related proteins.
• Methods Enzymol. 1995; 255: 65-82

• McCormick F
• Ras-related proteins in signal transduction and growth control.
• Mol Reprod Dev. 1995; 42: 500-6
• Display abstract

• Ras proteins are members of a superfamily of small GTPases that are involved in many aspects of cell growth control. The ras p21 protooncogene products, H-ras, K-ras, and N-ras, transmit signals from growth factor receptors to a cascade of protein kinases that begins with the Raf protooncogene product, and leads to alterations in transcription factors and cell cycle proteins in the nucleus. This cascade is controlled at several points: Ras p21 proteins are regulated by GAPs and by exchange factors, whose activities are altered by growth factor receptor activation (Boguski and McCormick, 1993: Nature 366:643-654). Transmission of signals from Ras to Raf is regulated by the Ras-related protein Rap1 (a protein capable of reverting cell transformation) and by cAMP. Other aspects of Ras p21 regulation will be discussed, including the existence of RasGDl proteins that inhibit GDP dissociation from Ras, and may thus regulate the level of active Ras in the cell. The role of Ras in activation of Raf kinase appears to be limited to the recruitment of Raf to the plasma membrane, at which time Raf becomes stably modified to render it active (Leevers et al., 1994: Nature 369:411-414; Stokoe et al., 1994: Science 264:1463-1467). The nature of these modifications is unclear. Raf in the plasma membrane becomes associated with insoluble structural cell components that may be part of the activation. Furthermore, Raf is associated with proteins of the 14-3-3 family that appear necessary for kinase activation. The 14-3-3 proteins interact with all three conserved regions of Raf, including the kinase domain. In addition to Raf, Ras proteins interact with two known classes of proteins in a manner consistent with effector functions: these are the GAPs and regulators of the Ras-related protein Ral referred to as RalGDS. These biochemical data suggest that other functional pathways are regulated by Ras, including, perhaps, pathways involved in regulating cell shape and motility. The protein R-Ras p21 is about 50% identical to the Ras p21 protooncogene product. This protein is incapable of transforming cells, even though it interacts with Raf and other putative Ras effectors (Fernandez-Sarabia and Bischoff, 1993: Nature 366:274-275). On the other hand, it has recently been shown that R-Ras binds to the protooncogene product Bcl-2, a protein that transforms B cells by blocking apoptosis. R-Ras is regulated by the same GAP molecules as H-Ras and the other Ras protooncogene products, and may therefore be activated in a manner co-ordinate with these growth-promoting proteins. The possible connection between R-Ras and apoptosis will be discussed.

• Nilsen-Hamilton M, Buss J, Hamilton RT
• Intracellular signaling from Ras to genes: an MCDB/ISU symposium held at Iowa State University, Ames, September 22-25, 1994.Introduction.
• Mol Reprod Dev. 1995; 42: 457-8

• Tocque B, Janicot M, Kenigsberg M
• Oocyte microinjection assay for evaluation of Ras-induced signaling pathways.
• Methods Enzymol. 1995; 255: 426-35

• Schweins T, Langen R, Warshel A
• Why have mutagenesis studies not located the general base in ras p21.
• Nat Struct Biol. 1994; 1: 476-84
• Display abstract

• Ras p21 plays a major role in the control of cell growth, and oncogenic mutations of this protein have been found in human cancers. Unfortunately, the detailed mode of action of Ras p21 is still unclear, in spite of the great interest in this protein and the availability of its X-ray crystal structure. In particular, mutagenesis studies of different active site residues could not identify the general base for GTP hydrolysis. Here we tackle this question using a computer simulation approach with clear and reliable energy considerations and conclude that the most likely general base is the bound GTP itself. Obviously, the identification of such a general base cannot be easily accomplished by mutagenesis experiments.

• Pawson T
• Regulation of the Ras signalling pathway by protein-tyrosine kinases.
• Biochem Soc Trans. 1994; 22: 455-60

• Wittinghofer A
• The structure of transducin G alpha t: more to view than just ras.
• Cell. 1994; 76: 201-4

• Schmidt K
• A puzzle: how similar signals yield different effects.
• Science. 1994; 266: 566-7

• Frech M et al.
• Role of glutamine-61 in the hydrolysis of GTP by p21H-ras: an experimental and theoretical study.
• Biochemistry. 1994; 33: 3237-44
• Display abstract

• The active GTP-bound form of p21ras is converted to the biologically inactive GDP-bound form by enzymatic hydrolysis and this function serves to regulate the wild-type ras protein. The side chain of the amino acid at position 61 may play a key role in this hydrolysis of GTP by p21. Experimental studies that define properties of the Q61E mutant of p21H-ras are presented along with supporting molecular dynamics simulations. We find that under saturating concentrations of GTP the Q61E mutant of p21H-ras has a 20-fold greater rate of intrinsic hydrolysis (kcat = 0.57 min-1) than the wild type. The affinity of the Q61E variant for GTP (Kd = 115 microM) is much lower than that of the wild type. GTPase activating protein does not activate the variant. From molecular dynamics simulations, we find that both the wild type and Q61E mutant have the residue 61 side chain in transient contact with a water molecule that is well-positioned for hydrolytic attack on the gamma phosphate. Thr-35 also is found to form a transient hydrogen bond with this critical water. These elements may define the catalytic complex for hydrolysis of the GTP [Pai et al. (1990) EMBO J. 9, 2351]. Similarly, the G12P mutant, which also has an intrinsic hydrolysis rate similar to the wild type, is found to form the same complex in simulation. In contrast, molecular dynamics analysis of the mutants G12R, G12V, and Q61L, which have much lower intrinsic rates than the wild-type p21, do not show this complex.(ABSTRACT TRUNCATED AT 250 WORDS)

