guanosine-diphosphate and Cell-Transformation--Neoplastic

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Genes, ras; Growth Substances; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Models, Biological; Oncogene Protein p21(ras); Signal Transduction; Transfection

Heterotrimeric G proteins are molecular switches that control signal transduction, and their dysregulation can promote oncogenesis. Somatic mutations in GNAS, GNAI2 and GNAQ genes induce oncogenesis by rendering Gα subunits constitutively activated. Recently the first somatic mutation, arginine(243) → histidine (R243H) in the GNAO1 (Gαo) gene was identified in breast carcinomas and shown to promote oncogenic transformation when introduced into cells. Here, we provide the molecular basis for the oncogenic properties of the Gαo R243H mutant. Using limited proteolysis assays, nucleotide-binding assays, and single-turnover and steady-state GTPase assays, we demonstrate that the oncogenic R234H mutation renders Gαo constitutively active by accelerating the rate of nucleotide exchange; however, this mutation does not affect Gαo's ability to become deactivated by GTPase-activating proteins (GAPs) or by its intrinsic GTPase activity. This mechanism differs from that of previously reported oncogenic mutations that impair GTPase activity and GAP sensitivity without affecting nucleotide exchange. The constitutively active Gαo R243H mutant also enhances Src-STAT3 signaling in NIH-3T3 cells, a pathway previously shown to be directly triggered by active Gαo proteins to promote cellular transformation. Based on structural analyses, we propose that the enhanced rate of nucleotide exchange in Gαo R243H results from loss of the highly conserved electrostatic interaction of R243 with E43, located in the in the P-loop that represents the binding site for the α- and β-phosphates of the nucleotide. We conclude that the novel R234H mutation imparts oncogenic properties to Gαo by accelerating nucleotide exchange and rendering it constitutively active, thereby enhancing signaling pathways, for example, src-STAT3, responsible for neoplastic transformation.

Topics: Adenosine Triphosphatases; Amino Acid Sequence; Amino Acid Substitution; Animals; Arginine; Binding Sites; Biocatalysis; Cell Line, Tumor; Cell Transformation, Neoplastic; GTP-Binding Protein alpha Subunits, Gi-Go; GTPase-Activating Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Histidine; Humans; Mice; Models, Molecular; Mutation; NIH 3T3 Cells; Protein Structure, Secondary; Protein Structure, Tertiary; Proto-Oncogene Proteins pp60(c-src); Sequence Homology, Amino Acid; Signal Transduction; STAT3 Transcription Factor

This paper reports the synthesis of a panel of small molecules with arylamides and arylsulfonamides groups and their biological activity in inhibiting nucleotide exchange on human Ras. The design of these molecules was guided by experimental and molecular modelling data previously collected on similar compounds. Aim of this work is the validation of the hypothesis that a phenyl hydroxylamine group linked to a second aromatic moiety generates a pharmacophore capable to interact with Ras and to inhibit its activation. In vitro experiments on purified human Ras clearly show that the presence of an aromatic hydroxylamine and a sulfonamide group in the same molecule is a necessary condition for Ras binding and nucleotide exchange inhibition. The inhibitor potency is lower in molecules in which either the hydroxylamine has been replaced by other functional groups or the sulfonamide has been replaced by an amide. In the case both these moieties, the hydroxylamine and sulfonamide are absent, inactive compounds are obtained.

Topics: Animals; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p21; Drug Design; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Hydroxylamines; Inhibitory Concentration 50; Mice; Molecular Structure; Mutation; NIH 3T3 Cells; ras Proteins; ras-GRF1; Structure-Activity Relationship; Sulfonamides; Two-Hybrid System Techniques

To investigate the unregulated Ras activation associated with cancer, we developed and validated a mathematical model of Ras signaling. The model-based predictions and associated experiments help explain why only one of two classes of activating Ras point mutations with in vitro transformation potential is commonly found in cancers. Model-based analysis of these mutants uncovered a systems-level process that contributes to total Ras activation in cells. This predicted behavior was supported by experimental observations. We also used the model to identify a strategy in which a drug could cause stronger inhibition on the cancerous Ras network than on the wild-type network. This system-level analysis of the oncogenic Ras network provides new insights and potential therapeutic strategies.

Topics: Antineoplastic Agents; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Computer Simulation; Extracellular Signal-Regulated MAP Kinases; Genes, ras; GTP Phosphohydrolases; GTPase-Activating Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Mathematics; Metabolic Networks and Pathways; Models, Biological; Neoplasms; Phosphorylation; Point Mutation; ras Proteins; Signal Transduction

Cdc42, a Ras-related GTP-binding protein, has been implicated in the regulation of the actin cytoskeleton, membrane trafficking, cell-cycle progression, and malignant transformation. We have shown previously that a Cdc42 mutant (Cdc42(F28L)), capable of spontaneously exchanging GDP for GTP (referred to as "fast-cycling"), transformed NIH 3T3 cells because of its ability to interfere with epidermal growth factor receptor (EGFR)-Cbl interactions and EGFR down-regulation. To further examine the link between the hyperactivation of Cdc42 and its ability to alter EGFR signaling and thereby cause cellular transformation, we examined the effects of expressing different forms of the Cdc42-specific guanine nucleotide exchange factor, intersectin-L, in fibroblasts. Full-length intersectin-L exhibited little ability to stimulate nucleotide exchange on Cdc42, whereas a truncated version that contained five Src homology 3 (SH3) domains, the Dbl and pleckstrin homology domains (DH and PH domains, respectively), and a C2 domain (designated as SH3A-C2) showed modest guanine nucleotide exchange factor activity, whereas a form containing just the DH, PH, and C2 domains (DH-C2) strongly activated Cdc42. However, DH-C2 showed little ability to stimulate growth in low serum or colony formation in soft agar, whereas SH3A-C2 gave rise to a much stronger stimulation of cell growth in low serum and was highly effective in stimulating colony formation. Moreover, although SH3A-C2 strongly transformed fibroblasts, it differed from the actions of the Cdc42(F28L) mutant, as SH3A-C2 showed little ability to alter EGFR levels or the lifetime of EGF-coupled signaling through ERK. Rather, we found that SH3A-C2 exhibited strong transforming activity through its ability to mediate cooperation between Ras and Cdc42.

Topics: Actins; Adaptor Proteins, Vesicular Transport; Agar; Animals; cdc42 GTP-Binding Protein; Cell Cycle; Cell Line; Cell Line, Transformed; Cell Transformation, Neoplastic; COS Cells; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Immunoblotting; Immunoprecipitation; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mice; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase Kinases; Models, Biological; Mutation; NIH 3T3 Cells; Phosphatidylinositol 3-Kinases; Plasmids; Protein Binding; Protein Structure, Tertiary; ras Proteins; Signal Transduction; src Homology Domains; Time Factors; Transfection

Lysophosphatidic acid (LPA), a major G protein coupled receptor (GPCR)-activating ligand present in serum, elicits growth factor like responses by stimulating specific GPCRs coupled to heterotrimeric G proteins such as G(i), G(q), and G12/13. Previous studies have shown that the overexpression of wild-type Galpha12 (Galpha12WT) results in the oncogenic transformation of NIH3T3 cells (Galpha12WT-NIH3T3) in a serum-dependent manner. Based on the potent growth-stimulating activity of LPA and the presence of LPA and LPA-like molecules in the serum, we hypothesized that the serum-dependent neoplastic transformation of Galpha12WT-NIH3T3 cells was mediated by the stimulation of LPA-receptors (LPARs) by LPA in the serum. In the present study, using guanine nucleotide exchange assay and GST-TPR binding assay, we show that the treatment of Galpha12WT-NIH3T3 with 2 muM LPA leads to the activation of Galpha12. Stimulation of these cells with LPA promotes JNK-activation, a critical component of Galpha12-response and cell proliferation. We also show that LPA can substitute for serum in stimulating JNK-activity, DNA synthesis, and proliferation of Galpha12WT-NIH3T3 cells. LPA-mediated proliferative response in NIH3T3 cells involves Galpha12, but not the closely related Galpha13. Pretreatment of Galpha12WT-NIH3T3 cells with suramin (100 microM), a receptor-uncoupling agent, inhibited LPA-stimulated proliferation of these cells by 55% demonstrating the signal coupling between cell surface LPAR and Galpha12 in the neoplastic proliferation of NIH3T3 cells. As LPA and LPAR mediated mitogenic pathways have been shown to play a major role in tumor genesis and progression, a mechanistic understanding of the signal coupling between LPAR, Galpha12, and the downstream effectors is likely to unravel additional targets for novel cancer chemotherapies.

