guanosine-triphosphate and Cell-Transformation--Neoplastic

The RAS genes encode for members of a large superfamily of guanosine-5'-triphosphate (GTP)-binding proteins that control diverse intracellular signaling pathways to promote cell proliferation. Somatic mutations in the RAS oncogenes are the most common activating lesions found in human cancers. These mutations invariably result in the gain-of-function of RAS by impairing GTP hydrolysis and are frequently associated with poor responses to standard cancer therapies. In this review, we summarize key findings of past and present landmark studies that have deepened our understanding of the RAS biology in the context of oncogenesis. We also discuss how emerging areas of research could further bolster a renewed global effort to target the largely undruggable oncogenic RAS and/or its activated downstream effector signaling cascades to achieve better treatment outcomes for RAS-mutant cancer patients.

Topics: Animals; Autophagy; Cell Transformation, Neoplastic; Guanosine Triphosphate; Humans; Mutation; Neoplasms; ras Proteins; Signal Transduction

Physical processes in living cells were not taken into consideration among the essentials of biological activity, regardless of the fact that they establish a state far from thermodynamic equilibrium. In biological system chemical energy is transformed into the work of physical forces for various biological functions. The energy transformation pathway is very likely connected with generation of the endogenous electrodynamic field as suggested by experimentally proved electrodynamic activity of biological systems connected with mitochondrial and microtubule functions. Besides production of ATP and GTP (adenosine and guanosine triphosphate) mitochondria form a proton space charge layer, strong static electric field, and water ordering around them in cytosol - that are necessary conditions for generation of coherent electrodynamic field by microtubules. Electrodynamic forces are of a long-range nature in comparison with bond and cohesive forces. Mitochondrial dysfunction leads to disturbances of the electromagnetic field; its power and coherence may be diminished, and frequency spectrum altered. Consequently, defective electrodynamic interaction forces between cancer and healthy cells may result in local invasion of cancer cells. Further deformation of interaction forces connected with experimentally disclosed spatial disarrangement of the cytoskeleton and disordered electrodynamic field condition metastatic process. Cancer therapeutic strategy targeting mitochondria may restore normal physiological functions of mitochondria and open the apoptotic pathway. Apoptosis of too much damaged cancer cells was observed. Considerable experience with DCA (dichloroacetate) cancer treatment in humans was accumulated. Clinical trials should assess DCA therapeutic potential and collect data for development of novel more effective drugs for mitochondrial restoration of various cancers.

Topics: Adenosine Triphosphate; Cell Transformation, Neoplastic; Dichloroacetic Acid; Electrophysiological Phenomena; Energy Metabolism; Guanosine Triphosphate; Humans; Microtubules; Mitochondria; Neoplasms

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder whose major feature is the occurrence of multiple neurofibromas, which are benign tumors of the nerve sheath. It affects an estimated one in 3000 to 4000 individuals. In addition to neurofibromas, there are many other clinical manifestations, including malignant tumors such as gliomas or malignant peripheral nerve sheath tumors, and nontumor effects such as skeletal dysplasia and learning disability. Diagnosis is established on the basis of clinical criteria. Molecular genetic testing is feasible, but the large size of the gene and wide range of pathogenic mutations have so far impeded the development of a clinical diagnostic test. Insights into pathogenesis have followed from identification of the NF1 gene and the development of animal models. The major function of the gene product appears to be regulation of the ras protein. Tumors are believed to arise by the loss of function of the NF1 protein, suggesting that NF1 behaves as a tumor suppressor gene. Heterozygous effects on some cell types are also likely, however. The role of ras in the pathogenesis of tumors in NF1 has suggested an approach to treatment using ras inhibitors, some of which are likely to begin in clinical trials in NF1 patients in the near future.

Topics: Animals; Brain Neoplasms; Cafe-au-Lait Spots; Cell Transformation, Neoplastic; Female; Genes, Dominant; Genes, Neurofibromatosis 1; Glioma; Guanosine Triphosphate; Humans; Hypertension; Learning Disabilities; Leukemia; Male; Mice; Mice, Knockout; Neurofibroma; Neurofibromatosis 1; Neurofibromin 1; Protein Structure, Tertiary; ras Proteins; Rhabdomyosarcoma; Scoliosis

As a result of its transforming abilities, activated Ras is expressed in a great number of cancers. The ras mutation frequency varies between 95% in pancreatic cancer and 5% in breast cancer. In leukemia, the highest frequency (30%) is found in acute myeloid leukemia. The presence of ras mutations has been correlated with a poor prognosis and negative clinical outcome. This suggests that mutated Ras activates mechanisms, which favor tumor growth, enhance the metastatic capacity of tumors or modulate tumor-specific immune responses. Several new functions of Ras, such as downregulation of major histocompatibility complex molecules, upregulation of certain cytokines, growth factors and degradative enzymes have been uncovered in the last decade. Additionally, mutated Ras can also serve as a primary target for the development of immunotherapy or drug therapy. This review will discuss the mechanisms by which Ras expressing tumors are able to evade destruction by the immune system and enhance their growth and metastatic potential. It will further elaborate on the attempts to develop successful immunotherapy and drug therapy targeting Ras expressing tumors.

Topics: Alkyl and Aryl Transferases; Animals; Antigen Presentation; Antineoplastic Agents; Cell Adhesion Molecules; Cell Transformation, Neoplastic; Cytokines; Drug Design; Endopeptidases; Enzyme Activation; Farnesyltranstransferase; Fungal Proteins; Fusion Proteins, bcr-abl; Genes, ras; Growth Substances; Guanosine Triphosphate; Humans; Immune System; Immunotherapy; Leukemia; Mice; Models, Biological; Mutation; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Neurofibromin 1; Oligonucleotides, Antisense; Proteins; Proto-Oncogene Proteins p21(ras); Reoviridae Infections; Repressor Proteins; Signal Transduction; SOS1 Protein; T-Lymphocyte Subsets

The search for proteins which interact with the active GTP-bound form of Ras in order to transmit signals for proliferation, differentiation and oncogenesis has been a long one. Now there are several strong candidates for Ras effectors that include protein kinases, lipid kinases and guanine nucleotide exchange factors. Structural information on how one Ras-binding domain in an effector interacts with Ras.GTP has recently been obtained. Recent data show that transformation by Ras oncoproteins requires the activation of several signal transduction pathways, including those which transmit signals via members of the Rho family of GTPases.

Topics: Animals; Binding Sites; Cell Transformation, Neoplastic; Guanosine Triphosphate; Humans; Oncogene Protein p21(ras); Signal Transduction

The notion that ras proteins are required for the stimulation of mitogenesis by different receptor tyrosine kinases (RTKs) has spurred researchers to investigate the precise role of p21ras in signal transduction. A large number of stimuli can drive p21ras in the active conformation, and several proteins that play an important role in regulating the GTP/GDP balance on p21ras have been identified. Indeed, activation of p21ras has been demonstrated to occur by stimulation of guanine nucleotide-releasing proteins (GNRPs) or inhibition of GTPase-activating proteins (GAPs). Moreover, a number of SH2-containing proteins have been implicated in this signaling pathway, such as shc and sem-5/grb2. On the other hand, downstream signaling from p21ras involves an important protein kinase cascade. This pathway seems to be conserved in evolution, and analogous routes have been described in organisms such as yeast, nematodes, and fruit flies. Nevertheless, the direct effector molecule of p21ras that could couple to this kinase cascade is still unknown. Some indications have been obtained that suggest that this function might be partially performed by p120GAP. This review gives an overview of the role of p21ras in signaling from diverse RTKs. Elucidation of this pathway will improve our understanding of mitogenic signaling pathways and the basis of cancer.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Drosophila melanogaster; Genes, ras; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Mice; Phospholipases; Protein Kinases; Proto-Oncogene Proteins p21(ras); Rabbits; Receptor Protein-Tyrosine Kinases; Signal Transduction; Transcription Factors

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

It is now established that ras oncogenes can induce metastatic characteristics in primary diploid fibroblasts, nonsenescing fibroblasts and nonmetastasizing tumors. The issue of whether ras is directly involved in maintaining the metastatic phenotype through the expression and action of its gene product has been examined by analyzing the relationship to ras expression and to the production of the p21 ras-GTP complex, which is thought to mediate ras-transforming activity. While these expression and mutation studies support the idea that p21 ras directly regulates metastasis formation, it is also evident that there are many examples of human and murine cancers which show no differences in ras expression in primary and metastatic tumor cells. This may be partially explained by the ability of protein kinase-encoding oncogenes to also induce metastatic potential. In addition, the ability of ras to induce metastasis may be dependent on the regulation of its activity by other genes. Furthermore, transformation does not occur as an isolated genetic event, but is rather the result of interaction of two or more oncogenes. We suggest that the nature of these gene interactions will ultimately determine whether a cell is a benign transformant or a malignant and metastatic cancer.

Topics: Animals; Cell Transformation, Neoplastic; Gene Expression; Genes, ras; Guanosine Triphosphate; Humans; Neoplasm Metastasis; Oncogene Protein p21(ras); Oncogenes

Oncogenes were first identified in neoplastic tissues and were thought to be disseminated by retroviruses. They were shown to cause neoplastic transformation of cells in vitro and were therefore regarded as "the genes that cause cancer." They occur in all eukaryotes including humans. More than 30 oncogenes have been identified, and 28 have already been mapped to the human karyotype. When the protein products of the oncogenes were investigated, they were found to be very similar to known substances that are normally involved in the control of cell division--growth factors, plasma-membrane receptors, modulators of the transduction of exogenous signals through the plasma membrane, and nuclear DNA-binding substances. These discoveries changed the original concept of oncogenes. They are now regarded as playing vital roles in the normal control of mitosis, and they should properly be called mitogenes rather than "oncogenes." Like all other genes, if a mitogene is mutated, rearranged, translocated, or otherwise deranged, its effects will change; it may then become an oncogene that will promote disorderly cell division and thus contribute to the pathogenesis of cancer. In addition to providing new insights into the normal control of mitosis and its neoplastic transformation, the discovery of the mitogenes has suggested new approaches to the early diagnosis of cancer, to a more rational classification of neoplasms based on pathogenesis rather than morphology, and, perhaps, to more rational forms of therapy.

Topics: Animals; Avian Sarcoma Viruses; Base Sequence; Cell Transformation, Neoplastic; Chromosomes, Human; DNA, Neoplasm; Gene Amplification; Guanosine Triphosphate; Humans; Neoplasms, Experimental; Oncogenes; Oncogenic Viruses; Platelet-Derived Growth Factor; Protein Biosynthesis; Protein Kinases; Transcription, Genetic

Topics: Adenosine Triphosphate; Animals; Biological Transport; Blood Cells; Cell Transformation, Neoplastic; Cyclic AMP; Cyclic GMP; Female; Guanosine Triphosphate; Humans; Kidney; Liver; Male; Neoplasms; Neoplasms, Experimental; Nucleotides, Cyclic; Rats; Receptors, Cell Surface

As key regulators of the actin cytoskeleton, RHO GTPase expression and/or activity are deregulated in tumorigenesis and metastatic progression. Nevertheless, the vast majority of experiments supporting this conclusion was conducted on cell lines but not on human tumor samples that were mostly studied at the expression level only. Up to now, the activity of RHO proteins remains poorly investigated in human tumors. In this article, we present the development of a robust nanobody-based ELISA assay, with a high selectivity that allows an accurate quantification of RHO protein GTP-bound state in the nanomolar range (1 nM; 20 μg/L), not only in cell lines after treatment but also in tumor samples. Of note, we present here a fine analysis of RHOA-like and RAC1 active state in tumor samples with the most comprehensive study of RHOA-GTP and RHOC-GTP levels performed on human breast tumor samples. We revealed increased GTP-bound RHOA and RHOC protein activities in tumors compared to normal tissue counterparts, and demonstrated that the RHO active state and RHO expression are two independent parameters among different breast cancer subtypes. Our results further highlight the regulation of RHO protein activation in tumor samples and the relevance of directly studying RHO GTPase activities involvement in molecular pathways.