• Low A, Sprinzl M, Limmer S
• Nucleotide binding and GTP hydrolysis by the 21-kDa product of the c-H-ras gene as monitored by proton-NMR spectroscopy.
• Eur J Biochem. 1993; 213: 781-8
• Display abstract

• Proton-NMR signals in the downfield region (below approximately 10 ppm) have been shown to provide a useful spectroscopic window to monitor the binding of guanine nucleotides to the active site of GTP/GDP-binding proteins via H-bonds, as specified here by the 21-kDa product of the c-H-ras gene (p21). The time course of the intensity change of certain peaks upon addition of GTP to nucleotide-free p21 corresponds to the GTP hydrolysis rate as determined by HPLC. Though there are fewer potential H-bond acceptors in the GDP-bound protein than in the GTP complex, more downfield peaks are found in the former complex, suggesting tighter binding of GDP. Moreover, inspection of the downfield proton-NMR spectra permits rapid detection of subtle changes of the active site induced by complexation with slowly hydrolyzing GTP analogues resulting from mutations of the amino acid sequence, especially in the phosphate binding loop. Our studies strongly suggest that no major conformational change of the phosphate-binding region occurs upon nucleotide complexation that precedes the catalytic step. Besides, it is suspected that the Ser17 hydroxyl group is involved in nucleotide binding and GTP hydrolysis.

• John J et al.
• Kinetic and structural analysis of the Mg(2+)-binding site of the guanine nucleotide-binding protein p21H-ras.
• J Biol Chem. 1993; 268: 923-9
• Display abstract

• The coordination and binding of the Mg2+ ion in the nucleotide-binding site of p21 have been investigated using site-directed mutagenesis, kinetic methods, and phosphorous NMR. Mg2+ in the p21.nucleotide.Mg2+ complex appears to be in fast equilibrium with the solvent. The dissociation constant between Mg2+ and the p21.GDP complex was determined to be 2.8 microM. It decreases 30- or 16-fold on substituting Ser-17 or Asp-57 with alanine, respectively, whereas the T35A mutation has no effect. All three mutations influence the dissociation constants and the association and dissociation rate constants of the interaction between guanine nucleotides and p21, but to a different degree. We conclude that Thr-35 is only complexed to Mg2+ in the GTP conformation and both Asp-57 and Ser-17 appear to be critical for both GDP and GTP binding. 31P NMR spectra of the GDP and Gpp(NH)p (guanosine-5'-(beta,gamma-imido)triphosphate) complexes of mutated p21 show a remarkable perturbation of the guanine nucleotide-binding site compared to wild-type protein. The mutant proteins show reduced GTPase rates, which are not stimulated by the GTPase-activating protein GAP. p21(S17A) has been reported to function just as p21(S17N) as a dominant negative inhibitor of normal p21. We find that it inhibits oncogenic p21-induced survival of primary neurons.

• Franken SM et al.
• Three-dimensional structures and properties of a transforming and a nontransforming glycine-12 mutant of p21H-ras.
• Biochemistry. 1993; 32: 8411-20
• Display abstract

• The three-dimensional structures and biochemical properties of two mutants of the G-domain (residues 1-166) of p21H-ras, p21 (G12D) and p21 (G12P), have been determined in the triphosphate-bound form using guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp). They correspond to the most frequent oncogenic and the only nononcogenic mutation of Gly-12, respectively. The G12D mutation is the only mutant analyzed so far that crystallizes in a space group different from wild type, and the atomic model of the protein shows the most drastic changes of structure around the active site as compared to wild-type p21. This is due to the interactions of the aspartic acid side chain with Tyr-32, Gln-61, and the gamma-phosphate, which result in reduced mobility of these structural elements. The interaction between the carboxylate group of Asp-12 and the gamma-phosphate is mediated by a shared proton, which we show by 31P NMR measurements to exist in solution as well. The structure of p21 (G12P) is remarkably similar to that of wild-type p21 in the active site, including the position of the nucleophilic water. The pyrrolidine ring of Pro-12 points outward and seems to be responsible for the weaker affinity toward GAP (GTPase-activating protein) and the failure of GAP to stimulate GTP hydrolysis.

• Miller AF, Papastavros MZ, Redfield AG
• NMR studies of the conformational change in human N-p21ras produced by replacement of bound GDP with the GTP analog GTP gamma S.
• Biochemistry. 1992; 31: 10208-16
• Display abstract

• 1H-Detected 15N-edited NMR in solution was used to study the conformational differences between the GDP- and GTP gamma S-bound forms of human N-p21ras. The amide protons of 15N-labeled glycine and isoleucine were observed. Resonances were assigned to residues of particular interest, glycines-60 and -75 and isoleucines-21 and -36, by incorporating various 13C-labeled amino acids in addition to [15N]glycine and [15N]iosleucine and by replacing Mg2+ by Co2+. When GTP gamma S replaced GDP in the active site of p21ras, only 5 of the 14 glycine amide resonances show major shifts, indicating that the conformational effects are fairly localized. Responsive glycines-10, -12, -13, and -15 are in the active site. Gly-75, located at the far end of a conformationally-active loop and helix, also responds to substitution of GTP gamma S for GDP, while Gly-77 does not, supporting a role for Gly-75 as a swivel point for the conformational change. The amide proton resonances of isoleucines-36 and -21 and a third unidentified isoleucine also undergo major shifts upon replacement of GDP by GTP gamma S. Thus, the effector-binding loop containing Ile-36 is confirmed to be involved in the conformational change, and the alpha-helix containing Ile-21 is also shown to be affected.