Topics: 3T3 Cells; Animals; Cell Proliferation; Cell Transformation, Neoplastic; Enzyme Activation; GTP-Binding Protein alpha Subunits, G12-G13; Guanosine Diphosphate; Guanosine Triphosphate; JNK Mitogen-Activated Protein Kinases; Lysophospholipids; Mice; Pertussis Toxin; Receptors, Lysophosphatidic Acid; Signal Transduction; Suramin

Cdc25Mm is a mammalian Ras-specific guanine nucleotide exchange factor (GEF). By homology modeling we show that it shares with Sos-GEF the structure of the putative catalytic HI hairpin where the dominant negative T1184E mutation is located. Similarly to Cdc25MmT1184E, the isolated wild-type and mutant hairpins retain the ability to displace Ras-bound nucleotide, originate a stable Ras/GEF complex and downregulate the Ras pathway in vivo. These results indicate that nucleotide re-entry and Ras/GEF dissociation--final steps in the GEF catalytic cycle--require GEF regions different from the HI hairpin. GEF down-sizing could lead to development of novel Ras inhibitors.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Buffers; Catalysis; Catalytic Domain; Cell Line, Transformed; Cell Transformation, Neoplastic; Crystallography, X-Ray; Down-Regulation; Escherichia coli; Fibroblasts; Genes, Dominant; Genes, ras; Genes, Reporter; Glutamic Acid; Guanosine Diphosphate; Guanosine Triphosphate; Homozygote; Luciferases; Mice; Models, Molecular; Molecular Sequence Data; NIH 3T3 Cells; ortho-Aminobenzoates; Protein Structure, Secondary; ras-GRF1; Sequence Homology, Amino Acid; Temperature

Embryonic stem (ES) cells are pluripotent cells derived from early mammalian embryos. Their immortality and rapid growth make them attractive sources for stem cell therapies; however, they produce tumours (teratomas) when transplanted, which could preclude their therapeutic usage. Why ES cells, which lack chromosomal abnormalities, possess tumour-like properties is largely unknown. Here we show that mouse ES cells specifically express a Ras-like gene, which we have named ERas. We show that human HRasp, which is a recognized pseudogene, does not contain reported base substitutions and instead encodes the human orthologue of ERas. This protein contains amino-acid residues identical to those present in active mutants of Ras and causes oncogenic transformation in NIH 3T3 cells. ERas interacts with phosphatidylinositol-3-OH kinase but not with Raf. ERas-null ES cells maintain pluripotency but show significantly reduced growth and tumorigenicity, which are rescued by expression of ERas complementary DNA or by activated phosphatidylinositol-3-OH kinase. We conclude that the transforming oncogene ERas is important in the tumour-like growth properties of ES cells.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Cell Division; Cell Transformation, Neoplastic; Cloning, Molecular; Embryo, Mammalian; Genes, ras; Guanosine Diphosphate; Guanosine Triphosphate; Mice; Molecular Sequence Data; Neoplasms; Oncogene Protein p21(ras); Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-raf; Pseudogenes; Stem Cells

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) that exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. Like many RhoGEFs, the in vitro catalytic activity of Dbs is not limited to a single substrate. It can catalyze the exchange of GDP for GTP on RhoA and Cdc42, both of which are expressed in most cell types. This lack of substrate specificity, which is relatively common among members of the RhoGEF family, complicates efforts to determine the molecular basis of their transforming activity. We have recently determined crystal structures of several RhoGEFs bound to their cognate GTPases and have used these complexes to predict structural determinants dictating the specificities of coupling between RhoGEFs and GTPases. Guided by this information, we mutated Dbs to alter significantly its relative exchange activity for RhoA versus Cdc42 and show that the transformation potential of Dbs correlates with exchange on RhoA but not Cdc42. Supporting this conclusion, oncogenic Dbs activates endogenous RhoA but not endogenous Cdc42 in NIH 3T3 cells. Similarly, a competitive inhibitor that blocks RhoA activation also blocks Dbs-mediated transformation. In conclusion, this study highlights the usefulness of specificity mutants of RhoGEFs as tools to genetically dissect the multiple signaling pathways potentially activated by overexpressed or oncogenic RhoGEFs. These ideas are exemplified for Dbs, which is strongly implicated in the transformation of NIH 3T3 cells via RhoA and not Cdc42.

Topics: 3T3 Cells; Animals; cdc42 GTP-Binding Protein; Cell Transformation, Neoplastic; Fibroblasts; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; Mice; Models, Molecular; Mutagenesis, Site-Directed; Protein Binding; Protein Structure, Tertiary; Rho Guanine Nucleotide Exchange Factors; rhoA GTP-Binding Protein; Structure-Activity Relationship; Substrate Specificity; Transfection

Cdc42 is a small GTP-binding protein which has been implicated in a number of cellular activities, including cell morphology, motility, cell-cycle progression, and malignant transformation. While GTPase-defective forms of Cdc42 inhibit cell growth, a mutation [Cdc42(F28L)] that allows the constitutive exchange of GDP for GTP and is GTPase-competent induces cellular transformation. These results suggest that Cdc42 must cycle between its GTP- and GDP-bound states to stimulate cell growth. In attempting to design Cdc42 molecules with more potent transforming activity, we set out to generate other types of Cdc42 mutants capable of constitutive GDP-GTP exchange. Here, we describe one such mutant, generated by changing a conserved aspartic acid residue at position 118 to an asparagine. The Cdc42(D118N) protein exchanges GDP for GTP more rapidly than wild-type Cdc42, but significantly more slowly than the Cdc42(F28L) mutant. Despite its slower rate of activation, the Cdc42(D118N) mutant is more potent at inducing cellular transformation than the Cdc42(F28L) protein, and causes a significant loss in actin stress fibers, reminiscent of what is observed with fibroblasts transformed by oncogenic Ras mutants. Effector-loop mutations made within the D118N background inhibit Cdc42-induced transformation and Cdc42-mediated antiapoptotic (survival) activity to similar extents. In addition, mutating aspartic acid 121 (to asparagine), which forms part of a caspase cleavage site (DLRD, residues 118-121 of Cdc42), in combination with the F28L mutation generates a Cdc42 molecule [Cdc42(F28L/D121N)] with transforming activity significantly stronger than that of Cdc42(F28L). Thus, mutations that combine some capacity for cycling between the GTP- and GDP-bound states with increased survival against apoptotic signals yield Cdc42 molecules with the maximum capability for inducing cellular transformation.