Topics: Breast Neoplasms; Cell Transformation, Neoplastic; Female; Guanosine Triphosphate; Humans; rhoA GTP-Binding Protein; rhoC GTP-Binding Protein

RAS proteins function as molecular switches that transmit signals from cell surface receptors into specific cellular responses via activation of defined signaling pathways (Fang, 2015). Aberrant constitutive RAS activation occurs with high incidence in different types of cancer (Bos, 1989). Thus, inhibition of RAS-mediated signaling is extremely important for therapeutic approaches against cancer. Here we showed that the ribonuclease (RNase) binase, directly interacts with endogenous KRAS. Further, molecular structure models suggested an inhibitory nature of binase-RAS interaction involving regions of RAS that are important for different aspects of its function. Consistent with these models, phosphorylation analysis of effectors of RAS-mediated signaling revealed that binase inhibits the MAPK/ERK signaling pathway. Interestingly, RAS activation assays using a non-hydrolysable GTP analog (GTPγS) demonstrated that binase interferes with the exchange of GDP by GTP. Furthermore, we showed that binase reduced the interaction of RAS with the guanine nucleotide exchange factor (GEF), SOS1. Our data support a model in which binase-KRAS interaction interferes with the function of GEFs and stabilizes the inactive GDP-bound conformation of RAS thereby inhibiting MAPK/ERK signaling. This model plausibly explains the previously reported, antitumor-effect of binase specific towards RAS-transformed cells and suggests the development of anticancer therapies based on this ribonuclease.

Topics: Animals; Cell Line; Cell Movement; Cell Transformation, Neoplastic; Endoribonucleases; Enzyme Stability; Extracellular Signal-Regulated MAP Kinases; Guanosine Triphosphate; Hydrolysis; Mice; Models, Molecular; Phosphorylation; Protein Binding; Protein Interaction Domains and Motifs; Proto-Oncogene Proteins p21(ras); Signal Transduction; SOS1 Protein; Structure-Activity Relationship; Time Factors; Transfection

Although guanine nucleotides are essential for cell growth, how their levels are sensed in mammalian cells is unknown. Sumita et al. show that PIP4K2B, a phosphoinositide kinase, is a molecular sensor that transduces changes in GTP into changes in the levels of the phosphoinositide PtdIns5P to modulate tumour cell growth.

Topics: Adenosine Triphosphate; Animals; Binding Sites; Cell Line, Transformed; Cell Proliferation; Cell Transformation, Neoplastic; Fibroblasts; Gene Expression Regulation, Neoplastic; Guanosine Triphosphate; Humans; Isoenzymes; Kinetics; Mice; Mice, Nude; Mutation; Phosphatidylinositol Phosphates; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Signal Transduction

NRAS mutation at codons 12, 13, or 61 is associated with transformation; yet, in melanoma, such alterations are nearly exclusive to codon 61. Here, we compared the melanoma susceptibility of an NrasQ61R knock-in allele to similarly designed KrasG12D and NrasG12D alleles. With concomitant p16INK4a inactivation, KrasG12D or NrasQ61R expression efficiently promoted melanoma in vivo, whereas NrasG12D did not. In addition, NrasQ61R mutation potently cooperated with Lkb1/Stk11 loss to induce highly metastatic disease. Functional comparisons of NrasQ61R and NrasG12D revealed little difference in the ability of these proteins to engage PI3K or RAF. Instead, NrasQ61R showed enhanced nucleotide binding, decreased intrinsic GTPase activity, and increased stability when compared with NrasG12D. This work identifies a faithful model of human NRAS-mutant melanoma, and suggests that the increased melanomagenecity of NrasQ61R over NrasG12D is due to heightened abundance of the active, GTP-bound form rather than differences in the engagement of downstream effector pathways.. This work explains the curious predominance in human melanoma of mutations of codon 61 of NRAS over other oncogenic NRAS mutations. Using conditional "knock-in" mouse models, we show that physiologic expression of NRASQ61R, but not NRASG12D, drives melanoma formation.

Topics: Alleles; AMP-Activated Protein Kinase Kinases; Animals; Cell Line, Tumor; Cell Transformation, Neoplastic; Codon; Extracellular Signal-Regulated MAP Kinases; Gene Deletion; Gene Order; Genes, ras; Genetic Loci; Genotype; Guanosine Triphosphate; Humans; Melanoma; Mice; Mitogen-Activated Protein Kinases; Mutation; Neoplasm Metastasis; Oncogene Proteins, Fusion; Phosphatidylinositol 3-Kinases; Protein Binding; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins B-raf; Tumor Burden

The Hippo pathway, a cascade of protein kinases that inhibits the oncogenic transcriptional coactivators YAP and TAZ, was discovered in Drosophila as a major determinant of organ size in development. Known modes of regulation involve surface proteins that mediate cell-cell contact or determine epithelial cell polarity which, in a tissue-specific manner, use intracellular complexes containing FERM domain and actin-binding proteins to modulate the kinase activities or directly sequester YAP. Unexpectedly, recent work demonstrates that GPCRs, especially those signaling through Galpha12/13 such as the protease activated receptor PAR1, cause potent YAP dephosphorylation and activation. This response requires active RhoA GTPase and increased assembly of filamentous (F-)actin. Morever, cell architectures that promote F-actin assembly per se also activate YAP by kinase-dependent and independent mechanisms. These findings unveil the ability of GPCRs to activate the YAP oncogene through a newly recognized signaling function of the actin cytoskeleton, likely to be especially important for normal and cancerous stem cells.

Topics: Actin Cytoskeleton; Actins; Animals; Cell Polarity; Cell Transformation, Neoplastic; Drosophila melanogaster; Drosophila Proteins; Gene Expression Regulation; GTP-Binding Protein alpha Subunits, G12-G13; Guanosine Triphosphate; Humans; Intracellular Signaling Peptides and Proteins; Neoplastic Stem Cells; Nuclear Proteins; Phosphorylation; Protein Isoforms; Protein Serine-Threonine Kinases; Signal Transduction; Trans-Activators; YAP-Signaling Proteins

The expression of proinflammatory protein tissue transglutaminase 2 (TG2) is frequently upregulated in multiple cancer cell types. However, the exact role of TG2 in cancer cells is not well-understood. We recently initiated studies to determine the significance of TG2 in cancer cells and observed that sustained expression of TG2 resulted in epithelial-to-mesenchymal transition (EMT) and promoted cancer stem cell (CSC) traits in mammary epithelial cells. These results suggested that TG2 could serve as a promising therapeutic target for overcoming chemoresistance and inhibiting metastatic spread of cancer cells.. Using various mutant constructs, we analyzed the activity of TG2 that is essential for promoting the EMT-CSC phenotype.. Our results suggest that catalytically inactive TG2 (TG2-C277S) is as effective as wild-type TG2 (TG2-WT) in inducing the EMT-CSC in mammary epithelial cells. In contrast, overexpression of a GTP-binding-deficient mutant (TG2-R580A) was completely incompetent in this regard. Moreover, TG2-dependent activation of the proinflammatory transcription factor NF-κB is deemed essential for promoting the EMT-CSC phenotype in mammary epithelial cells.. Our results suggest that the transamidation activity of TG2 is not essential for promoting its oncogenic functions and provide a strong rationale for developing small-molecule inhibitors to block GTP-binding pockets of TG2. Such inhibitors may have great potential for inhibiting the TG2-regulated pathways, reversing drug resistance and inhibiting the metastasis of cancer cells.

Topics: Breast Neoplasms; Catalytic Domain; Cell Movement; Cell Survival; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression; Gene Expression Regulation, Neoplastic; GTP-Binding Proteins; Guanosine Triphosphate; Homeodomain Proteins; Humans; Mammary Glands, Human; Neoplasm Invasiveness; Neoplastic Stem Cells; NF-kappa B; Nuclear Proteins; Phenotype; Protein Binding; Protein Glutamine gamma Glutamyltransferase 2; Signal Transduction; Snail Family Transcription Factors; Spheroids, Cellular; Transcription Factors; Transglutaminases; Twist-Related Protein 1; Zinc Finger E-box-Binding Homeobox 1

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

Transforming growth factor beta-1 (TGF-beta) acts as both a tumour suppressor and a tumour promoter in a context-dependent manner. The tumour-promoting activities of TGF-beta are likely to result from a combination of Smad and non-Smad signalling pathways but remain poorly understood. Here we show that TGF-beta-mediated activation of RhoA is dependent on the kinase activity of ALK5 and that continuous ALK5 activity maintains basal RhoA-ROCK signalling, cell morphology and actin dynamics in serum-starved rodent fibroblasts independently of Smad2, Smad3 and Smad4. In immortalized human diploid fibroblasts, we show that oncogenic rewiring by transduction of (V12)HaRas instigates regulation of RhoA-ROCK signalling through an autocrine TGF-beta1-ALK5 pathway. Furthermore, we show that ALK5-mediated activation of RhoA is required for efficient (V12)HaRas, V-Raf and (V600E)BRAF transformation and (V12)HaRas-mediated anchorage-independent growth. These findings identify a new pro-oncogenic activity of TGF-beta and indicate that tumours harbouring (V12)HaRas and (V600E)BRAF mutations may be susceptible to TGF-beta signalling inhibitors.

Topics: Actins; Animals; Benzamides; Blotting, Western; Cell Transformation, Neoplastic; Cells, Cultured; Cytoskeleton; Dioxoles; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Fluorescent Antibody Technique; Genes, ras; Guanosine Triphosphate; Humans; Mice; NIH 3T3 Cells; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins B-raf; Rats; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; rho-Associated Kinases; rhoA GTP-Binding Protein; Signal Transduction; Smad3 Protein; Smad4 Protein; Transfection; Transforming Growth Factor beta

Rheb (Ras-homolog enriched in brain) is a component of the phosphatidylinositol 3-kinase (PI3K) target of rapamycin (TOR) signaling pathway, functioning as a positive regulator of TOR. Constitutively active mutants of Rheb induce oncogenic transformation in cell culture. The transformed cells are larger and contain more protein than their normal counterparts. They show constitutive phosphorylation of the ribosomal protein S6 kinase and the eukaryotic initiation factor 4E-binding protein 1, two downstream targets of TOR. The TOR-specific inhibitor rapamycin strongly interferes with transformation induced by constitutively active Rheb, suggesting that TOR activity is essential for the oncogenic effects of mutant Rheb. Rheb-induced transformation is also dependent on a C-terminal farnesylation signal that mediates localization to a cellular membrane. An engineered N-terminal myristylation signal can substitute for the farnesylation. Immunofluorescence localizes wild-type and mutant Rheb to vesicular structures in the cytoplasm, overlapping with the endoplasmic reticulum.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; DNA-Binding Proteins; Forkhead Box Protein O1; Forkhead Transcription Factors; Guanosine Triphosphate; Humans; Monomeric GTP-Binding Proteins; Neuropeptides; Nuclear Proteins; Phosphatidylinositol 3-Kinases; Prenylation; Protein Kinases; Ras Homolog Enriched in Brain Protein; Signal Transduction; TOR Serine-Threonine Kinases; Y-Box-Binding Protein 1