• Langen R, Schweins T, Warshel A
• On the mechanism of guanosine triphosphate hydrolysis in ras p21 proteins.
• Biochemistry. 1992; 31: 8691-6
• Display abstract

• The residue Gln61 is assumed to play a major role in the mechanism of ras p21, and mutations of this residue are often found in human tumors. Such mutations lead to a major reduction in the rate of GTP hydrolysis by the complex of ras p21 and the GTPase activating protein (GAP) and lock the protein in a growth-promoting state. This work examines the role of Gln61 in ras p21 by using computer simulation approaches to correlate the structure and energetics of this system. Free energy perturbation calculations and simpler electrostatic considerations demonstrate that Gln61 is unlikely to serve as the general base in the intrinsic GAP-independent reaction of p21. Glutamine is already a very weak base in water, and surprisingly the GlnH+ OH-reaction intermediate is even less stable in the protein active site than in the corresponding reaction in water. The electrostatic field of Glu63, which could in principle stabilize the protonated Gln61, is found to be largely shielded by the surrounding solvent. However, it is still possible that Gln61 is a general base in the GAP/ras p21 complex since this system could enhance the electrostatic effect of Glu63. It is also possible that the gamma-phosphate acts as general base and that Gln61 accelerates the reaction by stabilizing the OH- nucleophile. If such a mechanism is operative, then GAP may enhance the effect of Gln61 by preorienting its hydrogen bonds in the transition-state configuration.

• Gandarillas A, Renart J, Quintanilla M
• Biochemical characterization of Artemia ras p21.
• Mol Cell Biochem. 1992; 112: 29-33
• Display abstract

• The biochemical properties of Artemia ras proteins (p21) have been studied after immunoprecipitation with the monoclonal antibody Y13-259. The ras products bind GTP and GDP, and have GTPase activity. Artemia p21 was unable to hydrolyze Gp4G, although this dinucleotide exhibits high affinity for the protein. Our results demonstrate that the protein(s) recognized by the Y13-259 antibody in this crustacean behave as typical mammalian ras p21s.

• Prive GG et al.
• X-ray crystal structures of transforming p21 ras mutants suggest a transition-state stabilization mechanism for GTP hydrolysis.
• Proc Natl Acad Sci U S A. 1992; 89: 3649-53
• Display abstract

• RAS genes isolated from human tumors often have mutations at positions corresponding to amino acid 12 or 61 of the encoded protein (p21), while retroviral ras-encoded p21 contains substitutions at both positions 12 and 59. These mutant proteins are deficient in their GTP hydrolysis activity, and this loss of activity is linked to their transforming potential. The crystal structures of the mutant proteins are presented here as either GDP-bound or GTP-analogue-bound complexes. Based on these structures, a mechanism for the p21 GTPase reaction is proposed that is consistent with the observed structural and biochemical data. The central feature of this mechanism is a specific stabilization complex formed between the Gln-61 side-chain and the pentavalent gamma-phosphate of the GTP transition state. Amino acids other than glutamine at position 61 cannot stabilize the transition state, and amino acids larger than glycine at position 12 would interfere with the transition-state complex. Thr-59 disrupts the normal position of residue 61, thus preventing its participation in the transition-state complex.

• Dykes DC, Brandt-Rauf P, Luster SM, Chung D, Friedman FK, Pincus MR
• Activated conformations of the ras-gene-encoded p21 protein. 1. An energy-refined structure for the normal p21 protein complexed with GDP.
• J Biomol Struct Dyn. 1992; 9: 1025-44
• Display abstract

• A complete three-dimensional structure for the ras-gene-encoded p21 protein with Gly 12 and Gln 61, bound to GDP, has been constructed in four stages using the available alpha-carbon coordinates as deposited in the Brookhaven National Laboratories Protein Data Bank. No all-atom structure has been made available despite the fact that the first crystallographic structure for the p21 protein was reported almost four years ago. In the p21 protein, if amino acid substitutions are made at any one of a number of different positions in the amino acid sequence, the protein becomes permanently activated and causes malignant transformation of normal cells or, in some cell lines, differentiation and maturation. For example, all amino acids except Gly and Pro at position 12 result in an oncogenic protein; all amino acids except Gln, Glu and Pro at position 61 likewise cause malignant transformation of cells. We have constructed our all-atom structure of the non-oncogenic protein from the x-ray structure in order to determine how oncogenic amino acid substitutions affect the three-dimensional structure of this protein. In Stage 1 we generated a poly-alanine backbone (except at Gly and Pro residues) through the alpha-carbon structure, requiring the individual Ala, Pro or Gly residues to conform to standard amino acid geometry and to form trans-planar peptide bonds. Since no alpha-carbon coordinates for residues 60-65 have been determined, these residues were modeled by generating them in the extended conformation and then subjecting them to molecular dynamics using the computer application DISCOVER and energy minimization using DISCOVER and the ECEPP (Empirical Conformational Energies for Peptides Program). In Stage 2, the positions of residues that are homologous to corresponding residues of bacterial elongation factor Tu (EF-Tu) to which p21 bears an overall 40% sequence homology, were determined from their corresponding positions in a high-resolution structure of EF-Tu. Non-homologous loops were taken from the structure generated in Stage 1 and were placed between the appropriate homologous segments so as to connect them. In Stage 3, all bad contacts that occurred in this resulting structure were removed, and the coordinates of the alpha-carbon atoms were forced to superimpose as closely as possible on the corresponding atoms of the reference (x-ray) structure. Then the side chain positions of residues of the non-homologous loop regions were modeled using a combination of molecular dynamics and energy minimization using DISCOVER and ECEPP respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