Topics: 3T3 Cells; Amino Acid Substitution; Animals; Apoptosis; Asparagine; Aspartic Acid; cdc42 GTP-Binding Protein; Cell Division; Cell Transformation, Neoplastic; COS Cells; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Mice; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Protein Binding; Trans-Activators; Transfection

Guanine nucleotide exchange factors from the Dbl family are proto-oncogenic proteins that activate small GTPases of the Rho family. Here we report the characterization of GEF720, a novel Dbl-like protein related to p115Rho-GEF. GEF720 activated RhoA both in our recently developed Yeast Exchange Assay and in biochemical in vitro exchange assays. GEF720 induced RhoA dependent assembly of actin stress fibers in REF52 fibroblastic cells. In NIH3T3 cells this Dbl-like protein elicited formation of transformation foci with a morphology similar to RhoA-V14 induced foci. In the PC12 neuron-like cell line, expression of GEF720, whose mRNA is brain specific, inhibited NGF-induced neurite outgrowth. Finally, GEF720 gene is located on human chromosome 1 on band 1p36, between Tumor Protein 73 and Tumor Necrosis Factor Receptor 12, two genes rearranged in many neuroblastoma cell lines. Together, these results show that this new Dbl related protein, GEF720, is an exchange factor that can directly activate RhoA in vivo and is potentially involved in the control of neuronal cell differentiation. GEF720 is also a new candidate gene involved in the progression of neuroblastoma and developmental abnormalities associated with rearrangements in the 1p36 chromosomal region.

Topics: 3T3 Cells; Actins; Amino Acid Sequence; Animals; Base Sequence; Brain; Brain Chemistry; Cell Differentiation; Cell Line, Transformed; Cell Transformation, Neoplastic; Chromosome Mapping; Chromosomes, Human, Pair 1; Disease Progression; Enzyme Activation; Exons; Fibroblasts; Genes; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Mice; Molecular Sequence Data; Multigene Family; Nerve Tissue Proteins; Neurites; Neuroblastoma; PC12 Cells; Protein Binding; Protein Structure, Tertiary; Rats; Recombinant Fusion Proteins; rhoA GTP-Binding Protein; Saccharomyces cerevisiae Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Stress Fibers; Transfection; Tumor Cells, Cultured

Ras plays an important role in a variety of cellular functions, including growth, differentiation, and oncogenic transformation. For instance, Ras participates in the activation of Raf, which phosphorylates and activates mitogen-activated protein kinase kinase (MEK), which then phosphorylates and activates extracellular signal-regulated kinase (ERK), a mitogen-activated protein (MAP) kinase. Activation of MAP kinase appears to be essential for propagating a wide variety of extracellular signals from the plasma membrane to the nucleus. N17Ras, a GDP-bound dominant negative mutant, is used widely as an interfering mutant to assess Ras function in vivo. Surprisingly, we observed that expression of N17Ras inhibited the activity and phosphorylation of Elk-1, a physiological substrate of MAP kinases, in response to phorbol myristate acetate. The activity and phosphorylation of the MAP kinase hemagglutinin epitope (HA)-ERK1 were not affected by N17Ras in response to the same stimulus. Additionally, expression of N17Ras, but not L61S186Ras, a GTP-bound interfering mutant, inhibited MEK-induced Elk-1 phosphorylation, suggesting that inhibition of Elk-1 may be unique to GDP-bound Ras mutants. Finally, we observed that V12Ras-induced focus formation in NIH3T3 cells is inhibited by coexpression of GDP-bound Ras mutants, such as N17, A15, and N17N69. Therefore, N17Ras and V12 Ras may be codominant with respect to Elk-1 activation and cellular transformation. These results indicate that N17Ras appears to have at least two distinguishable functions: interference with endogenous Ras activation and inhibition of Elk-1 and transfomation. Furthermore, our data imply the possibility that GDP-bound Ras, like N17Ras, may have a direct role in signal transduction.

Topics: Cell Transformation, Neoplastic; DNA-Binding Proteins; ets-Domain Protein Elk-1; Genes, ras; Growth Substances; Guanosine Diphosphate; Guanosine Triphosphate; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Mutation; Phosphorylation; Proto-Oncogene Proteins; ras Proteins; Ribosomal Protein S6 Kinases, 90-kDa; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription Factors; Transcriptional Activation

Cph was isolated from neoplastic Syrian hamster embryo fibroblasts initiated by 3-methylcholanthrene (MCA), and was shown to be a single copy gene in the hamster genome, conserved from yeast to human cells, expressed in fetal cells and most adult tissues, and acting synergistically with H-ras in the transformation of murine NIH3T3 fibroblasts. We have now isolated Syrian hamster full-length cDNAs for the cph oncogene and proto-oncogene. Nucleotide sequence analysis revealed that cph was activated in MCA-treated cells by a point-mutational deletion at codon 214, which caused a shift in the normal open reading frame (ORF) and brought a translation termination codon 33 amino acids downstream. While proto-cph encodes a protein (pcph) of 469 amino acids, cph encodes a truncated protein (cph) of 246 amino acids with a new, hydrophobic C-terminus. Similar mechanisms activated cph in other MCA-treated Syrian hamster cells. The cph and proto-cph proteins have partial sequence homology with two protein families: GDP/GTP exchange factors and nucleotide phosphohydrolases. In vitro translated, gel-purified cph proteins did not catalyze nucleotide exchange for H-ras, but were able to bind nucleotide phosphates, in particular ribonucleotide diphosphates such as UDP and GDP. Steady-state levels of cph mRNA increased 6.7-fold in hamster neoplastic cells, relative to a 2.2-fold increase in normal cells, when they were subjected to a nutritional stress such as serum deprivation. Moreover, cph-transformed NIH3T3 cells showed increased survival to various forms of stress (serum starvation, hyperthermia, ionizing radiation), strongly suggesting that cph participates in cellular mechanisms of response to stress.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Base Sequence; Catalysis; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Cloning, Molecular; Cricetinae; DNA, Complementary; Gene Expression Regulation, Neoplastic; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; Mesocricetus; Mice; Molecular Sequence Data; Nucleotides; Oncogene Protein p21(ras); Oncogene Proteins; Oncogenes; Point Mutation; Proteins; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogenes; ras Guanine Nucleotide Exchange Factors; Sequence Homology, Amino Acid

RasGRP, a guanyl nucleotide-releasing protein for the small guanosine triphosphatase Ras, was characterized. Besides the catalytic domain, RasGRP has an atypical pair of "EF hands" that bind calcium and a diacylglycerol (DAG)-binding domain. RasGRP activated Ras and caused transformation in fibroblasts. A DAG analog caused sustained activation of Ras-Erk signaling and changes in cell morphology. Signaling was associated with partitioning of RasGRP protein into the membrane fraction. Sustained ligand-induced signaling and membrane partitioning were absent when the DAG-binding domain was deleted. RasGRP is expressed in the nervous system, where it may couple changes in DAG and possibly calcium concentrations to Ras activation.

Topics: Amino Acid Sequence; Animals; Brain; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Catalysis; Cell Cycle Proteins; Cell Line; Cell Membrane; Cell Size; Cell Transformation, Neoplastic; Cloning, Molecular; Diglycerides; DNA-Binding Proteins; DNA, Complementary; Genes, ras; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; Molecular Sequence Data; Neurons; Phosphoprotein Phosphatases; ras Proteins; ras-GRF1; Rats; Recombinant Fusion Proteins; Signal Transduction

Like Ras, constitutively activated mutants of the Ras-related protein R-Ras cause tumorigenic transformation of NIH3T3 cells. However, since R-Ras causes a transformed phenotype distinct from that induced by Ras, it is likely that R-Ras controls signaling pathways and cellular processes distinct from those regulated by Ras. To address this possibility, we determined if R-Ras is regulated by activators and effectors distinct from those that regulate Ras function. We observed that Ras guanine nucleotide exchange factors failed to activate R-Ras in vivo, indicating that R-Ras is activated by distinct GEFs. Consistent with this, mutants of R-Ras with mutations analogous to the Ras(15A)/(17N) dominant negative proteins did not antagonize Ras GEF function and lacked the growth inhibitory activity seen with these mutant Ras proteins. Thus, R-Ras, but not Ras, is dispensable for the viability of NIH3T3 cells. Finally, whereas constitutively activated Ras can overcome the growth inhibitory action of the Ras(17N) dominant negative protein via Raf-dependent and -independent activities, transforming mutants of R-Ras failed to do so. This inability was consistent with our observation that Ras-, but not R-Ras-transformed, NIH3T3 cells possessed constitutively upregulated Raf kinase activities. Thus, R-Ras and Ras are regulators of distinct signaling pathways and cellular processes.