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

The BRAF(V600E) mutation is found in approximately 6% of human cancers and mimics the phosphorylation of the kinase domain activation segment. In wild-type B-Raf (B-Raf(wt)), activation segment phosphorylation is thought to cooperate with negative charges within the N-region for full activation. In contrast to Raf-1, the N-region of B-Raf is constitutively negatively charged owing to the presence of residues D447/D448 and the phosphorylation of S446. Therefore, it has been suggested that this hallmark predisposes B-Raf for oncogenic activation. In this study, we demonstrate that neutralizing mutations of these residues (in particular S446 and S447), or uncoupling of B-Raf from Ras-guanine 5'-triphosphate (GTP), strongly reduce the biological activity of B-Raf in a PC12 cell differentiation assay. We also confirm that S365 is a 14-3-3 binding site, and determine that mutation of this residue rescues the impaired biological activity of B-Raf proteins with a neutralized N-region, suggesting that the N-region opposes a 14-3-3-mediated transition into an inactive conformation. However, in the case of B-Raf(V600E), although complete N-region neutralization resulted in a 2.5-fold reduction in kinase activity in vitro, this oncoprotein strongly induced PC12 differentiation or transformation and epithelial-mesenchymal transition of MCF-10A cells regardless of its N-region charge. Furthermore, the biological activity of B-Raf(V600E) was independent of its ability to bind Ras-GTP. Our analysis identifies important regulatory differences between B-Raf(wt) and B-Raf(V600E) and suggests that B-Raf(V600E) cannot be inhibited by strategies aimed at blocking S446 phosphorylation or Ras activation.

Topics: 14-3-3 Proteins; Amino Acid Substitution; Animals; Binding Sites; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Chickens; Enzyme Activation; Epithelial Cells; Feedback, Physiological; Guanosine Triphosphate; Humans; MAP Kinase Signaling System; Mesoderm; Mice; Models, Biological; Mutation, Missense; NIH 3T3 Cells; Oncogene Protein p21(ras); PC12 Cells; Phosphorylation; Point Mutation; Protein Interaction Mapping; Protein Processing, Post-Translational; Protein Structure, Tertiary; Proto-Oncogene Proteins B-raf; Rats; Recombinant Fusion Proteins; Structure-Activity Relationship; Transfection

The small GTPase Cdc42 has been implicated in a number of cellular responses ranging from the regulation of the actin cytoskeletal architecture to intracellular trafficking and cell cycle progression. Cdc42 mutants that constitutively exchange GDP for GTP but still hydrolyze GTP (called 'fast-cycling' mutants) promote cellular transformation, whereas Cdc42 mutants that are unable to hydrolyze GTP and are irreversibly trapped in the GTP-bound state often inhibit cell growth. In this work, we have set out to further establish that Cdc42 needs to cycle between its 'on' and 'off' states to stimulate cell growth, by examining the consequences of manipulating its GTP-binding/GTP hydrolytic cycle in two different ways. One approach was to examine whether substitutions that act in a manner opposite to the 'fast cyclers', and extend the lifetime of the activated GTP-bound state by slowing the GTP hydrolytic reaction (i.e., 'slow-cycling' mutations), positively influence cell growth. Indeed we show that one such slow-cycling mutant, Cdc42[Y32A], which is insensitive to Cdc42GAP but still exhibits a measurable intrinsic GTP hydrolytic activity, gives rise to increased levels of activated Cdc42 in NIH 3T3 cells. We go on to show that the Y32A mutant stimulates the actin cytoskeletal changes that lead to filopodia formation, confer growth advantages to fibroblasts under low serum conditions, and enable cells to grow to high densities when exposed to normal levels of serum. The second approach was to determine whether the transforming activity of the fast-cycling Cdc42[F28L] mutant can be reversed by compensating for its accelerated nucleotide exchange reaction through the expression of the GTPase-activating protein (Cdc42GAP) and the ensuing stimulation of GTP hydrolytic activity. We showed that expression of the limit functional domain of Cdc42GAP inhibited Cdc42[F28L]-induced transformation, as well as selectively reversed the transformed phenotypes caused by the hyperactivation of wild-type Cdc42 in cells expressing the oncogenic version of Dbl (for Diffuse B cell lymphoma), a guanine nucleotide exchange factor for Cdc42 and the related Rac and Rho GTPases. Overall, the results reported here establish the requirement for Cdc42 to cycle between its signaling-on and -off states in order to positively influence cell growth and highlight how the Cdc42GAP can play an important role in regulating cell proliferation.

Topics: Animals; cdc42 GTP-Binding Protein; Cell Transformation, Neoplastic; Extracellular Signal-Regulated MAP Kinases; GTPase-Activating Proteins; Guanosine Triphosphate; Mice; NIH 3T3 Cells; Signal Transduction

Topics: Acylation; Animals; Cell Differentiation; Cell Membrane; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Cytosol; Dogs; Fluorescence Resonance Energy Transfer; Genes, ras; Golgi Apparatus; Guanosine Triphosphate; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Models, Biological; Palmitic Acid; Protein Isoforms; Proto-Oncogene Proteins p21(ras); ras Proteins

HiTrap-syndecan-2/p120-GAP and HiTrap-syndecan-2/RACK1 affinity columns were applied to reveal that Src tyrosine kinase was highly expressed in BALB/3T3 cells transfected with plasmids pcDNA3.1-[S-ras(Q(61)K)] of shrimp Penaeus japonicus. Both columns were effective to isolate Src tyrosine kinase. The selective molecular affinity for Src was found to be stronger with HiTrap-syndecan-2/RACK1, as revealed with competitive RACK1 to dislodge Src from HiTrap-syndecan-2/p120-GAP. We thus challenged the syndecan-2/p120-GAP and syndecan-2/RACK1 with GTP-K(B)-Ras(Q(61)K). The reaction between RACK1 and syndecan-2 was sustained in the presence of mutant Ras proteins, but not the reaction between p120-GAP and syndecan-2. In the presence of syndecan-2, GTP-K(B)-Ras(Q(61)K) exhibited sufficient reactivity with p120-GAP to discontinue the reaction between p120-GAP and syndecan-2. But the interference of mutant Ras disappeared when Src tyrosine kinase was introduced to stabilize the syndecan-2/p120-GAP complex. On the other hand, in the absence of syndecan-2, GTP-K(B)-Ras(Q(61)K) was found to react with RACK1. The reaction between GTP-K(B)-Ras(Q(61)K) and RACK1 could provide a mechanism to deprive RACK1 for the organization of syndecan-2/RACK1 complex and to facilitate the formation of syndecan-2/p120-GAP complex, as well as to provide docking sites for Src signaling upon transformation with oncogenic ras.

Topics: Animals; BALB 3T3 Cells; Cell Transformation, Neoplastic; Electrophoresis, Polyacrylamide Gel; Guanosine Triphosphate; Membrane Glycoproteins; Mice; Mutation; p120 GTPase Activating Protein; Protein Binding; Proteoglycans; ras Proteins; Receptors for Activated C Kinase; Receptors, Cell Surface; Recombinant Fusion Proteins; Signal Transduction; src-Family Kinases; Syndecan-2; Transfection

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

The novel small GTPases Rin and Rit are close relatives of Ras, and recent studies show that they play a role in mediating neuronal differentiation. However, the direct effectors of Rin and Rit have yet to be fully characterized. Here we showed that Rin and Rit directly bind to the PDZ domain of PAR6, a cell polarity-regulating protein, in a GTP-dependent manner both in vivo and in vitro. Moreover, Rin and Rit can form a ternary complex consisting of PAR6 and Rac/Cdc42, members of the Rho family of small GTPases modulating cell growth and polarity. This ternary complex synergistically potentiates cell transformation in NIH3T3 cells, and the interaction between Rin/Rit and the PDZ domain of PAR6 is important for this effect. These results suggest that the Rin/Rit-PAR6-Rac/Cdc42 ternary complex may work physiologically in the cells, such as in tumorigenesis.

Topics: Animals; beta-Galactosidase; Blotting, Western; cdc42 GTP-Binding Protein; Cell Transformation, Neoplastic; COS Cells; Glutathione Transferase; Guanosine Triphosphate; Immunoprecipitation; Mice; NIH 3T3 Cells; Protein Binding; Protein Structure, Tertiary; Proteins; ras Proteins; Recombinant Proteins; Transfection; Two-Hybrid System Techniques

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

RalGEFs were recently shown to be critical for Ras-mediated transformed and tumorigenic growth of human cells. We now show that the oncogenic activity of these proteins is propagated by activation of one RalGEF substrate, RalA, but blunted by another closely related substrate, RalB, and that the oncogenic signaling requires binding of the RalBP1 and exocyst subunit effector proteins. Knockdown of RalA expression impeded, if not abolished, the ability of human cancer cells to form tumors. RalA was also commonly activated in a panel of cell lines from pancreatic cancers, a disease characterized by activation of Ras. Activation of RalA signaling thus appears to be a critical step in Ras-induced transformation and tumorigenesis of human cells.

Topics: Animals; ATP-Binding Cassette Transporters; Carrier Proteins; Cell Line; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Gene Expression; GTPase-Activating Proteins; Guanosine Triphosphate; Humans; Mice; Mice, SCID; Neoplasm Transplantation; Neoplasms; Pancreatic Neoplasms; Protein Binding; Protein Transport; Proto-Oncogene Proteins p21(ras); ral GTP-Binding Proteins; ral Guanine Nucleotide Exchange Factor; rho GTP-Binding Proteins; RNA, Small Interfering; Transfection; Vesicular Transport Proteins

Stem cell factor (SCF) delays differentiation and enhances the expansion of erythroid progenitors. Previously, we performed expression-profiling experiments to link signaling pathways to target genes using polysome-bound mRNA. SCF-induced phosphoinositide-3-kinase (PI3K) appeared to control polysome recruitment of specific mRNAs associated with neoplastic transformation. To evaluate the role of mRNA translation in the regulation of expansion versus differentiation of erythroid progenitors, we examined the function of the eukaryote initiation factor 4E (eIF4E) in these cells. SCF induced a rapid and complete phosphorylation of eIF4E-binding protein (4E-BP). Overexpression of eIF4E did not induce factor-independent growth but specifically impaired differentiation into mature erythrocytes. Overexpression of eIF4E rendered polysome recruitment of mRNAs with structured 5' untranslated regions largely independent of growth factor and resistant to the PI3K inhibitor LY294002. In addition, overexpression of eIF4E rendered progenitors insensitive to the differentiation-inducing effect of LY294002, indicating that control of mRNA translation is a major pathway downstream of PI3K in the regulation of progenitor expansion.