• Fujita-Yoshigaki J et al.
• Guanine-nucleotide binding activity, interaction with GTPase-activating protein and solution conformation of the human c-Ha-Ras protein catalytic domain are retained upon deletion of C-terminal 18 amino acid residues.
• J Protein Chem. 1992; 11: 731-9
• Display abstract

• A truncated human c-Ha-Ras protein that lacks the C-terminal 18 amino acid residues and the truncated Ras protein with the amino acid substitution Gly-->Val in position 12 were prepared by an E. coli overexpression system. The truncated Ras protein showed the same guanine-nucleotide binding activity and GTPase activity as those of the full-length Ras protein. Further, the same extent of GTPase activity enhancement due to GTPase-activating protein was observed for the truncated and full-length Ras proteins. In fact, two-dimensional proton NMR analyses indicated that the tertiary structure of the truncated Ras protein (GDP-bound or GMPPNP-bound) was nearly the same as that of the corresponding catalytic domain of the full-length Ras protein. Moreover, a conformational change around the effector region upon GDP-->GMPPNP exchange occurred in the same manner for both proteins. These observations indicate that the C-terminal flanking region (18 amino acid residues) of the Ras protein does not appreciably interact with the catalytic domain. Therefore, the truncated Ras protein is suitable for studying the molecular mechanism involved in the GTPase activity and the interaction with the GTPase-activating protein. On the other hand, an active form of the truncated Ras protein, unlike that of the full-length Ras protein, did not induce neurite outgrowth of rat pheochromocytoma PC12 cells. Thus, membrane anchoring of the Ras protein through its C-terminal four residues is not required for the interaction of Ras and GAP, but may be essential for the following binding of the Ras-GAP complex with the putative downstream target.

• Wittinghofer F, Krengel U, John J, Kabsch W, Pai EF
• Three-dimensional structure of p21 in the active conformation and analysis of an oncogenic mutant.
• Environ Health Perspect. 1991; 93: 11-5
• Display abstract

• The three-dimensional structure of the active guanosine triphosphate (GTP)-analogue-containing complex of the H-ras-encoded p21 has been determined. It was necessary to correct the topology of p21 as published earlier. The structure analysis shows all of the interactions between protein and GTP and how the important cofactor Mg2+ is bound. From the oncogenic mutants of p21 crystallized, a Gly12 to Arg mutation has been analyzed in detail. It shows that the overall structure of the mutant is not perturbed and that the side chain of Arg12 is coming close to the gamma-phosphate for an interaction.

• Jurnak F, Heffron S, Schick B, Delaria K
• Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu.
• Biochim Biophys Acta. 1990; 1050: 209-14
• Display abstract

• 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.

• Masters SB, Landis CA, Bourne HR
• GTPase-inhibiting mutations in the alpha subunit of Gs.
• Adv Second Messenger Phosphoprotein Res. 1990; 24: 70-5

• Chen JM, Lee G, Brandt-Rauf PW, Murphy RB, Rackovsky S, Pincus MR
• Comparison of the predicted structure for the activated form of the P21 protein with the X-ray crystal structure.
• J Protein Chem. 1990; 9: 543-7
• Display abstract

• The predicted conformation and position of the central transforming region (residues 55-67) of the p21 protein are compared with the conformation and position of this segment in a recently determined X-ray crystal structure of residues 1-166 of this protein in the activated state bound to a nonhydrolyzable GTP derivative. We previously predicted that this segment of the protein would adopt a roughly extended conformation from Ile 55-Thr 58, a reverse turn at Ala 59-Gln 61, followed by an alpha-helix from Glu 62-Met 67. We further predicted that this region of the activated protein occupies a position that is virtually identical to corresponding regions in the homologous purine nucleotide-binding proteins, bacterial elongation factor (EF-tu), and adenylate kinase (ADK). We find that there is a close correspondence between the conformation and position of our predicted structure and those found in the X-ray crystal structure. A mechanism for activation of the protein is proposed and is corroborated by X-ray crystallographic data.

• Neal SE, Eccleston JF, Webb MR
• Hydrolysis of GTP by p21NRAS, the NRAS protooncogene product, is accompanied by a conformational change in the wild-type protein: use of a single fluorescent probe at the catalytic site.
• Proc Natl Acad Sci U S A. 1990; 87: 3562-5
• Display abstract

• 2'(3')-O-(N-Methyl)anthraniloylguanosine 5'-triphosphate (mantGTP) is a fluorescent analogue of GTP that has similar properties to the physiological substrate in terms of its binding constant and the kinetics of its interactions with p21NRAS, the NRAS protooncogene product. There is a 3-fold increase in fluorescence intensity when mantGTP binds to p21NRAS. The rate constant for the cleavage of mantGTP complexed with the protein is similar to that of GTP, and cleavage is accompanied by a fluorescence intensity change in the wild-type protein complex. A two-phase fluorescence change also occurs when the nonhydrolyzable analogue 2'(3')-O-(N-methyl)anthraniloylguanosine 5'-[beta, gamma-imido]triphosphate (mantp[NH]ppG) binds to wild-type p21NRAS. The second phase occurs at the same rate as the second phase observed after mantGTP binding. Thus this second phase is probably a conformation change of the p21NRAS nucleotiside triphosphate complex and that the change controls the rate of GTP hydrolysis on the protein. With a transforming mutant, [Asp12]-p21NRAS, there is no second phase of the fluorescence change after mantGTP or mantp[NH]ppG binding, even though mantGTP is hydrolyzed. This shows that an equivalent conformational change does not occur and thus the mutant may stay in a "GTP-like" conformation throughout the GTPase cycle. These results are discussed in terms of the proposed role of p21NRAS in signal transduction and the transforming properties of the mutant.