Topics: 3T3 Cells; Animals; Cell Division; Cell Transformation, Neoplastic; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Mice; Mutation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; rap GTP-Binding Proteins; ras Proteins

The three-dimensional structure of the complex between human H-Ras bound to guanosine diphosphate and the guanosine triphosphatase (GTPase)-activating domain of the human GTPase-activating protein p120GAP (GAP-334) in the presence of aluminum fluoride was solved at a resolution of 2.5 angstroms. The structure shows the partly hydrophilic and partly hydrophobic nature of the communication between the two molecules, which explains the sensitivity of the interaction toward both salts and lipids. An arginine side chain (arginine-789) of GAP-334 is supplied into the active site of Ras to neutralize developing charges in the transition state. The switch II region of Ras is stabilized by GAP-334, thus allowing glutamine-61 of Ras, mutation of which activates the oncogenic potential, to participate in catalysis. The structural arrangement in the active site is consistent with a mostly associative mechanism of phosphoryl transfer and provides an explanation for the activation of Ras by glycine-12 and glutamine-61 mutations. Glycine-12 in the transition state mimic is within van der Waals distance of both arginine-789 of GAP-334 and glutamine-61 of Ras, and even its mutation to alanine would disturb the arrangements of residues in the transition state.

Topics: Aluminum Compounds; Amino Acid Sequence; Binding Sites; Catalysis; Cell Transformation, Neoplastic; Crystallography, X-Ray; Enzyme Activation; Fluorides; GTP Phosphohydrolases; GTP-Binding Proteins; GTPase-Activating Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Models, Molecular; Molecular Sequence Data; Mutation; Protein Conformation; Protein Structure, Secondary; Proteins; ras GTPase-Activating Proteins; ras Proteins; Signal Transduction

To investigate the role of the cellular ras gene product in neoplastic transformation by the SV40 large tumor antigen (SVLT), murine C3H10T1/2 cells were rendered deficient in Ras activity by transfection with inducible or constitutive antisense ras gene constructs or through the introduction of the dominant-negative mutant, ras(asn17). Consistent with previous results, SVLT-induced morphological transformation was unaffected by the down-regulation of c-ras gene product activity. On the other hand, colony formation in soft agar and tumorigenicity in nude mice were drastically reduced in c-Ras-deficient cells. In addition, SVLT expression in C3H10T1/2 cells led to increased c-Ras activity, as determined by an increase in the Ras-bound GTP/GTP + GDP ratio. These results suggest that c-Ras is required for full neoplastic transformation by SVLT.

Topics: Animals; Antigens, Polyomavirus Transforming; Cell Line, Transformed; Cell Transformation, Neoplastic; Fibroblasts; Gene Expression Regulation, Neoplastic; Genes, ras; Guanosine Diphosphate; Guanosine Triphosphate; Mice; Mice, Nude; Mutation; Neoplasms, Experimental; Oncogene Proteins v-raf; ras Proteins; Retroviridae Proteins, Oncogenic; RNA, Antisense; Simian virus 40; Transfection

Cdc42Hs is a small GTPase of the Rho-subfamily, which regulates signaling pathways that influence cell morphology and polarity, cell-cycle progression and transcription. An essential role for Cdc42Hs in cell growth regulation has been suggested by the finding that the Dbl oncoprotein is an upstream activator-a guanine nucleotide exchange factor (GEF)-for Cdc42Hs, and that activated mutants of the closely related GTPases Rac and Rho are transforming. As we were unable to obtain significant over-expression of GTPase-defective Cdc42Hs mutants, we have generated a mutant, Cdc42Hs(F28L), which can undergo spontaneous GTP-GDP exchange while maintaining full GTPase activity, and thus should exhibit functional activities normally imparted by Dbl. In cultured fibroblasts, Cdc42Hs(F28L) activated the c-Jun kinase (JNK1) and stimulated filopodia formation. Cells stably expressing Cdc42Hs(F28L) also exhibited several hallmarks of transformation-reduced contact inhibition, lower dependence on serum for growth, and anchorage-independent growth. Our findings indicate that Cdc42Hs plays a role in cell proliferation, and is a likely physiological mediator of Dbl-induced transformation.

Topics: 3T3 Cells; Animals; Calcium-Calmodulin-Dependent Protein Kinases; cdc42 GTP-Binding Protein; Cell Cycle Proteins; Cell Division; Cell Transformation, Neoplastic; Enzyme Activation; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; JNK Mitogen-Activated Protein Kinases; Mice; Mitogen-Activated Protein Kinases; Mutation

While Ras proteins are activated by stimulated GDP release, which enables acquisition of the active GTP-bound state, little is known about how guanine nucleotide exchange factors (GEFs) interact with Ras to promote this exchange reaction. Here we report that mutations within the switch 2 domain of Ras (residues 62-69) inhibit activation of Ras by the mammalian GEFs, Sos1, and GRF/CDC25Mm. While mutations in the 62-69 region blocked upstream activation of Ras, they did not disrupt Ras effector functions, including transcriptional activation and transformation of NIH 3T3 cells. Biochemical analysis indicated that the loss of GEF responsiveness of a Ras(69N) mutant was due to a loss of GEF binding, with no change in intrinsic nucleotide exchange activity. Furthermore, structural analysis of Ras(69N) using NMR spectroscopy indicated that mutation of residue 69 had a very localized effect on Ras structure that was limited to alpha-helix 2 of the switch 2 domain. Together, these results suggest that the switch 2 domain of Ras forms a direct interaction with GEFs.

Topics: 3T3 Cells; Animals; Binding Sites; Cell Cycle Proteins; Cell Transformation, Neoplastic; Cloning, Molecular; Escherichia coli; Fungal Proteins; Genes, ras; GTP-Binding Proteins; GTPase-Activating Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Kinetics; Magnetic Resonance Spectroscopy; Mammals; Mice; Models, Structural; Mutagenesis, Site-Directed; Phosphoprotein Phosphatases; Point Mutation; Protein Structure, Secondary; Proteins; ras GTPase-Activating Proteins; ras Proteins; ras-GRF1; Recombinant Fusion Proteins; Repressor Proteins; SOS1 Protein; Transcriptional Activation

Although the Ras-related protein TC21/R-Ras2 has only 55% amino acid identity with Ras proteins, mutated forms of TC21 exhibit the same potent transforming activity as constitutively activated forms of Ras. Therefore, like Ras, TC21 may activate signaling pathways that control normal cell growth and differentiation. To address this possibility, we determined if regulators and effectors of Ras are also important for controlling TC21 activity. First, we determined that Ras guanine nucleotide exchange factors (SOS1 and RasGRF/CDC25) synergistically enhanced wild-type TC21 activity in vivo and that Ras GTPase-activating proteins (GAPs; p120-GAP and NF1-GAP) stimulated wild-type TC21 GTP hydrolysis in vitro. Thus, extracellular signals that activate Ras via SOS1 activation may cause coordinate activation of Ras and TC21. Second, we determined if Raf kinases were effectors for TC21 transformation. Unexpectedly, yeast two-hybrid binding analyses showed that although both Ras and TC21 could interact with the isolated Ras-binding domain of Raf-1, only Ras interacted with full-length Raf-1, A-Raf, or B-Raf. Consistent with this observation, we found that Ras- but not TC21-transformed NIH 3T3 cells possessed constitutively elevated Raf-1 and B-Raf kinase activity. Thus, Raf kinases are effectors for Ras, but not TC21, signaling and transformation. We conclude that common upstream signals cause activation of Ras and TC21, but activated TC21 controls cell growth via distinct Raf-independent downstream signaling pathways.