Topics: 5' Untranslated Regions; Animals; Blotting, Western; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Chromones; DNA Primers; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Erythrocytes; Eukaryotic Initiation Factor-4E; Flow Cytometry; Genetic Vectors; Guanosine Triphosphate; Hemoglobins; Immunoprecipitation; Mice; Microscopy, Fluorescence; Morpholines; Phosphatidylinositol 3-Kinases; Phosphorylation; Polyribosomes; Protein Biosynthesis; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Sepharose; Signal Transduction; Stem Cell Factor; Stem Cells; Time Factors

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

Accumulating evidence suggests that p21(Cip1) located in the cytoplasm might play a role in promoting transformation and tumor progression. Here we show that oncogenic H-RasV12 contributes to the loss of actin stress fibers by inducing cytoplasmic localization of p21(Cip1), which uncouples Rho-GTP from stress fiber formation by inhibiting Rho kinase (ROCK). Concomitant with the loss of stress fibers in Ras-transformed cells, there is a decrease in the phosphorylation level of cofilin, which is indicative of a compromised ROCK/LIMK/cofilin pathway. Inhibition of MEK in Ras-transformed NIH3T3 results in restoration of actin stress fibers accompanied by a loss of cytoplasmic p21(Cip1), and increased phosphorylation of cofilin. Ectopic expression of cytoplasmic but not nuclear p21(Cip1) in Ras-transformed cells was effective in preventing stress fibers from being restored upon MEK inhibition and inhibited phosphorylation of cofilin. p21(Cip1) was also found to form a complex with ROCK in Ras-transformed cells in vivo. Furthermore, inhibition of the PI 3-kinase pathway resulted in loss of p21(Cip1) expression accompanied by restoration of phosphocofilin, which was not accompanied by stress fiber formation. These results suggest that restoration of cofilin phosphorylation in Ras-transformed cells is necessary but not sufficient for stress fiber formation. Our findings define a novel mechanism for coupling cytoplasmic p21(Cip1) to the control of actin polymerization by compromising the Rho/ROCK/LIMK/cofilin pathway by oncogenic Ras. These studies suggest that localization of p21(Cip1) to the cytoplasm in transformed cells contributes to pathways that favor not only cell proliferation, but also cell motility thereby contributing to invasion and metastasis.

Topics: Actin Depolymerizing Factors; Actins; Animals; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cytoplasm; Genes, ras; Guanosine Triphosphate; Intracellular Signaling Peptides and Proteins; Lim Kinases; MAP Kinase Kinase Kinase 1; MAP Kinase Kinase Kinases; Mice; Microfilament Proteins; NIH 3T3 Cells; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; rho GTP-Binding Proteins; rho-Associated Kinases; Stress Fibers

The Ras homolog enriched in striatum, Rhes, is the product of a thyroid hormone-regulated gene during brain development. Rhes and the dexamethasone-induced Dexras1 define a novel distinct subfamily of proteins within the Ras family, characterized by an extended variable domain in the carboxyl terminal region. We have carried this study because there is a complete lack of knowledge on Rhes signaling. We show that in PC12 cells, Rhes is targeted to the plasma membrane by farnesylation. We demonstrate that about 30% of the native Rhes protein is bound to GTP and this proportion is unaltered by typical Ras family nucleotide exchange factors. However, Rhes is not transforming in murine fibroblasts. We have also examined the role of Rhes in cell signaling. Rhes does not stimulate the ERK pathway. By contrast, it binds to and activates PI3K. On the other hand, we demonstrate that Rhes impairs the activation of the cAMP/PKA pathway by thyroid-stimulating hormone, and by an activated beta2 adrenergic receptor by a mechanism that suggests uncoupling of the receptor to its cognate heterotrimeric complex. Overall, our results provide the initial insights into the role in signal transduction of this novel Ras family member.

Topics: Animals; Carbachol; Cell Transformation, Neoplastic; Cyclic AMP; Enzyme Activation; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Mice; Mitogen-Activated Protein Kinases; NIH 3T3 Cells; PC12 Cells; Phosphatidylinositol 3-Kinases; Phylogeny; Protein Transport; Proto-Oncogene Proteins c-raf; Rats; Receptors, G-Protein-Coupled; Receptors, Muscarinic; Receptors, Thyrotropin; RNA, Messenger; Signal Transduction

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Cell Line, Transformed; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Genes, ras; Guanosine Triphosphate; Inhibitory Concentration 50; Kinetics; Magnetic Resonance Spectroscopy; Mitogen-Activated Protein Kinases; Models, Molecular; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-raf; ras Proteins; Signal Transduction; Spectrometry, Fluorescence; Sulindac

The Ras small GTPase functions as a signaling node and is activated by extracellular stimuli. Upon activation, Ras interacts with a spectrum of functionally diverse downstream effectors and stimulates multiple cytoplasmic signaling cascades that regulate cellular proliferation, differentiation, and apoptosis. In addition to the association of Ras with the plasma membrane, recent studies have established an association of Ras with Golgi membranes. Whereas the effectors of signal transduction by activated, plasma membrane-localized Ras are well characterized, very little is known about the effectors used by Golgi-localized Ras. In this study, we report the identification of a novel Ras-interacting protein, Rain, that may serve as an effector for endomembrane-associated Ras. Rain does not share significant sequence similarity with any known mammalian proteins, but contains a Ras-associating domain that is found in RalGDS, AF-6, and other characterized Ras effectors. Rain interacts with Ras in a GTP-dependent manner in vitro and in vivo, requires an intact Ras core effector-binding domain for this interaction, and thus fits the definition of a Ras effector. Unlike other Ras effectors, however, Rain is localized to perinuclear, juxta-Golgi vesicles in intact cells and is recruited to the Golgi by activated Ras. Finally, we found that Rain cooperates with activated Raf and causes synergistic transformation of NIH3T3 cells. Taken together, these observations support a role for Rain as a novel protein that can serve as an effector of endomembrane-localized Ras.

Topics: Amino Acid Sequence; Animals; Apoptosis; Carrier Proteins; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; COS Cells; Cytoplasm; DNA, Complementary; Glutathione Transferase; Golgi Apparatus; Guanosine Triphosphate; Humans; Intracellular Signaling Peptides and Proteins; Mice; Microscopy, Fluorescence; Models, Genetic; Molecular Sequence Data; NIH 3T3 Cells; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; ras Proteins; RNA, Messenger; Sequence Homology, Amino Acid; Signal Transduction; Subcellular Fractions; Transfection; Two-Hybrid System Techniques

Multiple myeloma (MM) is an incurable plasma cell malignancy. To investigate biochemical lesions associated with MM, we constructed an expression cDNA library from the OPM-2 human myeloma line. A highly transforming H-Ras mutant was identified by transfection analysis using NIH 3T3 cells. DNA sequencing demonstrated a single-point mutation at position 117 located in the guanine nucleotide-binding site resulting in a lysine-to-glutamic acid substitution. This mutant, H-Ras (K117E), was found to be constitutively activated in terms of GTP binding. We compared the biological effects of H-Ras (K117E) and H-Ras (G12V) in 32D murine hematopoietic progenitor cells. Whereas both Ras proteins are constitutively activated, 32D cells expressing H-Ras (G12V) are still dependent on IL-3 for survival and proliferation while cells carrying H-Ras (K117E) become IL-3 independent. Similar experiments conducted with the B9 line, an IL-6-dependent hybridoma, also demonstrated that B9/H-Ras (K117E) became IL-6-independent. Expression of H-Ras (K117E) in the human IL-6-dependent ANBL-6 myeloma line resulted in enhanced proliferation at suboptimal concentrations of IL-6. These observations suggest that H-Ras mutations at the binding site for the GTP nucleotide ring structure may also represent activating lesions and have additional biological effects when compared to previously described Ras mutants.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Genes, ras; Growth Substances; Guanosine Triphosphate; Humans; Mice; Multiple Myeloma; Mutation; Tumor Cells, Cultured

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

The BCR/ABL oncogene causes chronic myelogenous leukemia (CML) in humans and a CML-like disease, as well as lymphoid leukemia, in mice. p210 BCR/ABL is an activated tyrosine kinase that phosphorylates itself and several cellular signaling proteins. The autophosphorylation site tyrosine 177 binds the adaptor Grb2 and helps determine the lineage and severity of BCR/ABL disease: Tyr177 mutation (BCR/ABL-Y177F) dramatically impairs myeloid leukemogenesis, while diminishing lymphoid leukemogenesis. The critical signal(s) from Tyr177 has remained unclear. We report that Tyr177 recruits the scaffolding adaptor Gab2 via a Grb2/Gab2 complex. Compared to BCR/ABL-expressing Ba/F3 cells, BCR/ABL-Y177F cells exhibit markedly reduced Gab2 tyrosine phosphorylation and association of phosphatidylinositol-3 kinase (PI3K) and Shp2 with Gab2 and BCR/ABL, and decreased PI3K/Akt and Ras/Erk activation, cell proliferation, and spontaneous migration. Remarkably, bone marrow myeloid progenitors from Gab2 (-/-) mice are resistant to transformation by BCR/ABL, whereas lymphoid transformation is diminished as a consequence of markedly increased apoptosis. BCR/ABL-evoked PI3K/Akt and Ras/Erk activation also are impaired in Gab2 (-/-) primary myeloid and lymphoid cells. Our results identify Gab2 and its associated proteins as key determinants of the lineage and severity of BCR/ABL transformation.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Benzamides; Cell Division; Cell Movement; Cell Transformation, Neoplastic; Erythroid Precursor Cells; Fusion Proteins, bcr-abl; GRB2 Adaptor Protein; Guanosine Triphosphate; Helminth Proteins; Humans; Imatinib Mesylate; Immunoblotting; Leukemia, Lymphoid; Mice; Mice, Knockout; Phosphatidylinositol 3-Kinases; Phosphorylation; Piperazines; Precipitin Tests; Proteins; Pyrimidines; Signal Transduction; Tyrosine

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

Ras proteins are molecular switches that control signaling pathways critical in the onset of a variety of human cancers. The signaling pathways activated by Ras proteins are those controlled by its direct effectors such as the serine-threonine protein kinase Raf-1, the exchange factor for other GTPases Ral-GDS, and the lipid kinase PI3K. As a consequence of Ras activation, a number of additional enzymes are affected, including several members of the serine-threonine intracellular proteins kinases as well as enzymes related to phospholipid metabolism regulation such as phospholipases A2 and D, and choline kinase. The precise mechanisms by which ras oncogenes impinge into these later molecules and their relevance to the onset of the carcinogenic process is still not fully understood. Here we have investigated the mechanism of regulation of choline kinase by Ras proteins and found no direct link between PLD and choline kinase activation. We provide evidence that Ras proteins regulate the activity of choline kinase through its direct effectors Ral-GDS and PI3K, while the Raf pathways seems to be not relevant in this process. The importance of Ras-dependent activation of choline kinase is discussed.

Topics: 3T3 Cells; Amino Acid Substitution; Animals; Cell Line; Cell Transformation, Neoplastic; Choline Kinase; Embryo, Mammalian; Enzyme Activation; Enzyme Induction; Genes, ras; Guanosine Triphosphate; Humans; Isoenzymes; Kidney; Mice; Mutation, Missense; Phospholipase D; Phosphorylcholine; Protein Structure, Tertiary; Proto-Oncogene Proteins c-raf; Proto-Oncogene Proteins p21(ras); ral Guanine Nucleotide Exchange Factor; ras Proteins; Recombinant Fusion Proteins; Second Messenger Systems; Transfection

Tissue transglutaminase (tTGase, tTG) is known as being implicated in the intracellular cross-linking of proteins occurring in a growing series of physiological conditions including--just to mention the most relevant ones--programmed cell death (apoptosis), cell adhesion, growth, spreading and differentiation, tumor growth, metastasis, cell adhesion, proliferation, differentiation, extra cellular matrix (ECM) stabilization. In the current work we investigated tTG activity and expression of "normal" and potential transformed cytosolic tTG antigens in mammalian cells. Most cell lines studied showed low tTG activity, which in all cases could be enhanced considerably by treating cell cytosol homogenates with trypsin. The results suggested the existence -in transformed cells- of inactive types of tTGase. We purified cytosolic tTG antigens from these cells utilizing a GTP-agarose resin, and we can therefore conclude that "normal" molecular weight (mw) tTG antigens, but also high molecular weight (hmw) and low molecular weight (lmw) tTG antigens from transformed cells, retain GTP-binding ability. The initial results from our study also allowed us to hypothesize that transformed hmw- and lmw- tTG antigens should not be considered as the result of post-translational modifications of normal mw, cytosolic tTG. The potentially low or absent transamidating functionality of cytosolic tTG species in transformed mammalian cells could be responsible for decreased or even abolished programmed cell death, whereas the unaffected GTP-binding functionality of such proteins in these cells might lead to increased signal transduction and possibly proliferation.