• Pai EF, Krengel U, Petsko GA, Goody RS, Kabsch W, Wittinghofer A
• Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis.
• EMBO J. 1990; 9: 2351-9
• Display abstract

• The crystal structure of the H-ras oncogene protein p21 complexed to the slowly hydrolysing GTP analogue GppNp has been determined at 1.35 A resolution. 211 water molecules have been built into the electron density. The structure has been refined to a final R-factor of 19.8% for all data between 6 A and 1.35 A. The binding sites of the nucleotide and the magnesium ion are revealed in high detail. For the stretch of amino acid residues 61-65, the temperature factors of backbone atoms are four times the average value of 16.1 A2 due to the multiple conformations. In one of these conformations, the side chain of Gln61 makes contact with a water molecule, which is perfectly placed to be the nucleophile attacking the gamma-phosphate of GTP. Based on this observation, we propose a mechanism for GTP hydrolysis involving mainly Gln61 and Glu63 as activating species for in-line attack of water. Nucleophilic displacement is facilitated by hydrogen bonds from residues Thr35, Gly60 and Lys16. A mechanism for rate enhancement by GAP is also proposed.

• Sanders DA
• A guide to low molecular weight GTPases.
• Cell Growth Differ. 1990; 1: 251-8

• Milburn MV et al.
• Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins.
• Science. 1990; 247: 939-45
• Display abstract

• Ras proteins participate as a molecular switch in the early steps of the signal transduction pathway that is associated with cell growth and differentiation. When the protein is in its GTP complexed form it is active in signal transduction, whereas it is inactive in its GDP complexed form. A comparison of eight three-dimensional structures of ras proteins in four different crystal lattices, five with a nonhydrolyzable GTP analog and three with GDP, reveals that the "on" and "off" states of the switch are distinguished by conformational differences that span a length of more than 40 A, and are induced by the gamma-phosphate. The most significant differences are localized in two regions: residues 30 to 38 (the switch I region) in the second loop and residues 60 to 76 (the switch II region) consisting of the fourth loop and the short alpha-helix that follows the loop. Both regions are highly exposed and form a continuous strip on the molecular surface most likely to be the recognition sites for the effector and receptor molecule(or molecules). The conformational differences also provide a structural basis for understanding the biological and biochemical changes of the proteins due to oncogenic mutations, autophosphorylation, and GTP hydrolysis, and for understanding the interactions with other proteins.

• Hall A
• ras and GAP--who's controlling whom?
• Cell. 1990; 61: 921-3

• Isomura M, Kawata M, Takai Y
• [ras p21/ras p21-like small G proteins and their regulatory proteins]
• Nippon Rinsho. 1990; 48: 61-70

• Brunger AT et al.
• Crystal structure of an active form of RAS protein, a complex of a GTP analog and the HRAS p21 catalytic domain.
• Proc Natl Acad Sci U S A. 1990; 87: 4849-53
• Display abstract

• Normal RAS proteins play a key role of molecular switch in the transduction of the growth signal from extracellular to intracellular space. The state of the switch is "on" when GTP is bound and "off" when GDP is bound to the protein. The crystal structure of a complex between a nonhydrolyzable GTP analog and the catalytic domain of a RAS protein has been determined by a rotation-translation search method. The orientations and positions of four independent molecules have been determined using a single molecule as a probe in the search. The crystal structure reveals that the gamma phosphate of the GTP analog induces extensive conformational changes on two loop regions of the protein.

• Johnson LN
• Crystallography. Following proteins in time.
• Nature. 1990; 345: 294-5

• Tong LA et al.
• Structural differences between a ras oncogene protein and the normal protein.
• Nature. 1989; 337: 90-3
• Display abstract

• One of the most commonly found transforming ras oncogenes in human tumours has a valine codon replacing the glycine codon at position 12 of the normal c-Ha-ras gene. To understand the structural reasons behind cell transformation arising from this single amino acid substitution, we have determined the crystal structure of the GDP-bound form of the mutant protein, p21(Val-12), encoded by this oncogene. We report here the overall structure of p21(Val-12) at 2.2 A resolution and compare it with the structure of the normal c-Ha-ras protein. One of the major differences is that the loop of the transforming ras protein that binds the beta-phosphate of the guanine nucleotide is enlarged. Such a change in the 'catalytic site' conformation could explain the reduced GTPase activity of the mutant, which keeps the protein in the GTP bound 'signal on' state for a prolonged period time, ultimately causing cell transformation.

• Grand RJ, Levine BA, Byrd PJ, Gallimore PH
• The binding of guanine nucleotide to N-ras p21--a phosphorous and proton magnetic resonance study.
• Oncogene. 1989; 4: 355-61
• Display abstract

• One dimensional [1H] and [31P] nuclear magnetic resonance (NMR) studies have been carried out on purified wild type and mutant (Gly-12----Asp) N-ras protein expressed at high level in E. coli. Both proteins were isolated as stable 1:1 molar complexes with GDP with the upper limit for the first order rate constant for nucleotide dissociation 3 x 10(-4)s-1. From observation of the [31P] NMR spectrum after the addition of GTP it was concluded that the rate of nucleotide hydrolysis is appreciably greater than that of nucleotide exchange. Differences in the [31P] spectra of mutant and wild type proteins suggest that the mutation has a direct influence on the catalytic step. [1H] NMR spectra obtained for both mutant and wild type p21 were consistent with proteins of considerable stability and the addition of urea to concentrations of 4M appeared to cause little disruption in secondary structure. Additionally, the protein environment of the bound nucleotide remained well defined in the presence of a number of added reagents and over the pH range 5.8-9.5. The data are discussed in the light of the known crystal structure for H-ras p21 and indicate that the transforming mutation of aspartate for glycine-12 results in structural perturbations near the nucleotide binding site.