Topics: 3T3 Cells; Animals; Cell Cycle Proteins; Cell Transformation, Neoplastic; Female; Fungal Proteins; Genes, ras; GTP-Binding Proteins; GTPase-Activating Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Male; Membrane Proteins; Mice; Monomeric GTP-Binding Proteins; Organ Specificity; Phosphoprotein Phosphatases; Pregnancy; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; ras GTPase-Activating Proteins; ras Proteins; ras-GRF1; Recombinant Proteins; Repressor Proteins; Signal Transduction; SOS1 Protein; Transcriptional Activation; Transfection

We examined the effect of two rhesus papillomavirus 1 (RhPV) oncogenes on cytokine-induced signal transduction pathways leading to the possible activation of Ras protein (p21ras) and phosphatidylinositol kinase. p21ras in both the activated (GTP-bound) and inactivated (GDP-bound) states were quantitated. NIH 3T3 cell lines expressing the RhPV 1 E5 gene or epidermal growth factor receptor cDNA had about a sixfold higher ratio of p21ras-bound GTP to p21ras-bound GDP as compared with parental NIH 3T3 cells or a cell line expressing the RhPV 1 E7 gene under normal culture conditions, yet expressed similar levels of p21ras. Quiescent cells had dramatically reduced levels of activated p21ras, except those containing RhPV 1 E7. Levels were restored by stimulation with epidermal growth factor or platelet-derived growth factor. Both epidermal growth factor and platelet-derived growth factor receptor of RhPV 1 E5- and E7-containing cells responded to cytokine stimulation. Endogenous phosphatidylinositol-3'-kinase was up-regulated in NIH 3T3 cells transformed with the E5 genes of RhPV 1 and bovine papillomavirus 1. These results suggest that E5 genes of papillomaviruses play a major role in the regulation of transduction pathways.

Topics: 3T3 Cells; Animals; Cattle; Cell Transformation, Neoplastic; Enzyme Activation; ErbB Receptors; Genes, Viral; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Macaca; Mice; Papillomaviridae; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins p21(ras); Recombinant Proteins; Transfection; Viral Proteins

Ras p21s are known as molecular switch for signal transduction pathways. They act as intracellular signal transducers of extracellular signals for growth and differentiation. Ras activities are regulated by the rotation between active GTP-bound form and inactive GDP-bound form. This cycle is regulated by the GDP/GTP exchange reaction and intrinsic GTPase activity of ras p21. The N116Y, v-H-ras mutant substituted the asparagine-116 with tyrosine, has dominant negative activity toward normal ras p21, and suppresses ras dependent transformed phenotypes. To investigate the effects of N116Y on ras-mediated signals for transformation, I constructed an inducible vector by recombination of the N116Y mutant to the downstream of human metallothionein promoter, and transfected it into an NIH3T3 cell line transformed by LTR linked normal c-H-ras, 18A. I isolated two 18A cell clones T1 and T6. Both of the cell lines were able to induce the N116Y mutant after the heavy metal treatment. These clones displayed flat reversion within 24 hours. In addition, these clones also inhibited colony formation in soft agar by epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or serum stimulation. The N116Y mutant blocked GDP/GTP exchange reaction by each growth stimulation. On the other hand, this mutant could not have sufficient influence upon extracellular signal-regulated kinase 2 (ERK2) phosphorylation, which located downstream of ras-mediated signal transduction, provoked by PDGF and serum stimulation. These results suggest that ERK2 activation is not necessary and sufficient for ras-dependent transformation. There could be a divergency in signal transduction between cell growth and transformation. The signal suppressed by the N116Y mutant may play an important role in cellular transformation.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Gene Expression Regulation, Neoplastic; Genes, ras; GTP Phosphohydrolases; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Mice; Mutation; Signal Transduction

The lbc oncogene is tumorigenic in nude mice, transforms NIH 3T3 fibroblasts, and encodes a Dbl homology domain found in several transforming gene products including the dbl oncogene product. While both lbc- and dbl-transformed NIH 3T3 foci exhibited a comparable gross appearance, lbc-transformed cell morphology was clearly distinct from that of dbl-transformed cells. Given these differences, we investigated the biochemical activity and target specificity of the Lbc oncoprotein both in vivo and in vitro. Here we show that Lbc associates specifically with the GTP-binding protein Rho in vivo, but not with the Ras, Rac, or Cdc42Hs GTP-binding proteins, and that recombinant, affinity-purified Lbc specifically catalyzes the guanine-nucleotide exchange activity of Rho in vitro. Consistent with an in vivo role for Lbc in Rho regulation, we further demonstrate that micro-injected onco-lbc potently induces actin stress fiber formation in quiescent Swiss 3T3 fibroblasts indistinguishable from that induced by Rho. Finally, lbc-induced NIH 3T3 focus formation is inhibited by co-transfection with a rho dominant-negative mutant. These results strongly indicate that the lbc oncogene encodes a specific guanine nucleotide exchange factor for Rho and causes cellular transformation through activation of the Rho signaling pathway.

Topics: 3T3 Cells; A Kinase Anchor Proteins; Actins; Adaptor Proteins, Signal Transducing; Animals; Cell Transformation, Neoplastic; GTP-Binding Proteins; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Membrane Proteins; Mice; Minor Histocompatibility Antigens; Proteins; Proto-Oncogene Proteins; Proto-Oncogenes; ras Guanine Nucleotide Exchange Factors; rhoB GTP-Binding Protein; Transfection

Rho-like GTPases have been implicated in the regulation of the actin cytoskeleton which controls the morphology, adhesion and motility of cells. Like Ras proteins, they become activated when bound GDP is exchanged for GTP, a process catalysed by GDP-dissociation stimulator (GDS) proteins. Several GDS proteins specific for Rho-like GTPases have been identified. Most of these contain a conserved catalytic domain, the DBL-homology (DH) domain, and activate Cdc42 or Rho but not Rac. We have isolated the invasion-inducing Tiam1 gene, which also encodes a protein with a DH domain. Here we show that Tiam1 is a GDS protein for Rho-like GTPases in vitro. In fibroblasts, Tiam1 induces a similar phenotype as constitutively activated (V12)Rac1, including membrane ruffling, and this is inhibited by dominant negative (N17)Rac1. Moreover, T-lymphoma cells expressing V12Rac1 become invasive, indicating that the Tiam1-Rac signalling pathway could be operating in the invasion and metastasis of tumour cells.

Topics: 3T3 Cells; Animals; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Fibroblasts; Glutathione Transferase; GTP Phosphohydrolases; GTP-Binding Proteins; Guanine Nucleotide Exchange Factors; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Proteins; rac GTP-Binding Proteins; rap GTP-Binding Proteins; Recombinant Fusion Proteins; rhoA GTP-Binding Protein; T-Lymphoma Invasion and Metastasis-inducing Protein 1; Transfection; Tumor Cells, Cultured

Although Ras residue phenylalanine-156 (F156) is strictly conserved in all members of the Ras superfamily of proteins, it is located outside of the consensus GDP/GTP-binding pocket. Its location within the hydrophobic core of Ras suggests that its strict conservation reflects a crucial role in structural stability. However, mutation of the equivalent residue (F157L) in the Drosophila Ras-related protein Rap results in a gain-of-function phenotype, suggesting an alternative role for this residue. Therefore, we have introduced an F156L mutation into Ras to evaluate the role of this residue in Ras structure and function. Whereas introduction of this mutation activated the transforming potential of wild-type Ras, it did not impair that of oncogenic Ras. Further, Ras (156L) exhibited an extremely rapid off rate for bound GDP/GTP in vitro and showed increased levels of Ras.GTP in vivo. To determine the structural basis for these altered properties, we used high-resolution nuclear magnetic resonance spectroscopy. The F156L mutation caused loss of contact with residues 6, 23, 55, and 79, resulting in disruption of secondary structure in alpha-helix 1 and in beta-sheets 1-5. These major structural changes contrast with the isolated alterations induced by oncogenic mutation (residues 12 or 61) that perturb GTPase activity, and instead, weaken Ras contacts with Mg2+ and its guanine nucleotide substrate and result in increased rates of GDP/GTP dissociation. Altogether, these observations demonstrate the essential role of this conserved residue in Ras structure and its function as a regulated GDP/GTP switch.