Topics: 3T3 Cells; Animals; Antibodies, Monoclonal; Blotting, Western; Cell Transformation, Neoplastic; Chromatography, Affinity; Cytosol; Electrophoresis, Polyacrylamide Gel; Guanosine Triphosphate; Humans; Immunohistochemistry; Isoenzymes; Mammals; Mice; Molecular Weight; Neoplasms; Phenotype; Transglutaminases

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

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation state depends on the competing action of GTPase Activating Proteins (GAP) and Guanine nucleotide Exchange Factors (GEF). A tryptophan residue (Trp1056 in CDC25Mm-GEF), conserved in all ras-specific GEFs identified so far has been previously shown to be essential for GEF activity. Its substitution with glutamic acid results in a catalytically inactive mutant, which is able to efficiently displace wild-type GEF from p21ras and to originate a stable ras/GEF binary complex due to the reduced affinity of the nucleotide-free ras/GEF complex for the incoming nucleotide. We show here that this 'ras-sequestering property' can be utilized to attenuate ras signal transduction pathways in mouse fibroblasts transformed by oncogenic ras. In fact overexpression of the dominant negative GEFW1056E in stable transfected cells strongly reduces intracellular ras-GTP levels in k-ras transformed fibroblasts. Accordingly, the transfected fibroblasts revert to wild-type phenotype on the basis of morphology, cell cycle and anchorage independent growth. The reversion of the transformed phenotype is accompanied by DNA endoreduplication. The possible use of dominant negative ras-specific GEFs as a tool to down-regulate tumor growth is discussed.

Topics: Animals; Carcinogenicity Tests; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Down-Regulation; Female; Fibroblasts; Genes, Dominant; Genes, ras; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Mice; Mice, Nude; Mutation, Missense; ras Proteins; ras-GRF1; Signal Transduction

The Ras-related GTP-binding protein Cdc42 is implicated in a variety of biological activities including the establishment of cell polarity in yeast, the regulation of cell morphology, motility and cell-cycle progression in mammalian cells and the induction of malignant transformation. We identified a Cdc42 mutant (Cdc42F28L) which binds GTP in the absence of a guanine nucleotide exchange factor, but still hydrolyses GTP with a turnover number identical to that for wild-type Cdc42. Expression of this mutant in NIH 3T3 fibroblasts causes cellular transformation, mimicking many of the characteristics of cells transformed by the Dbl oncoprotein, a known guanine nucleotide exchange factor for Cdc42. Here we searched for new Cdc42 targets in an effort to understand how Cdc42 mediates cellular transformation. We identified the gamma-subunit of the coatomer complex (gammaCOP) as a specific binding partner for activated Cdc42. The binding of Cdc42 to gammaCOP is essential for a transforming signal distinct from those elicited by Ras.

Topics: 3T3 Cells; Animals; cdc42 GTP-Binding Protein; Cell Transformation, Neoplastic; Coat Protein Complex I; COS Cells; Enzyme Activation; Guanosine Triphosphate; Mice; Mutation; Protein Binding; Recombinant Fusion Proteins

Codon 12 mutations are frequent in the Ki-ras oncogene in human lung adenocarcinomas, but the effects of these alterations have not been well characterized in lung epithelial cells. Murine primary lung tumors derived from peripheral epithelial cells also may present Ki-ras mutations and are useful models for study of early phases of tumor development. One hypothesis is that Ki-ras mutation and/or a Ki-ras p21 increase could enhance Ki-ras p21-GTP and cell-cycle stimulation through raf-1 and extracellularly regulated protein kinases (Erks). We examined lung tumors 1-7 mm in largest dimension initiated in male Swiss mice by N-nitrosodimethylamine for pathologic type, Ki-ras mutations and levels of total Ki-ras p21, Ki-ras p21 bound to GTP, raf-1, Erk1 and Erk2 and their phosphorylated (activated) forms, and proliferating cell nuclear antigen. Total Ki-ras p21 and activated ras-GTP were not significantly greater in tumors than in normal lung or in tumors with versus those without Ki-ras mutations. Carcinomas with Ki-ras mutations were significantly smaller than those without mutations. Carcinomas were significantly larger than adenomas only for tumors without mutations. High levels of Erk2 and correlation of Erk2 amount with ras-GTP were specific characteristics of tumors with Ki-ras mutations. Size of all tumors correlated with ras-GTP but not with proliferating cell nuclear antigen. Raf-1 was expressed mainly in alveolar macrophages in normal lung but was focally upregulated in papillary areas of some tumors. The results indicate that Ki-ras influences the characteristics of lung tumors, but a linear ras-raf-Erk-cell-cycle control sequence does not adequately characterize tumorigenic events in this model. Mol. Carcinog. 28:156-167, 2000.

Topics: Adenoma; Animals; Apoptosis; Carcinoma; Cell Cycle; Cell Transformation, Neoplastic; Codon; Dimethylnitrosamine; DNA Mutational Analysis; DNA, Neoplasm; Genes, ras; Guanosine Triphosphate; Humans; Lung Neoplasms; Male; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-raf; Proto-Oncogene Proteins p21(ras); Species Specificity

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

Based on the previous experiments with the N17 mutant of CDC42, it has been speculated, but not proved as yet, that CDC42 is required for Ras-induced malignant transformation of fibroblasts. However, since this inhibitor could sequester many GDP-dissociation stimulators (GDSs), such as DBL, OST and Tiam-1 which activate not only CDC42, but also Rho or Rac, in fact it is not a specific inhibitor that inactivates only CDC42. Thus, we have taken the minimum CDC42-binding domain (residues 504 - 545, called ACK42) of the Tyr-kinase ACK-1 that binds only CDC42 in the GTP-bound form, and thereby blocking the interactions of CDC42-GTP with its downstream effectors such as ACKs, PAKs and N-WASP. First of all, using the ACK42-GST fusion protein as a specific ligand for the GTP-CDC42 complex, we have revealed that CDC42 is activated by oncogenic Ras mutants such as v-Ha-Ras in NIH3T3 fibroblasts, and similarly in PC12 cells by both NGF (Nerve Growth Factor) and EGF (Epidermal Growth Factor) which activate the endogenous normal Ras, providing the first direct evidence that CDC42 acts downstream of Ras and NGF/EGF. Furthermore, over-expression of ACK42 completely reversed Ras-induced malignant phenotypes such as focus formation and anchorage/serum-independent growth of the fibroblasts, and a cell-permeable derivative of ACK42 called WR-ACK42 strongly inhibited the growth of Ras transformants, with little effect on the parental normal cell growth, and also abolished Ras-induced filopodium/microspike formation of the fibroblasts which is CDC42-dependent. These observations unambiguously proved for the first time that the RAS-induced activation of CDC42 is indeed essential for Ras to transform the fibroblasts, and furthermore suggest that ACK42 or its peptidomimetics are potentially useful for genotherapy or chemotherapy of Ras-associated cancer.

Topics: 3T3 Cells; Animals; Base Sequence; cdc42 GTP-Binding Protein; Cell Transformation, Neoplastic; Genes, ras; Guanosine Triphosphate; Mice; Molecular Sequence Data; PC12 Cells; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Rats; Recombinant Fusion Proteins; Templates, Genetic; Thrombin; Transfection

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

It has been reported that expression of the active mutant of heterotrimeric GTP-binding protein alpha subunit G alpha i2 transforms Rat-1 cells. However, the G alpha i2-mediated mitogenic signaling pathways remain to be elucidated. Here, we demonstrate that inducible expression of the active mutant of G alpha i2 (G alpha i2(Q205L)) activates Ras and c-Jun N-terminal kinase (JNK) in addition to extracellular signal-regulated kinase (ERK) in Rat-1 cells. Our findings suggest that Ras may play a critical role in the G alpha i2-induced transformation and G alpha i2 can transduce signals from the Gi-coupled receptor to JNK and ERK in certain types of mammalian cells.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cell Transformation, Neoplastic; Enzyme Activation; Epidermal Growth Factor; Escherichia coli; Fibroblasts; Flavonoids; GTP-Binding Protein alpha Subunit, Gi2; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Proteins; Guanosine Triphosphate; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinases; Mitogens; Mutation; Phosphorylation; Proto-Oncogene Proteins; ras Proteins; Rats; Recombinant Fusion Proteins; Signal Transduction; Trypsin

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

ras proteins are positively regulated by nucleotide exchange factors and negatively regulated by GTPase-activating proteins (GAPs). Two GAPs have been found in mammalian cells, p120GAP and neurofibromin, the product of the type 1 neurofibromatosis (NF1) gene. A library of substitutions in the effector loop region of ras in an Escherichia coli plasmid expression system was screened for c-Ha-ras species with altered GAP interactions. Several substitutions preferentially disrupted the interaction of ras with p120GAP as compared with the interaction with the recombinant GAP-related domain of neurofibromin (NF1-GRD). The most extreme example, Tyr32His, encoded a ras species that was unaffected by p120GAP but was stimulated normally by NF1-GRD. Tyr32His was weakly transforming in Rat2 cells. Tyr32His ras was primarily GDP-bound in quiescent Rat2 cells, although it rapidly associated with GTP after treatment of cells with epidermal growth factor. These results show that the NF1 product has less stringent requirements than p120GAP for ras effector domain structure and that negative regulation of ras can be achieved in rat fibroblasts by the product of NF1.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Growth Factor; GTP-Binding Proteins; GTPase-Activating Proteins; Guanine Nucleotides; Guanosine Triphosphate; Humans; Neurofibromin 1; Point Mutation; Proteins; Proto-Oncogene Proteins p21(ras); ras GTPase-Activating Proteins; Rats; Recombinant Fusion Proteins; Signal Transduction; Structure-Activity Relationship

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

An amino-truncated variant form of the epidermal growth factor receptor (EGFRvIII) has been identified in human brain, breast, lung and ovarian tumors. We have found that overexpression of this mutant EGF receptor in NIH3T3 cells results in transformation as a result of the activation of the receptor kinase via ligand-independent dimerization. Transformation was correlated with tyrosine phosphorylation of only a subset of the proteins observed in cells overexpressing the normal EGF receptor. This suggested that further studies on cells expressing the EGFRvIII might provide insights into the pathways most relevant to transformation. In clones expressing high levels of mutant EGF receptor, the levels of both Grb2 and SHC were decreased. Despite this decrease, much of the endogenous Grb2 immunoprecipitated with EGFRvIII. Interestingly, no increase in ras-GTP loading was found in clones expressing the EGFRvIII and MAP kinase assays indicated only a small increase in activity. These results indicate that high-level expression of the EGFRvIII induces down-regulation of the ras-MAP kinase pathway and that other components involved in EGF receptor signal transduction may play a greater role in neoplastic transformation by the EGFRvIII.