• Feuerstein J, Goody RS, Webb MR
• The mechanism of guanosine nucleotide hydrolysis by p21 c-Ha-ras. The stereochemical course of the GTPase reaction.
• J Biol Chem. 1989; 264: 6188-90
• Display abstract

• The use of guanosine 5'-O-(gamma-thio)triphosphate as a substrate for p21 c-Ha-ras was established. By using chirally labeled [gamma-17O,18O]guanosine 5'-O-(gamma-thio)triphosphate, the stereochemical course of the GTPase reaction was determined. The analysis shows that the hydrolysis occurs with inversion at the gamma-phosphorus. This shows that the most likely mechanism is a single step, in-line transfer, without a phosphoenzyme or other phosphorylated intermediate.

• Tong L, Milburn MV, de Vos AM, Kim SH
• Structure of ras proteins.
• Science. 1989; 245: 244-244

• Sigal IS, Gibbs JB
• Oncogenes.
• Curr Opin Cell Biol. 1989; 1: 286-90

• Kamei H
• Human lipocortins not similar to ras gene product.
• Cell Biol Int Rep. 1988; 12: 495-6

• Marx JL
• First portrait of an oncogene product.
• Science. 1988; 239: 863-863

• John J, Frech M, Wittinghofer A
• Biochemical properties of Ha-ras encoded p21 mutants and mechanism of the autophosphorylation reaction.
• J Biol Chem. 1988; 263: 11792-9
• Display abstract

• Kinetic studies performed on p21H guanine nucleotide complexes with and without Mg2+ show that point mutations at positions 12, 59, and 61 each have a different effect on the rate of nucleotide dissociation. Double mutants with a combination of these amino acid substitutions reveal that the effects of each mutation on these kinetics are interactive (nonadditive) for positions 12 and 59 and approximately additive for the positions 12 and 61. The magnitude and direction of the effects seen are dependent on the nature of the nucleotide and whether or not the complexes contain Mg2+. All the mutants have reduced GTPase activity. It is also shown that the autophosphorylation reaction velocity is of first order with respect to the protein concentration and that this reaction is an intramolecular one, which takes place as a side reaction of the GTPase reaction. The autophosphorylation is not reversible under the experimental conditions. The covalently bound phosphate does not decrease the nucleotide-binding ability of the protein nor does it change the relative affinity of the protein for GTP versus GDP. The results are discussed in terms of the structural model and function of p21H.

• Satoh T, Endo M, Nakamura S, Kaziro Y
• Analysis of guanine nucleotide bound to ras protein in PC12 cells.
• FEBS Lett. 1988; 236: 185-9
• Display abstract

• The ras gene product (p21) specifically binds GDP or GTP. In analogy with the reaction mechanism of other GTP-binding proteins, only the GTP-bound conformation is believed to be the biologically active one. Previously, we reported that not only oncogenic p21(Val-12) but also proto-oncogenic p21(Gly-12) could induce morphological differentiation in rat pheochromocytoma PC12 cells when microinjected in the complexed form with GTP gamma S [(1987) Mol. Cell. Biol. 7, 4553-4556]. In the present report we transformed PC12 cells with the oncogenic ras gene placed under the metallothionein I promoter. It was found that the transformed cells, when induced with Cd2+, differentiated in the absence of NGF. Then we analyzed the guanine nucleotide bound to p21 in the intact PC12 cells. It was found that conditionally induced p21(Val-12) was mostly present in the GTP-bound form, whereas the endogenous p21(Gly-12) was in the GDP-bound form. These results indicate again that p21.GTP induces the morphological differentiation of PC12 cells.

• Sigal IS
• The ras oncogene. A structure and some function.
• Nature. 1988; 332: 485-6

• Cales C, Hancock JF, Marshall CJ, Hall A
• The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product.
• Nature. 1988; 332: 548-51
• Display abstract

• About 30% of human tumours contain a mutation in one of the three ras genes leading to the production of p21ras oncoproteins that are thought to make a major contribution to the transformed phenotype of the tumour. The biochemical mode of action of the ras proteins is unknown but as they bind GTP and GDP and have an intrinsic GTPase activity, they may function like regulatory G proteins and control cell proliferation by regulating signal transduction pathways at the plasma membrane. It is assumed that an external signal is detected by a membrane molecule (or detector) that stimulates the conversion of p21.GDP to p21.GTP which then interacts with a target molecule (or effector) to generate an internal signal. Recently a cytoplasmic protein, GAP, has been identified that interacts with the ras proteins, dramatically increasing the GTPase activity of normal p21 but not of the oncoproteins. We report here that GAP appears to interact with p21ras at a site previously identified as the 'effector' site, strongly implicating GAP as the biological target for regulation by p21.