Topics: 3T3 Cells; Animals; Cell Transformation, Neoplastic; Genes, ras; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Mice; Models, Molecular; Mutagenesis, Site-Directed; Phenylalanine; Protein Structure, Secondary; Protein Structure, Tertiary; Proto-Oncogene Proteins p21(ras); Structure-Activity Relationship

The ras-oncogene-encoded p21 protein becomes oncogenic if amino acid substitutions occur at critical positions in the polypeptide chain. The most commonly found oncogenic forms contain Val in place of Gly 12 or Leu in place of Gln 61. To determine the effects of these substitutions on the three-dimensional structure of the whole p21 protein, we have performed molecular dynamics calculations on each of these three proteins bound to GDP and magnesium ion to compute the average structures of each of the three forms. Comparisons of the computed average structures shows that both oncogenic forms with Val 12 and Leu 61 differ substantially in structure from that of the wild type (containing Gly 12 and Gln 61) in discrete regions: residues 10-16, 32-47, 55-74, 85-89, 100-110, and 119-134. All of these regions occur in exposed loops, and several of them have already been found to be involved in the cellular functioning of the p21 protein. These regions have also previously been identified as the most flexible domains of the wild-type protein and have been bound to be the same ones that differ in conformation between transforming and nontransforming p21 mutant proteins neither of which binds nucleotide. The two oncogenic forms have similar conformations in their carboxyl-terminal domains, but differ in conformation at residues 32-47 and 55-74. The former region is known to be involved in the interaction with at least three downstream effector target proteins. Thus, differences in structure between the two oncogenic proteins may reflect different relative affinities of each oncogenic protein for each of these effector targets. The latter region, 55-74, is known to be a highly mobile segment of the protein. The results strongly suggest that critical oncogenic amino acid substitutions in the p21 protein cause changes in the structures of vital domains of this protein.

Topics: Cell Transformation, Neoplastic; Genes, ras; Guanosine Diphosphate; Oncogene Proteins; Protein Conformation; Proto-Oncogene Proteins p21(ras); Thermodynamics

Vav is a proto-oncogene specifically expressed in cells of hematopoietic origin. Its gene product contains a series of structural motifs, including SH2 and SH3 domains, suggestive of a role in signal transduction. The Vav protein also possesses a Dbl-homology (DH) domain previously found in regulators of the Ras superfamily of small GTP-binding proteins. Recently, Vav has been reported to be the major Ras GDP/GTP exchange factor (GEF) in hematopoietic cells [Gulbins et al., Science 260, 822 (1993); J. Immunol. 152, 2123 (1994)]. The following observations are inconsistent with such a role: (i) Vav proteins do not exhibit Ras GEF activity in standard GDP/GTP exchange assays; (ii) Cells overexpressing Vav do not have increased levels of GTP-bound Ras proteins; (iii) Overexpression of Vav does not overcome the growth inhibitory activity of RasN17, a mutant that blocks Ras signaling by inhibiting Ras GEFs; (iv) Transformation of NIH3T3 cells by Vav oncoproteins is not inhibited by a farnesyl transferase inhibitor that completely blocks transformation by both Ras and its well characterized GEF, RasCDC25 and (v) The morphology of Vav-transformed NIH3T3 cells is dramatically different from that induced by Ras and RasCDC25. Whereas these observations make it unlikely that Vav functions either as a RasGEF or as an upstream regulatory element of Ras, we have observed that Vav can cooperate with normal Ras proteins to transform NIH3T3 cells. These results suggest that Vav and Ras may mediate signal transduction by distinct, but interactive mitogenic pathways.

Topics: 3T3 Cells; Alkyl and Aryl Transferases; Animals; Cell Cycle Proteins; Cell Transformation, Neoplastic; Farnesyltranstransferase; Fibroblasts; Genes, ras; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; Mice; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-vav; Proto-Oncogene Proteins p21(ras); ras Guanine Nucleotide Exchange Factors; Signal Transduction; Transferases

The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth-factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13-259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G-protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces proliferative signals, in part, through downstream activation of p21 RAS. Furthermore, p21 RAS activity is linked to pathways that regulate c-jun and c-fos expression.

Topics: Animals; Benzoquinones; Blotting, Northern; Bone Marrow; Cell Line; Cell Transformation, Neoplastic; DNA Probes; Fusion Proteins, bcr-abl; Gene Expression; Genes, ras; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Lactams, Macrocyclic; Mice; Oncogenes; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Quinones; Rifabutin; RNA; Signal Transduction; Transcription, Genetic

We report biochemical evidence that epidermal growth factor and platelet-derived growth factor stimulate the Ras guanine nucleotide exchange factor activity in quiescent NIH 3T3 cells. Moreover, the exchange activity is constitutively enhanced in NIH 3T3 cells transformed by Src and ErbB2 oncogenic tyrosine protein kinases (TPKs), whereas transformation by oncogenic Mos and Raf does not alter the activity. GTPase-activating protein activity was not affected under these conditions. Overexpression of pp60c-Src mutants containing activated and suppressor TPK mutations resulted in stimulation and inhibition of the exchange factor activity, respectively. A TPK inhibitor, genistein, prevented the activation of the exchange factor in epidermal growth factor/platelet-derived growth factor-treated cells and src-transformed cells. Furthermore, the exchange factor activity bound to an anti-phosphotyrosine antibody immunoaffinity column. These findings suggest that the guanine nucleotide exchange factor, but not GTPase-activating protein, plays a major role in the Ras activation in cell proliferation initiated by growth factor receptor TPKs and malignant transformation by oncogenic TPKs and that tyrosine phosphorylation of either the exchange factor or a tightly bound protein(s) may mediate the activation of the exchange factor by these TPKs.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Cell Line, Transformed; Cell Transformation, Neoplastic; Chromatography, Affinity; Chromatography, Ion Exchange; Epidermal Growth Factor; ErbB Receptors; Genes, src; Genistein; GTPase-Activating Proteins; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Isoflavones; Mice; Mutagenesis, Site-Directed; Phosphorylation; Platelet-Derived Growth Factor; Point Mutation; Protein-Tyrosine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogenes; ras GTPase-Activating Proteins; ras Guanine Nucleotide Exchange Factors; Receptor, ErbB-2; Receptors, Platelet-Derived Growth Factor

The best characterized yeast guanine nucleotide releasing factor is CDC25, which acts on RAS and thereby stimulates cAMP production in Saccharomyces cerevisiae. In order to determine if CDC25 could be a specific GDP-GTP releasing factor for the mammalian proteins Ha-ras, Ki-ras, and N-ras, its functions were studied both in vitro and in NIH3T3 cells. The 561 amino acid composing the C-terminal domain of CDC25 (CDC25 C-domain) released guanine nucleotides (both GDP and GTP) from Ha-, Ki-, and N-ras but not from Rap1A, Rab5, and Rab11. CDC25 acted on oncogenically activated Ha-ras even if the last 23 amino acids (167-189) of the Ras proteins were not present. CDC25 transformed NIH3T3 cells; its transforming capacity was enhanced by overexpression of wild-type Ha-ras. CDC25 C-domain probably exerts its effects through the activation of cellular Ras proteins. These data suggest that the CDC25 C-domain can function as an upstream activator of Ras proteins in a heterologous system and therefore could be a useful tool to study the regulation of Ras activation by growth factor receptors.