Topics: 3T3 Cells; Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Transformation, Neoplastic; ErbB Receptors; Female; GRB2 Adaptor Protein; Guanosine Triphosphate; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Sequence Data; Neoplasm Proteins; Neoplasm Transplantation; Phosphorylation; Protein Biosynthesis; Protein Processing, Post-Translational; Signal Transduction; Transfection

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

Conserved amino-acids of H-ras from residues 25 to 34 were mutated in human H-ras cDNA with a pre-existing valine-12 activating mutation ([V12]p21), and built into SV40-driven expression vectors. The influence of the introduced mutations was initially screened by transfection of Rat-1 cells to score foci of transformed cells. Non-conservative mutations of amino-acids 25 (tryptophan for glutamine), 27 (asparagine for histidine) and 34 (alanine for proline) did not abrogate the transforming potential of [V12]p21. The conservative mutation of phenylalanine-28 to tryptophan ([V12W28]p21) was also still transforming. Significantly, in the absence of the valine-12 activating mutation, tryptophan-28-ras ([W28]p21) was weakly transforming while, in contrast, [V12D28]p21 was unable to transform Rat-1 cells and retarded cell growth. Analysis of the binding and dissociation of GTP and GDP to normal and mutated p21 expressed in Escherichia coli showed that [V12D28]p21 and [D28]p21 do not bind GTP. The dissociation rate of both GTP and GDP bound to [W28]p21 is increased, suggesting a mechanism for its transforming potential in Rat-1 cells. These studies illustrate the importance of phenylalanine-28 in guanine nucleotide binding by p21H-ras. The mutations described could be valuable tools in investigations of cellular signal transduction involving small GTP-binding proteins.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Gene Expression; Guanosine Triphosphate; Humans; Phenylalanine; Proto-Oncogene Proteins p21(ras); Rats; Signal Transduction; Structure-Activity Relationship

The neurofibromatosis type 1 (NF1) gene encodes a protein, neurofibromin, containing GTPase-activating protein-related domain (GRD) that stimulates intrinsic GTPase activity of Ras protein. By screening a randomly mutagenized NF1-GRD library in Saccharomyces cerevisiae, we isolated two NF1-GRD mutants (NF201 and NF204) with single amino acid substitutions, which suppress the heat shock-sensitive phenotype of the RAS2(G19V) mutant. The NF1-GRD mutants also suppress the oncogenic Ras-induced transformation of NIH 3T3 mouse fibroblasts (Nakafuku, M., Nagamine, M., Ohtoshi, A., Tanaka, K., Toh-e, A., and Kaziro, Y. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6706-6710). In this paper, we investigated the molecular mechanism of inhibition of the transforming Ras-specific function by the NF1-GRD mutants in mammalian cells. In human embryonic kidney (HEK) 293 cells, the mutant NF1-GRDs attenuated the stimulation of mitogen-activated protein kinase by Ras(G12V), but not by platelet-derived growth factor. In cell-free systems, purified recombinant NF1-GRD mutants showed an inhibitory effect on the association of Ras.guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with Raf at several times lower concentrations than the wild type. Furthermore, it was revealed that the binding affinity of the mutant NF1-GRDs toward Ras.GTP gamma S is approximately 5-10 times higher than the wild type. These results suggest that the mutant NF1-GRDs tightly bind to an oncogenic Ras in its GTP-bound active conformation and block the interaction between Ras and its effector, Raf.

Topics: 3T3 Cells; Animals; Cell Line; Cell Transformation, Neoplastic; Cell-Free System; Guanosine Triphosphate; Humans; Kidney; Mice; Mutation; Neurofibromin 1; Oncogenes; Protein Binding; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; ras Proteins; Rats

BCR-ABL is a deregulated tyrosine kinase expressed in Philadelphia chromosome-positive human leukemias. Prolongation of hematopoietic cell survival by inhibition of apoptosis has been proposed to be an integral component of BCR-ABL-induced chronic myelogenous leukemia. BCR-ABL elicits transformation of both fibroblast and hematopoietic cells and blocks apoptosis following cytokine deprivation in various factor-dependent cells. To elucidate the mechanisms whereby BCR-ABL induces transformation and blocks apoptosis in hematopoietic cells, we examined the biological effects of expression of a series of BCR-ABL mutants. Single amino acid substitutions in the GRB2 binding site (Y177F), Src homology 2 domain (R552L), or an autophosphorylation site in the tyrosine kinase domain (Y793F) do not diminish the antiapoptotic and transforming properties of BCR-ABL in hematopoietic cells, although these mutations were previously shown to drastically reduce the transforming activity of BCR-ABL in fibroblasts. A BCR-ABL molecule containing all three mutations (Y177F/R552L/Y793F) exhibits a severe decrease in transforming and antiapoptotic activities compared with the wild-type BCR-ABL protein in 32D myeloid progenitor cells. Ras is activated, the SHC adapter protein is tyrosine phosphorylated and binds GRB2, and myc mRNA levels are increased following expression of all kinase active BCR-ABL proteins with the exception of the Y177F/R552L/Y793F BCR-ABL mutant in 32D cells. We propose that BCR-ABL uses multiple pathways to activate Ras in hematopoietic cells and that this activation is necessary for the transforming and antiapoptotic activities of BCR-ABL. However, Ras activation is not sufficient for BCR-ABL-mediated transformation. A BCR-ABL deletion mutant (delta 176-427) that activates Ras and blocks apoptosis but has severely impaired transforming ability in 32D cells has been identified. These data suggest that BCR-ABL requires additional signaling components to elicit tumorigenic growth which are distinct from those required to block apoptosis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; B-Lymphocytes; Base Sequence; Cell Line; Cell Transformation, Neoplastic; Erythroid Precursor Cells; Fusion Proteins, bcr-abl; GRB2 Adaptor Protein; Guanosine Triphosphate; Humans; Mice; Mice, Nude; Molecular Sequence Data; Mutation; Oncogene Protein p21(ras); Phosphorylation; Protein-Tyrosine Kinases; Proteins; Proto-Oncogene Proteins c-myc; RNA, Messenger; Signal Transduction

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 serine/threonine kinase Raf-1 functions downstream from Ras to activate mitogen-activated protein kinase kinase, but the mechanisms of Raf-1 activation are incompletely understood. To dissect these mechanisms, wild-type and mutant Raf-1 proteins were studied in an in vitro system with purified plasma membranes from v-Ras- and v-Src-transformed cells (transformed membranes). Wild-type (His)6- and FLAG-Raf-1 were activated in a Ras- and ATP-dependent manner by transformed membranes; however, Raf-1 proteins that are kinase defective (K375M), that lack an in vivo site(s) of regulatory tyrosine (YY340/341FF) or constitutive serine (S621A) phosphorylation, that do not bind Ras (R89L), or that lack an intact zinc finger (CC165/168SS) were not. Raf-1 proteins lacking putative regulatory sites for an unidentified kinase (S259A) or protein kinase C (S499A) were activated but with apparently reduced efficiency. The kinase(s) responsible for activation by Ras or Src may reside in the plasma membrane, since GTP loading of plasma membranes from quiescent NIH 3T3 cells (parental membranes) induced de novo capacity to activate Raf-1. Wild-type Raf-1, possessing only basal activity, was not activated by parental membranes in the absence of GTP loading. In contrast, Raf-1 Y340D, possessing significant activity, was, surprisingly, stimulated by parental membranes in a Ras-independent manner. The results suggest that activation of Raf-1 by phosphorylation may be permissive for further modulation by another membrane factor, such as a lipid. A factor(s) extracted with methanol-chloroform from transformed membranes or membranes from Sf9 cells coexpressing Ras and SrcY527F significantly enhanced the activity of Raf-1 Y340D or active Raf-1 but not that of inactive Raf-1. Our findings suggest a model for activation of Raf-1, wherein (i) Raf-1 associates with Ras-GTP, (ii) Raf-1 is activated by tyrosine and/or serine phosphorylation, and (iii) Raf-1 activity is further increased by a membrane cofactor.

Topics: 3T3 Cells; Adenosine Triphosphate; Animals; Cell Membrane; Cell Transformation, Neoplastic; Enzyme Activation; Guanosine Triphosphate; Mice; Mutation; Oligopeptides; Peptides; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; ras Proteins; Recombinant Fusion Proteins; Signal Transduction

Despite many advances in understanding the structure and function of GTP-binding proteins the mechanism by which these molecules switch from the GTP-bound on-state to the GDP-bound off-state is still poorly understood. Theoretical studies suggest that the activation of the nucleophilic water which hydrolyzes GTP needs a general base. Such a base could not be located in any of the many GTP-binding proteins. Here we present a unique type of linear free energy relationships that not only supports a mechanism for p21ras in which the substrate GTP itself acts as the catalytic base driving the GTPase reaction but can also help to explain why certain mutants of p21ras are oncogenic and others are not.

Topics: Amino Acid Sequence; Animals; Binding Sites; Catalysis; Cell Transformation, Neoplastic; Computer Simulation; Crystallography, X-Ray; Glutamine; GTP-Binding Proteins; GTPase-Activating Proteins; Guanosine Triphosphate; Hydrolysis; Insecta; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Binding; Protein Conformation; Proteins; Proto-Oncogene Proteins p21(ras); ras GTPase-Activating Proteins; Recombinant Fusion Proteins; Structure-Activity Relationship; Thermodynamics; Water

A key event for Ras transformation involves the direct physical association between Ras and the Raf-1 kinase. This interaction promotes both Raf translocation to the plasma membrane and activation of Raf kinase activity. Although substantial experimental evidence has demonstrated that Raf residues 51-131 alone are sufficient for Ras binding, conflicting observations have suggested that the Raf cysteine-rich domain (residues 139-184) may also be important for interaction with Ras. To clarify the role of the Raf cysteine-rich domain in Ras-Raf binding, we have compared the ability of two distinct Raf fragments to interact with Ras using both in vitro Ras binding and in vivo Ras inhibition assays. First, we determined that both Raf sequences 2-140 and 139-186 (designated Raf-Cys) showed preferential binding to active, GTP-bound Ras in vitro. Second, we observed that Raf-Cys antagonized oncogenic Ras(Q61L)-mediated transactivation of Ras-responsive elements and focus-forming activity in NIH 3T3 cells and insulin-induced germinal vesicle breakdown in Xenopus laevis oocytes in vivo. This inhibitory activity suggests that Raf-Cys can interact with Ras in vivo. Taken together, these results suggest that Ras interaction with two distinct domains of Raf-1 may be important in Ras-mediated activation of Raf kinase activity.

Topics: 3T3 Cells; Animals; Catalysis; Cell Transformation, Neoplastic; Cysteine; Guanosine Triphosphate; Mice; Protein Binding; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; ras Proteins; Signal Transduction; Xenopus laevis

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

Vav and Dbl are members of a novel class of oncogene proteins that share significant sequence identity in a approximately 250-amino-acid domain, designated the Dbl homology domain. Although Dbl functions as a guanine nucleotide exchange factor (GEF) and activator of Rho family proteins, recent evidence has demonstrated that Vav functions as a GEF for Ras proteins. Thus, transformation by Vav and Dbl may be a consequence of constitutive activation of Ras and Rho proteins, respectively. To address this possibility, we have compared the transforming activities of Vav and Dbl with that of the Ras GEF, GRF/CDC25. As expected, GRF-transformed cells exhibited the same reduction in actin stress fibers and focal adhesions as Ras-transformed cells. In contrast, Vav- and Dbl-transformed cells showed the same well-developed stress fibers and focal adhesions observed in normal or RhoA(63L)-transformed NIH 3T3 cells. Furthermore, neither Vav- or Dbl-transformed cells exhibited the elevated levels of Ras-GTP (60%) observed with GRF-transformed cells. Finally, GRF, but not Vav or Dbl, induced transcriptional activation from Ras-responsive DNA elements (ets/AP-1, fos promoter, and kappa B). However, like Ras- and GRF-transformed cells, both Vav- and Dbl-transformed cells exhibited constitutively activated mitogen-activated protein kinases (MAPKs) (primarily p42MAPK/ERK2). Since kinase-deficient forms of p42MAPK/ERK2 and p44MAPK/ERK1 inhibited Dbl transformation, MAPK activation may be an important component of its transforming activity. Taken together, our observations indicate that Vav and Dbl transformation is not a consequence of Ras activation and instead may involve the constitutive activation of MAPKs.