• Liotta LA
• H-ras p21 and the metastatic phenotype.
• J Natl Cancer Inst. 1988; 80: 468-9

• Pingoud A et al.
• Spectroscopic and hydrodynamic studies reveal structural differences in normal and transforming H-ras gene products.
• Biochemistry. 1988; 27: 4735-40
• Display abstract

• 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)

• Dolphin AC
• Is p21-ras a real G protein?
• Trends Neurosci. 1988; 11: 287-91

• Williams AR, Piris J, Wyllie AH
• ras p21 expression in the progression of breast cancer.
• Hum Pathol. 1988; 19: 1115-1115

• Thor A, Schlom J
• Monoclonal antibody RAP-5 and ras p21 expression.
• Hum Pathol. 1988; 19: 1119-20

• de Vos AM et al.
• Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21.
• Science. 1988; 239: 888-93
• Display abstract

• The crystal structure at 2.7 A resolution of the normal human c-H-ras oncogene protein lacking a flexible carboxyl-terminal 18 residue reveals that the protein consists of a six-stranded beta sheet, four alpha helices, and nine connecting loops. Four loops are involved in interactions with bound guanosine diphosphate: one with the phosphates, another with the ribose, and two with the guanine base. Most of the transforming proteins (in vivo and in vitro) have single amino acid substitutions at one of a few key positions in three of these four loops plus one additional loop. The biological functions of the remaining five loops and other exposed regions are at present unknown. However, one loop corresponds to the binding site for a neutralizing monoclonal antibody and another to a putative "effector region"; mutations in the latter region do not alter guanine nucleotide binding or guanosine triphosphatase activity but they do reduce the transforming activity of activated proteins. The data provide a structural basis for understanding the known biochemical properties of normal as well as activated ras oncogene proteins and indicate additional regions in the molecule that may possibly participate in other cellular functions.

• Feuerstein J, Goody RS, Wittinghofer A
• Preparation and characterization of nucleotide-free and metal ion-free p21 "apoprotein".
• J Biol Chem. 1987; 262: 8455-8
• Display abstract

• p21 isolated under nondenaturing conditions is obtained as a complex with guanosine nucleotides and magnesium ions. We have developed a high performance liquid chromatography method which removes greater than 95% of bound nucleotide and the metal ion very rapidly under mild conditions. At the same time, p21 is purified from minor protein impurities. The protein thus prepared is thermally much less stable than the complexed p21, but can be used for studying its interaction with nucleotides and metal ions at low temperatures. The association rate constant for p21 and GDP is 1.47 X 10(6) M-1 s-1 and for GTP is 2.9 X 10(6) M-1 s-1 at 0 degree C. By using appropriately determined dissociation rate constants we have determined the binding constant for p21.GDP and p21.GTP in the presence of excess Mg2+ to be 5.7 X 10(10) M-1 and 6.0 X 10(10) M-1, respectively, at 0 degree C.

• Brandt-Rauf PW, Pincus MR
• Oncogenes and oncogene proteins.
• Occup Med. 1987; 2: 27-38

• Clanton DJ, Lu YY, Blair DG, Shih TY
• Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis.
• Mol Cell Biol. 1987; 7: 3092-7
• Display abstract

• Point mutations of p21 proteins were constructed by oligonucleotide-directed mutagenesis of the v-rasH oncogene, which substituted amino acid residues within the nucleotide-binding consensus sequence, GXG GXGK. When the glycine residue at position 10, 13, or 15 was substituted with valine, the viral rasH product p21 lost its GTP-binding and autokinase activities. Other substitutions at position 33, 51, or 59 did not impair its binding activity. G418-resistant NIH 3T3 cell lines were derived by transfection with constructs obtained by inserting the mutant proviral DNA into the pSV2neo plasmid. Clones with a valine mutation at position 13 or 15 were incapable of transforming cells, while all other mutants with GTP-binding activity were competent. A mutant with a substitution of valine for glycine at position 10 which had lost its ability to bind GTP and its autokinase activity was fully capable of transforming NIH 3T3 cells. These cells grew in soft agar and rapidly formed tumors in nude mice. The p21 of cell lines derived from tumor explants still lacked the autokinase activity. These findings suggest that the glycine-rich consensus sequence is important in controlling p21 activities and that certain mutations may confer to p21 its active conformation without participation of ligand binding.

• Trahey M et al.
• Biochemical and biological properties of the human N-ras p21 protein.
• Mol Cell Biol. 1987; 7: 541-4
• Display abstract

• We characterized the normal (Gly-12) and two mutant (Asp-12 and Val-12) forms of human N-ras proteins produced by Escherichia coli. No significant differences were found between normal and mutant p21 proteins in their affinities for GTP or GDP. Examination of GTPase activities revealed significant differences between the mutant p21s: the Val-12 mutant retained 12% of wild-type GTPase activity, whereas the Asp-12 mutant retained 43%. Both mutant proteins, however, were equally potent in causing morphological transformation and increased cell motility after their microinjection into quiescent NIH 3T3 cells. This lack of correlation between transforming potency and GTPase activity or guanine nucleotide binding suggests that position 12 mutations affect other aspects of p21 function.

• Adam-Vizi V, Knight D, Hall A
• The ras protein is not associated with exocytosis.
• Nature. 1987; 328: 581-581

• Trahey M, McCormick F
• A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants.
• Science. 1987; 238: 542-5
• Display abstract

• The role of guanine nucleotides in ras p21 function was determined by using the ability of p21 protein to induce maturation of Xenopus oocytes as a quantitative assay for biological activity. Two oncogenic mutant human N-ras p21 proteins, Asp12 and Val12, actively induced maturation, whereas normal Gly12 p21 was relatively inactive in this assay. Both mutant proteins were found to be associated with guanosine triphosphate (GTP) in vivo. In contrast, Gly12 p21 was predominantly guanosine diphosphate (GDP)-bound because of a dramatic stimulation of Gly12 p21-associated guanosine triphosphatase (GTPase) activity. A cytoplasmic protein was shown to be responsible for this increase in activity. This protein stimulated GTP hydrolysis by purified Gly12 p21 more than 200-fold in vitro, but had no effect on Asp12 or Val12 mutants. A similar factor could be detected in extracts from mammalian cells. It thus appears that, in Xenopus oocytes, this protein maintains normal p21 in a biologically inactive, GDP-bound state through its effect on GTPase activity. Furthermore, it appears that the major effect of position 12 mutations is to prevent this protein from stimulating p21 GTPase activity, thereby allowing these mutants to remain in the active GTP-bound state.