Topics: 3T3 Cells; Animals; Cell Cycle Proteins; Cell Transformation, Neoplastic; Enhancer Elements, Genetic; Escherichia coli; Fungal Proteins; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Mice; Mice, Nude; Neoplasm Transplantation; Plasmids; Polyomavirus; Proto-Oncogene Proteins p21(ras); ras-GRF1; Recombinant Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transfection

The mechanisms of ras activation by mutations in residue 61 and in the NKXD guanine nucleotide-binding consensus sequence (ras residues 116-119) have been evaluated. Weakly transforming mutations that either reduce intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activities (61P) or enhance guanine nucleotide exchange rates (116H, 119E) were combined into the same H-ras proteins. The resulting double-mutant proteins exhibited significantly stronger transforming forming activities than are observed with each individual mutation, suggesting that the consequences of these two different mechanisms of activation favor maintenance of ras in the active form, which is GTP bound. In vivo nucleotide association analysis demonstrated a direct relationship between ras-GTP formation and transforming activity. Although both 61P and 61L mutations result in reduced intrinsic GTPase activity and loss of GAP stimulation in vitro, only H-ras(61L) exhibits strong transforming activity. While H-ras(61L) is found predominantly in the GTP-bound form, H-ras(61P) is predominantly complexed with GDP in vivo. Thus, in vitro GAP stimulation of GTPase activity does not directly correlate with transforming potential, suggesting that other ras-specific regulatory components may also be important in regulating the cycling of ras between CDP- and GTP-bound states.

Topics: 3T3 Cells; Animals; Cell Transformation, Neoplastic; DNA Mutational Analysis; Genes, ras; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Mice; Mutagenesis, Site-Directed; Proto-Oncogene Proteins p21(ras); Structure-Activity Relationship

The double mutation, D33H/P34S, reduced the transforming activity of oncogenic RasH proteins, G12V and Q61L, 400- and 20-fold, respectively. Remarkably, this same mutation did not reduce the transforming activity of normal RasH, nor did it impair the ability of the protein to restore a functional Ras pathway in cells whose endogenous Ras proteins were inhibited. Another mutation in this region, D38N, had similar effects. The mutations reduced downstream coupling efficiency of normal Ras as assessed by yeast adenylyl cyclase stimulation. However, this was offset by decreased GTPase activating protein (GAP) binding, since the latter resulted in elevated GTP-bound mutant Ras in cells. The mutations produced a similar decrease in downstream coupling efficiency of oncogenic Ras, but decreased GAP binding did not compensate because the GTPase activity of oncogenic Ras is not stimulated by GAP. These results imply that preferential inactivation of oncogenic Ras in human tumors may be achieved by reagents designed to inhibit the GAP-binding/"effector" domain of Ras proteins.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; GTP-Binding Proteins; GTPase-Activating Proteins; Guanosine Diphosphate; Guanosine Triphosphate; In Vitro Techniques; Mice; Microinjections; Mutation; Proteins; Proto-Oncogene Proteins p21(ras); ras GTPase-Activating Proteins; Signal Transduction; Structure-Activity Relationship

The ras gene product (p21) is a GTP-binding protein and has been thought to transduce signals regulating proliferation or differentiation of cells. Like other GTP-binding proteins, p21.GTP is an active conformation, which can transduce the signals downstream, whereas p21.GDP is an inactive one. Recently, we have shown that p21.GTP levels increased in cells treated with fetal bovine serum or platelet-derived growth factor to initiate DNA synthesis. In this paper, we report that epidermal growth factor can also increase the amounts of p21.GTP in the cells. Effects of epidermal growth factor and platelet-derived growth factor are not additive. In contrast, mutant [Val12]p21, which has transforming activity, responded neither to platelet-derived growth factor nor to epidermal growth factor. We also found that the ratio of p21.GTP to p21.GDP increased 3- to 4-fold in transformants carrying activated erbB-2/neu or v-src oncogenes. These results strongly suggest an important role of p21 in transduction of signals for both normal proliferation and malignant transformation through growth factor receptors with tyrosine kinase activity or related oncogene products.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; DNA Replication; Epidermal Growth Factor; Guanosine Diphosphate; Guanosine Triphosphate; Mice; Oncogene Proteins; Oncogenes; Platelet-Derived Growth Factor; Protein Binding; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras)

One- and two-dimensional nuclear magnetic resonance spectroscopy (1D and 2D NMR) and site-directed mutagenesis were used to study the influence of mutations on the conformation of the H-ras oncogene product p21. No severe structural differences between the different mutants, whether they were transforming or nontransforming, could be detected. Initially, selective incorporation of 3,5-deuterated tyrosyl residues into p21 and 2D NMR were used to identify the resonances representing the spin systems of the imidazole rings of the three histidyl residues in the protein, of six of the nine tyrosyl rings, and of four of the five phenylalanyl rings. The spin systems of the phenyl rings of Phe28, Phe78, and Phe82 could be assigned by using mutant proteins, since no severe structure-induced spectral changes in the aromatic part of the spectra of the mutant proteins were detected. Sequence-specific assignments of the histidine imidazole resonances could be obtained by comparison of the distance information obtained by nuclear Overhauser enhancement spectroscopy (NOESY) experiments with the crystal structure. The change in the chemical shift values of the Hl' proton and the alpha-phosphate of the bound GDP in the NMR spectra of the p21(F28L) mutant and the 28-fold increase in the GDP dissociation rate constants of this mutant suggest a strong interaction between Phe28 and the p21-bound nucleotide. In solution, the p21-bound GDP.Mg2+ has an anti conformation, and the phenyl ring of Phe28 is close to the ribose of the bound GDP.Mg2+.

Topics: Base Sequence; Cell Transformation, Neoplastic; DNA; Guanosine Diphosphate; Guanosine Triphosphate; Magnesium; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Mutation; Oncogene Protein p21(ras); Phenylalanine; Protein Conformation; Tyrosine

The 21-kD proteins encoded by ras oncogenes (p21Ras) are modified covalently by a palmitate attached to a cysteine residue near the carboxyl terminus. Changing cysteine at position 186 to serine in oncogenic forms produces a nonpalmitylated protein that fails to associate with membranes and does not transform NIH 3T3 cells. Nonpalmitylated p21Ras derivatives were constructed that contained myristic acid at their amino termini to determine if a different form of lipid modification could restore either membrane association or transforming activity. An activated p21Ras, altered in this way, exhibited both efficient membrane association and full transforming activity. Surprisingly, myristylated forms of normal cellular Ras were also transforming. This demonstrates that Ras must bind to membranes in order to transmit a signal for transformation, but that either myristate or palmitate can perform this role. However, the normal function of cellular Ras is diverted to transformation by myristate and therefore must be regulated ordinarily by some unique property of palmitate that myristate does not mimic. Myristylation thus represents a novel mechanism by which Ras can become transforming.

Topics: Animals; Cell Membrane; Cell Transformation, Neoplastic; Gene Products, gag; Guanosine Diphosphate; Guanosine Triphosphate; Humans; In Vitro Techniques; Mice; Myristic Acid; Myristic Acids; Protein Processing, Post-Translational; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Retroviridae Proteins

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)

Topics: Cell Transformation, Neoplastic; Circular Dichroism; Escherichia coli; Genes, ras; Guanosine Diphosphate; Humans; Peptide Elongation Factor Tu; Protein Binding; Protein Conformation; Protein Denaturation; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Thermodynamics

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.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Escherichia coli; GTP Phosphohydrolases; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Mice; Mutation; Oncogenes; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras)

Deletions of small sequences from the viral Harvey ras gene have been generated, and resulting ras p21 mutants have been expressed in Escherichia coli. Purification of each deleted protein allowed the in vitro characterization of GTP-binding, GTPase and autokinase activity of the proteins. Microinjection of the highly purified proteins into quiescent NIH/3T3 cells, as well as transfection experiments utilizing a long terminal repeat (LTR)-containing vector, were utilized to analyze the biological activity of the deleted proteins. Two small regions located at 6-23 and 152-165 residues are shown to be absolutely required for in vitro and in vivo activities of the ras product. By contrast, the variable region comprising amino acids 165-184 was shown not to be necessary for either in vitro or in vivo activities. Thus, we demonstrate that: (i) amino acid sequences at positions 5-23 and 152-165 of ras p21 protein are probably directly involved in the GTP-binding activity; (ii) GTP-binding is required for the transforming activity of ras p21 and by extension for the normal function of the proto-oncogene product; and (iii) the variable region at the C-terminal end of the ras p21 molecule from amino acids 165 to 184 is not required for transformation.