Topics: 3T3 Cells; Actins; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Adhesion; Cell Transformation, Neoplastic; Drosophila Proteins; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Membrane Proteins; Mice; Oncogene Proteins; Proteins; Proto-Oncogene Proteins c-vav; Proto-Oncogene Proteins p21(ras); ras Guanine Nucleotide Exchange Factors; ras-GRF1; Retroviridae Proteins, Oncogenic; Signal Transduction

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

Ras proteins in mammalian cells cycle between a GTP-bound 'on' state and a GDP-bound 'off' state. Activation of Ras p21 results from the dissociation of tightly bound GDP and the exchange of bound GDP for GTP. A guanine nucleotide exchange factor is required for this activation. Activation promotes interaction with effector molecules and allows the signal to be transduced. In Saccharomyces cerevisiae, the function of guanine nucleotide exchange has been ascribed to the product of the CDC25 gene. The C-terminus domain of SDC25, a homologue of CDC25, can substitute for the CDC25 protein in yeast. We have demonstrated that the SDC25 C-terminus domain promotes GTP binding to Ras p21 in CHO cells. In the present study, we found that the stable expression of the SDC25 C-terminus domain induced transformation of NIH3T3 cells. Ras proteins in these tumorigenic cells were GTP bound. In addition, the coexpression of wild-type Ha-Ras protein with the SDC25 C-terminus was found to enhance the tumorigenic properties of the NIH3T3 cells. These results imply that, in subsets of human tumours, cellular Ras p21 might be found in its GTP-bound active form as a consequence of an oncogenic activation of a mammalian Ras guanine nucleotide exchange factor.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Cell Transformation, Neoplastic; Fungal Proteins; Gene Expression Regulation; Genes, ras; GTP-Binding Proteins; Guanosine Triphosphate; Mice; Molecular Sequence Data; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); rap GTP-Binding Proteins; Saccharomyces cerevisiae

Single turnover and equilibrium binding measurements on the interaction of Gly-12 and Pro-12 Ras.GTP with the catalytic domains of the GTPase-activating proteins, p120-GAP and neurofibromin, have been made utilizing fluorescent 2'(3')O-(N-methylanthraniloyl)-nucleotides. These have enabled the equilibrium dissociation constants (Kd) for their initial binding and the rate constants of the hydrolysis step to be measured. p120-GAP binds to both Ras proteins with a Kd of 17 microM, whereas neurofibromin binds to both Ras proteins with a Kd of 1 microM. Both p120-GAP and neurofibromin increased the rate constant of the GTP hydrolysis step of Pro-12 Ras, but the maximal activation at 30 degrees C was 120-fold and 560-fold, as compared with 70,000- and 52,000-fold, with Gly-12 Ras. The affinity with which p120-GAP and neurofibromin binds to either Gly-12 or Pro-12 Ras protein was decreased dramatically by increasing ionic strength caused by addition of NaCl. The rate constant of the cleavage step of hydrolysis catalyzed by neurofibromin increases with increasing ionic strength, whereas that catalyzed by p120-GAP appears to be unaffected. The high ionic strength within the cell might result in a much lower overall GTPase-activating protein activity than is measured under conditions of low ionic strength in vitro, with p120-GAP being more severely inhibited. The GTP hydrolysis rate of Pro-12 Ras is 2-fold faster than that of normal Ras. The low oncogenicity of Pro-12 ras is explained by a model in which the intrinsic rates of hydrolysis and exchange, as well as GTPase-activating protein- and exchange factor-stimulated rates, are determinants of the biological activity of Ras proteins in fibroblasts.

Topics: Cell Transformation, Neoplastic; Genes, ras; Glycine; GTP Phosphohydrolases; GTPase-Activating Proteins; Guanosine Triphosphate; Humans; Hydrolysis; Kinetics; Mutation; Neurofibromin 1; Osmolar Concentration; Proline; Proteins; ras GTPase-Activating Proteins

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

Plasma membrane fractions from normal, regenerating liver and the AS-30D ascites hepatocarcinoma exhibited a high degree of enrichment when a set of plasma membrane enzyme markers were studied in comparison to the ones associated to the mitochondrial and cytosolic compartments. While the (Ca2+, Mg2+)-ATPase observed for the plasma membrane fraction isolated from normal liver showed an activity of 1.2 mumoles/mg/min, the regenerating liver and the AS-30D plasma membrane fractions presented a much lower ATPase activity (0.3 and 0.22 mumoles/mg/min respectively). Despite the differences in ATPase activity observed between models, the plasma membrane fraction from the AS-30D hepatocarcinoma presented a calcium transport activity similar to the value observed for the normal system (5.9 and 5.5 nmoles Ca2+/mg/10 min, respectively). Interestingly, the ATP in equilibrium with Pi exchange experiments carried out with the different plasma membrane fractions revealed that the (Ca2+, Mg2+)-ATPase contained in the plasma membrane from the AS-30D cells shows an exchange activity of 26 nmoles ATP in equilibrium with Pi/mg/min, similar to the one observed fo the enzyme from normal liver (30 nmoles ATP in equilibrium with Pi/mg/min). Our results suggest that the plasma membrane from the transformed model presents a more efficient mechanism to regulate the movement of calcium through the calcium pump, with an optimum expenditure of energy.

Topics: Adenosine Triphosphate; Animals; Biological Transport, Active; Ca(2+) Mg(2+)-ATPase; Calcium; Calcium-Transporting ATPases; Cell Membrane; Cell Transformation, Neoplastic; Cytidine Triphosphate; Detergents; Guanosine Triphosphate; Hydrogen-Ion Concentration; Inosine Triphosphate; Kinetics; Liver; Liver Neoplasms, Experimental; Liver Regeneration; Male; Octoxynol; Polyethylene Glycols; Rats; Rats, Inbred Strains; Sodium Chloride; Vanadates

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 products of ras genes may function as GTP-binding signal transducers, but the nature of their targets is largely unknown. To define genetically the cellular effector(s) of ras in rat fibroblast transformation, somatic variants that suppress the nontransforming phenotype of v-H-ras effector domain mutations were sought. Variant cell lines perturbed in the ras effector pathway were recovered, and the properties of one suggest that the primary target of ras action may be altered. In this cell variant, no single residue in the ras protein effector domain must be wild type to bring about transformation. In parental rat cells, conservative substitutions are tolerated in six of nine residues. Functional interaction with the target may not require a high degree of structural specificity in the ras protein effector domain.

Topics: Alleles; Amino Acid Sequence; Animals; Cell Division; Cell Transformation, Neoplastic; Genes, ras; Guanosine Triphosphate; Molecular Sequence Data; Mutation; Oncogene Protein p21(ras); Proviruses; Rats

We have introduced point mutations in v-rasH to study their effects on biochemical and biological properties of the ras-encoded protein p21. Several of these mutant proteins do not bind GTP and thus lack GTPase activity, while others were shown to have their GTP binding reduced. We have introduced these ras mutants into NIH 3T3 fibroblastoid cells to study major parameters of clinical importance which are associated with neoplastic transformation, particularly MHC expression in cells, metastasis and tumorigenesis in both nude mice and immune competent mice. Our data show that certain mutations in v-ras differentially affect the expression of the transformed phenotype. Mutant ras molecules deficient in GTP binding fail to generate rapidly progressing tumors in immune competent mice, and not all morphologically transformed cells were capable of experimental metastasis. Cells transformed by certain v-ras mutants form tumors in immunocompetent mice and show reduced expression of MHC class-I antigens. Other cells are morphologically transformed and tumorigenic in athymic nude mice, but fail to form tumors in normal mice and show levels of MHC class-I antigen expression similar to non-transformed 3T3 cells. The inverse relationship between MHC class-I-antigen expression and the degree of transformation in fibroblastoid cells suggests that the ras gene product could be involved in regulating MHC expression.

Topics: Animals; Blotting, Southern; Cell Line; Cell Transformation, Neoplastic; Clone Cells; DNA, Neoplasm; Fibroblasts; Gene Expression Regulation, Neoplastic; Genes, MHC Class I; Genes, ras; GTP Phosphohydrolases; Guanosine Triphosphate; Major Histocompatibility Complex; Mice; Mice, Inbred BALB C; Mice, Nude; Mutagenesis; Neoplasm Metastasis; Neoplasms, Experimental; Proto-Oncogene Proteins p21(ras); Transfection

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)

Site-directed mutagenesis of the conserved sequence motifs of p21 generated a group of mutant p21s defective in GTP binding. Some of these mutants were highly transforming, whereas others were transformation defective. Among the latter group, we found two mutants, derived from the v-H-ras oncogene by substituting the asparagine-116 with tyrosine and isoleucine, that exhibited a trans-dominant activity of suppressing the transformed phenotype of NIH3T3 cells induced by a long terminal repeat-linked c-H-ras and a wild-type v-H-ras. They caused reduction of the colony-forming efficiency in soft agar (78% in c-ras-transformed cells; 55% in v-ras cells) and morphological reversion of ras transformants. Subclones of revertants expressed a great excess of mutant p21 relative to the c-ras p21 present in these cells. These mutants were not lethal to NIH3T3 cells. Apparently, defective proteins encoded by suppressor mutants sequestered vital targets for ras function. Suppressor mutants also induced morphological reversion of NIH3T3 cells transformed by src, fes/flp, sis, and fms oncogenes, suggesting that these oncogenes function upstream to ras in the signaling pathways. Cells transformed by mos and a chemical carcinogen were unaffected.