• Tucker J, Sczakiel G, Feuerstein J, John J, Goody RS, Wittinghofer A
• Expression of p21 proteins in Escherichia coli and stereochemistry of the nucleotide-binding site.
• EMBO J. 1986; 5: 1351-8
• Display abstract

• v-Ha-ras encoded p21 protein (p21V), the cellular c-Ha-ras encoded protein (p21C) and its T24 mutant form p21T were produced in Escherichia coli under the control of the tac promoter. Large amounts of the authentic proteins in a soluble form can be extracted and purified without the use of denaturants or detergents. All three proteins are highly active in GDP binding, GTPase and, for p21V, autokinase activity. Inhibition of [3H]GDP binding to p21C by regio- and stereospecific phosphorothioate analogs of GDP and GTP was investigated to obtain a measure of the relative affinities of the three diphosphate and five triphosphate analogs of guanosine. p21 has a preference for the Sp isomers of GDP alpha S and GTP alpha S. It has low specificity for the Sp isomer of GTP beta S. Together with the data for GDP beta S and GTP gamma S these results are compared with those obtained for elongation factor (EF)Tu and transducin. This has enabled us to probe the structural relatedness of these proteins. We conclude that p21 seems to be more closely related to EF-Tu than to transducin.

• Colby WW, Hayflick JS, Clark SG, Levinson AD
• Biochemical characterization of polypeptides encoded by mutated human Ha-ras1 genes.
• Mol Cell Biol. 1986; 6: 730-4
• Display abstract

• We expressed six forms of p21-ras polypeptides in Escherichia coli with differing transformation potentials resulting from amino acid substitutions at position 12. The ability of the encoded p21's to autophosphorylate, bind guanine nucleotides, and hydrolyze GTP was assessed. All versions of p21 bound GTP equivalently; the kinase activity, while dependent upon residue 12, did not correlate with the transforming potential of the polypeptide. All transforming versions exhibited an impaired GTPase activity, while a novel nontransforming derivative [p21(pro-12)] possessed an enhanced GTPase activity. These results provide strong support for the proposal that an impairment of the cellular p21 GTPase activity can unmask its transforming potential.

• McCormick F, Levenson C, Cole G, Innis M, Clark R
• Genetic and biochemical analysis of ras p21 structure.
• Symp Fundam Cancer Res. 1986; 39: 137-42
• Display abstract

• We tested aspects of our model of the ras p21 structure using generic, biochemical, and immunologic approaches. First, we made a monoclonal antibody against a p21 region that is highly conserved and likely to be critical to p21 function. The antibody blocks p21 function in various cell systems. Its binding to p21 is completely blocked by guanine nucleotides, even though the region of p21 to which it binds does not seem to be part of the guanine nucleotide-binding site. We propose that the conformation of this critical region is modulated by nucleotide binding. Another interesting region of p21 includes amino acids 116 and 119, which seem to confer, in part, the specificity of p21 for guanine nucleotides. We made a series of mutants in this region and tested their ability to bind GTP, and such related purine nucleotides as XTP and diaminopurine nucleoside triphosphate. We were able to refine our model for guanine nucleotide interaction with p21 and to create mutant proteins with altered specificity for purine nucleotides. Finally, we tested rates of autophosphorylation of six position 12 mutants and conclude that amino acid 12 affects the positioning of bound nucleotides relative to sequences around amino acid 59.

• Lowy DR, Willumsen BM
• The ras gene family.
• Cancer Surv. 1986; 5: 275-89
• Display abstract

• Members of the ras multigene family have been found in virtually all eukaryotes, from yeast to mammals. ras is required for normal cell growth in the yeast Saccharomyces cerevisiae and in at least some mammalian cells. These genes induce tumorigenic transformation of established NIH 3T3 cells by increased expression of a normal ras gene, certain point mutations or amino acid deletion. In tumours, point mutation appears to be the most common mechanism of activation. The ras proteins are found at the plasma membrane, bind guanine nucleotides GDP and GTP and possess a GTPase activity. At least some ras proteins that have been activated by single amino acid substitutions possess a GTPase activity that is lower than that of the normal version. These results are consistent with the hypothesis that ras protein stimulates its putative target(s) when GTP is bound to it, as is true for the G regulatory proteins or elongation factor Tu. In Saccharomyces cerevisiae, ras has been shown to stimulate adenylate cyclase. However, there does not appear to be a direct interaction between ras and adenylate cyclase in mammalian cells.

• McGrath JP, Capon DJ, Goeddel DV, Levinson AD
• Comparative biochemical properties of normal and activated human ras p21 protein.
• Nature. 1984; 310: 644-9
• Display abstract

• Human Ha-ras1 cDNAs encoding normal and activated p21 polypeptides have been efficiently expressed in Escherichia coli and the biochemical activities associated with each polypeptide compared. In addition to the guanine nucleotide binding activity, normal p21 displays a GTPase activity which is selectively impaired by a mutation which activates its oncogenic potential.

© 2021 EMBL | Privacy Policy