Topics: Animals; Cell Transformation, Neoplastic; Chromosome Deletion; DNA Restriction Enzymes; Escherichia coli; Genes, Viral; GTP Phosphohydrolases; Guanosine Diphosphate; Guanosine Triphosphate; Harvey murine sarcoma virus; Kinetics; Mice; Mutation; Neoplasm Proteins; Oncogene Protein p21(ras); Oncogenes; Phosphorylation; Plasmids; Protein Binding; Proto-Oncogenes; Sarcoma Viruses, Murine

An Ala-to-Thr substitution at position 59 activates the transforming properties of the p21ras protein without impairment of GTPase activity, a biochemical alteration associated with other activating mutations. To investigate the basis for the transforming properties of the Thr-59 mutant, we characterized guanine nucleotide release. This reaction exhibited a slow rate and stringent temperature requirements. To further dissect the release reaction, we used monoclonal antibodies directed against different epitopes of the p21 molecule. One monoclonal specifically interfered with nucleotide release, while others which recognized different regions of the molecule blocked nucleotide binding. Mutants with the Thr-59 substitution exhibited a three- to ninefold-higher rate of GDP and GTP release than normal p21 or mutants with other activating lesions. This alteration in the Thr-59 mutant would have the effect of increasing its rate of nucleotide exchange. In an intracellular environment with a high GTP/GDP ratio, this would favor the association of GTP with the Thr-59 mutant. Consistent with knowledge of known G-regulatory proteins, these findings support a model in which the p21-GTP complex is the biologically active form of the p21 protein.

Topics: Cell Transformation, Neoplastic; Escherichia coli; Genes, Viral; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Plasmids; Proto-Oncogenes; Retroviridae; Thionucleotides

A systematic study has been performed using a series of differentiated rat thyroid epithelial cell lines either uninfected or infected with Kirsten murine sarcoma virus (KiMSV), to determine the levels of the p21 product of the v-ras-Ki oncogene in transformed and normal cell lines. The p21 levels have been assayed by SDS-polyacrylamide gel electrophoresis of immunoprecipitates of 35S-labelled cell extracts and by a GDP binding assay. All cell lines analysed showed a significant increase in the levels of p21 after transformation with KiMSV compared to the p21 levels of uninfected and untransformed differentiated thyroid cells. The results reported here confirm the potential ability of the v-ras-Ki oncogene product to transform epithelial cells. They show, furthermore, that not only is p21 present in some epithelial cells transformed by KiMSV, but also that it is functionally active, as has been shown for fibroblasts transformed by the same virus, and that its functioning is maintained after passaging in vivo of the transformed cells.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Epithelium; Genes, Viral; Guanosine Diphosphate; Kirsten murine sarcoma virus; Molecular Weight; Nucleic Acid Hybridization; Oncogenes; Rats; RNA, Viral; Sarcoma Viruses, Murine; Thyroid Gland; Viral Proteins

In the presence of ADP and GDP the tyrosine-phosphorylating activities of the viral as well as the cellular pp60src show a similar concentration-dependent inhibition in vitro. Addition of diadenosine 5',5"'-p1p4 tetraphosphate (Ap4A) to the kinase assay leads to an inhibition of the viral kinase activity, whereas the cellular kinase is not influenced.

Topics: Adenine Nucleotides; Adenosine Diphosphate; Animals; Avian Sarcoma Viruses; Cell Transformation, Neoplastic; Cell Transformation, Viral; Chick Embryo; Dinucleoside Phosphates; Guanosine Diphosphate; Mice; Nucleotides; Oncogene Protein pp60(v-src); Phosphorylation; Protein Kinase Inhibitors; Viral Proteins

We have isolated eight rat lymphocyte-myeloma hybrid cell lines producing monoclonal antibodies that react with the 21,000-dalton transforming protein (p21) encoded by the v-ras gene of Harvey murine sarcoma virus (Ha-MuSV). These antibodies specifically immunoprecipitate both phosphorylated and non-phosphorylated forms of p21 from lysates of cells transformed by Ha-MuSV. All eight react with the products of closely related ras genes expressed in cells transformed by two additional sarcoma viruses (rat sarcoma virus and BALB sarcoma virus) or by a cellular Harvey-ras gene placed under the control of a viral promoter. Three of the antibodies also react strongly with the p21 encoded by the v-ras gene of Kirsten MuSV. These same three antibodies immunoprecipitate the predominant p21 species synthesized normally in a variety of rodent cell lines, including the p21 produced at high levels in 416B murine hemopoietic cells. This suggests that an endogenous gene closely related to Kirsten-ras is expressed in these cells. The monoclonal antibodies have been used to confirm two properties associated with p21; localization at the inner surface of the membrane of Ha-MuSV-transformed cells, assayed by immunofluorescence microscopy, and binding of guanine nucleotides.

Topics: Animals; Antibodies, Monoclonal; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cell Transformation, Viral; Genes, Viral; Guanosine Diphosphate; Mice; Oncogenic Viruses; Rats; Sarcoma Viruses, Murine; Viral Proteins

In earlier studies, we molecularly cloned a normal cellular gene, c-rasH-1, homologous to the v-ras oncogene of Harvey murine sarcoma virus (v-rasH). By ligating a type c retroviral promotor to c-rasH-1, we could transform NIH 3T3 cells with the c-rasH-1 gene. The transformed cells contained high levels of a p21 protein coded for by the c-rasH-1 gene. In the current studies, we have purified extensively both v-rasH p21 and c-rasH p21 and compared the in vivo and in vitro biochemical properties of both these p21 molecules. The p21 proteins coded for by v-rasH and c-rasH-1 shared certain properties: each protein was synthesized as a precursor protein which subsequently became bound to the inner surface of the plasma membrane; each protein was associated with guanine nucleotide-binding activity, a property which copurified with p21 molecules on a high-pressure liquid chromatography molecular sizing column. In some other properties, the v-rasH and c-rasH p21 proteins differed. In vivo, approximately 20 to 30% of v-rasH p21 molecules were in the form of phosphothreonine-containing pp21 molecules, whereas in vivo only a minute fraction of c-rasH-1 p21 contained phosphate, and this phosphate was found on a serine residue. v-rasH pp21 molecules with an authentic phosphothreonine peptide could be synthesized in vitro in an autophosphorylation reaction in which the gamma phosphate of GTP was transferred to v-rasH p21. No autophosphorylating activity was associated with purified c-rasH-1 p21 in vitro. The results indicate a major qualitative difference between the p21 proteins coded for by v-rasH and c-rasH-1. The p21 coded for by a mouse-derived oncogenic virus, BALB murine sarcoma virus, resembled the p21 coded for by c-rasH-1 in that it bound guanine nucleotides but did not label appreciably with 32Pi. The forms of p21 coded for by other members of the ras gene family were compared, and the results indicate that the guanine nucleotide-binding activity is common to p21 molecules coded for by all known members of the ras gene family.

Topics: Animals; Blood Proteins; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Genes, Viral; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Mice; Oncogenes; Phosphoserine; Phosphothreonine; Receptors, Cell Surface; Sarcoma Viruses, Murine; Viral Proteins

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Genes, Viral; Guanine Nucleotides; Guanosine Diphosphate; Kirsten murine sarcoma virus; Mice; Rats; Sarcoma Viruses, Murine; Viral Proteins