Topics: Animals; Base Sequence; Cell Line; Cell Transformation, Neoplastic; Chlorocebus aethiops; Consensus Sequence; Ethyl Methanesulfonate; Fibroblasts; Genes, Dominant; Genes, ras; GTP-Binding Proteins; Guanosine Triphosphate; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Oncogene Protein p21(ras); Oncogene Proteins; Oncogenes; Proto-Oncogene Proteins p21(ras); Signal Transduction; Suppression, Genetic

Mutational replacement of glutamine-227 with a leucine residue in the GTP-binding domain of the alpha subunit of GS (Q227L alpha S) reduces its ability to hydrolyse GTP and causes constitutive activation of the mutant protein. Expression in Swiss 3T3 fibroblasts of Q227L alpha S caused markedly increased basal adenylyl cyclase activity, enhanced intracellular cyclic AMP (cAMP) accumulation and increased mitogenic sensitivity in response to forskolin and the potent phosphodiesterase inhibitor Ro 20-1724. These results support a role for cAMP in the regulation of cell proliferation, and suggest that alterations in a G protein can directly modify the ability of cells to respond mitogenically to extracellular factors.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Animals; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cyclic AMP; Enzyme Activation; Glutamine; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hydrolysis; Immunoblotting; Leucine; Mitogens; Molecular Sequence Data; Mutation; Thionucleotides; Transfection

Topics: Cell Transformation, Neoplastic; Chemical Phenomena; Chemistry, Physical; GTP Phosphohydrolases; Guanosine Triphosphate; Humans; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); X-Ray Diffraction

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

Conformational, GTP binding, and GTP hydrolytic studies are carried out with synthetically prepared N-terminal 34 residue segments (residues 2-35) of p21 ras oncogenic (12-Val) and non-oncogenic (12-Gly) proteins. It was found that these N-terminal regions bind nucleotides through their phosphate groups, and that substitution of valine for glycine produces a more pronounced alpha-helical structure and decreases the conformational flexibility. The glycine containing peptide, when compared to the valine containing analog, catalyses the hydrolysis of GTP 6 times more efficiently. Results suggest that restriction of conformational adaptation may contribute to the transforming capacity of the Val-12 p21 protein.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Cell Transformation, Neoplastic; Circular Dichroism; Glycine; Guanosine Triphosphate; Hydrolysis; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Neoplasm Proteins; Oncogene Protein p21(ras); Oncogene Proteins, Viral; Peptide Fragments; Protein Conformation; Structure-Activity Relationship; Valine

To identify the role of ras oncogene and p21 in the coupling mechanism of GTP-binding proteins to adenylate cyclase, we used v-Ki-ras transformed NIH/3T3 fibroblast cells. In the previous study, we investigated that NaF, cholera toxin and forskolin remarkably enhanced the adenylate cyclase activity in transformed cells compared to normal NIH/3T3 cells. In the present study, adenylate cyclase was more enhanced by GTP gamma S in transformed cells than in normal cells. It was considered that p21 plays enhancing role in coupling of GTP-binding proteins to adenylate cyclase. Further, as measured by the degree of [32P] ADP-ribosylation of GTP-binding proteins by cholera toxin and pertussis toxin respectively, the amount of Gs (46 kDa) was almost equal in both cells, while the amount of Gi (41 kDa) in transformant was about one third of that in normal cells. This difference seems to be reflected in either the biological situations or the quantities of Gi. Our data suggest that v-Ki-ras transformation resulted in the decrease of Gi protein so that the inhibitory regulation on adenylate cyclase relatively becomes low and then stimulatory influence of Gs seems to be enhanced.

Topics: Adenosine Diphosphate Ribose; Adenylyl Cyclases; Animals; Cell Line; Cell Transformation, Neoplastic; Fibroblasts; Genes, ras; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Kinetics; Mice; Molecular Weight; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Thionucleotides

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

Single amino acid changes were introduced into normal (non-oncogenic) and activated forms of the human H-ras protein at a position (residue 116) proposed on structural grounds to represent a contact site with guanine nucleotides. Substitutions at this site could significantly reduce the ability of both forms to bind and hydrolyze guanosine 5'-triphosphate; these substitutions, however, did not necessarily diminish the transforming capacity of activated derivatives. One substitution that severely impairs these functions activated the transforming potential of the otherwise normal polypeptide.

Topics: Amino Acid Sequence; Cell Transformation, Neoplastic; DNA; Guanosine Triphosphate; Humans; Neoplasm Proteins; Oncogene Protein p21(ras); Oncogenes

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

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Transformation, Neoplastic; Fibroblasts; Gene Expression Regulation; GTP Phosphohydrolases; Guanosine Triphosphate; Humans; Mice; Neoplasm Proteins; Protein Conformation; Proto-Oncogene Proteins p21(ras); Proto-Oncogenes; Transfection

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

As many as 40 distinct oncogenes of viral and cellular origin have been identified to date. Many of these genes can be grouped into functional classes on the basis of their effects on cellular phenotype. These groupings suggest a small number of mechanisms of action of the oncogene-encoded proteins. Some data suggest that, in the cytoplasm, these proteins may regulate levels of critical second messenger molecules; in the nucleus, these proteins may modulate the activity of the cell's transcriptional machinery. Many of the gene products can also be related to a signaling pathway that determines the cell's response to growth-stimulating factors. Because some of these genes are expressed in nongrowing, differentiated cells, the encoded proteins may in certain tissues mediate functions that are unrelated to cellular growth control.

Topics: Animals; Birds; Cell Nucleus; Cell Transformation, Neoplastic; Chickens; Cytoplasm; Deltaretrovirus; DNA Tumor Viruses; Drosophila; Epidermal Growth Factor; Growth Substances; Guanosine Triphosphate; Humans; Mutation; Neoplasms; Oncogenes; Platelet-Derived Growth Factor; Polyomavirus; Proto-Oncogenes; Rats; Repetitive Sequences, Nucleic Acid; Retroviridae; Simian virus 40; Transcription, Genetic

The high prevalence of ras oncogenes in human tumors has given increasing impetus to efforts aimed at elucidating the structure and function of their p21 products. To identify functionally important domains of the p21 protein, antibodies were generated against synthetic peptides corresponding to various regions of the protein. Antibodies directed against a synthetic peptide fragment corresponding to amino acid residues 161 to 176 in the carboxy-terminal region of the H-ras-encoded p21 molecule specifically recognized H-ras-encoded p21 proteins. This antibody was also shown to strikingly and specifically inhibit the guanine nucleotide-binding function of the p21 protein. The inability of p21 protein to bind guanine nucleotides was associated with a lack of autophosphorylation or GTPase activities. These studies suggest that a region toward its carboxy terminus is directly or indirectly involved in the guanine nucleotide-binding function of the p21 molecule.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Binding Sites; Cell Transformation, Neoplastic; Cells, Cultured; Genes; Guanosine Triphosphate; Mice; Oncogenes; Phosphorylation; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras)

An antibody (anti-p21ser) was raised against a ras p21-related synthetic peptide and was able to recognize specifically the substitution of serine for glycine at amino acid 12 of p21. This substitution causes oncogenic activation of p21. Anti-p21ser was found to immunoprecipitate v-Ki-ras p21 and to strongly inhibit its ability to autophosphorylate and to bind GTP in an immunoabsorption assay. Furthermore, binding of the antibody to p21 was specifically inhibited by GTP or GDP, suggesting that amino acids around position 12 are part of the GTP/GDP binding site. These results, taken together with the observation that the microinjection of anti-p21ser into cells transformed by v-Ki-ras p21 causes a transient reversion of the cells to a normal phenotype [Feramisco, J. R., Clark, R., Wong, G., Arnheim, N., Milley, R. & McCormick, F. (1985) Nature (London) 314, 639-642], support the idea that interaction of p21 with guanine nucleotides is crucial to the transforming function of this protein.

Topics: Amino Acid Sequence; Antibodies; Cell Transformation, Neoplastic; Glycine; Guanosine Triphosphate; Humans; Kinetics; Neoplasm Proteins; Oncogenes; Phenotype; Proto-Oncogene Proteins p21(ras); Serine

Topics: Adenylyl Cyclases; Animals; Cell Transformation, Neoplastic; Cyclic AMP; Drosophila; Guanosine Triphosphate; Humans; Oncogenes; Rodentia; Yeasts

Topics: Animals; Annexins; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA; Epidermal Growth Factor; ErbB Receptors; Guanosine Triphosphate; Humans; Membrane Proteins; Neoplasm Proteins; Nucleoproteins; Oncogene Protein pp60(v-src); Oncogenes; Phosphotyrosine; Platelet-Derived Growth Factor; Protein Binding; Protein Biosynthesis; Protein Kinases; Receptors, Cell Surface; Retroviridae; Tyrosine; 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

Spontaneous transformation of RL-PR-C hepatocytes leads to alterations in the adenylate cyclase complex which include a lower than normal basal level of activity, a loss of sensitivity to exogenous GTP, and a decreased sensitivity to isoproterenol. Both normal and transformed membranes possess substantial GTPase activity. Treatment of transformed hepatocyte membranes with either isoproterenol plus GMP or with cholera toxin, under conditions that displace tightly bound GDP, restored the GTP effect on adenylate cyclase, and eliminated the lag in the activation by guanyl-5'-yl-imidodiphosphate. Such pretreatment also enhanced guanine nucleotide effects on the adenylate cyclase of normal hepatocytes. These results are explainable on the basis that transformation increases adenylate cyclase-associated GTPase activity, and increases occupancy of nucleotide regulatory sites by inactive or inhibitory guanine nucleotides, e.g., GDP. Seemingly, both catecholamines and cholera toxin promote an exchange reaction at the regulatory sites, resulting in clearance of these sites of inhibitory nucleotides.

Topics: Adenylyl Cyclases; Animals; Cell Transformation, Neoplastic; Cholera Toxin; Clone Cells; Dose-Response Relationship, Drug; Guanine Nucleotides; Guanosine Monophosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Isoproterenol; Liver; Rats

Topics: Animals; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cloning, Molecular; Genes, Viral; Guanosine Triphosphate; Kirsten murine sarcoma virus; Phosphorylation; Rats; Recombination, Genetic; Retroviridae; RNA, Viral; Viral Proteins

Topics: Adenylyl Cyclases; Animals; Cell Line; Cell Transformation, Neoplastic; Epinephrine; Fatty Acids, Nonesterified; Fatty Acids, Unsaturated; Fibroblasts; Guanosine Triphosphate; Lysophosphatidylcholines; Mice; Moloney murine leukemia virus; Rats

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Animals; Cell Line; Cell Transformation, Neoplastic; Enzyme Activation; Fibroblasts; Guanosine Triphosphate; Kidney; Kinetics; Leukemia Virus, Murine; Magnesium; Manganese; Moloney murine leukemia virus; Phosphorus Radioisotopes; Prostaglandins; Protein Binding; Rats; Species Specificity; Time Factors

Topics: Animals; Binding Sites; Cattle; Cell Line; Cell Transformation, Neoplastic; DNA; DNA-Directed RNA Polymerases; DNA, Viral; Guanosine Triphosphate; Haplorhini; Kidney; Nucleic Acid Denaturation; Protein Binding; Simian virus 40; Templates, Genetic; Thymus Gland

Topics: Base Sequence; Cell Transformation, Neoplastic; Coliphages; Cyclic AMP; Genes; Genetics, Microbial; Guanosine Triphosphate; Molecular Biology; Nucleotides; Phosphoric Monoester Hydrolases; Protein Biosynthesis; RNA; RNA, Transfer; Transcription, Genetic; Virus Replication

Topics: Adenosine Triphosphate; Animals; Autoradiography; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Cytosine Nucleotides; Deoxyribonucleotides; Guanosine Triphosphate; Hot Temperature; Kidney; Polyomavirus; Radiation Effects; Temperature; Thymine Nucleotides; Tritium

The colchicine-binding protein in procine-brain tissue is a dimer of molecular weight 110,000 that is believed to be the subunit of neuronal microtubules. Conditions are established under which the dimers aggregate with reproducible kinetics. This aggregation reaction, which is monitored by development of turbidity, has the following characteristics: (a) Colchicine inhibits development of turbidity; (b) the reaction inhibited by colchicine is reversed by long-wave ultraviolet irradiation; (c) the aggregation is temperature-dependent; (d) the reaction is nucleotide triphosphate-specific, being stimulated by 1 mM GTP; (e) the reaction appears to be specific for microtubule subunits since in the presence of other added proteins and in curde cell extracts, only microtubule subunits aggregate. On the basis of these criteria, we conclude that we have established an in vitro system for the aggregation of microtubule subunits that shares some of the properties characteristic of the in vivo assembly of cytoplasmic and spindle microtubules.

Topics: Animals; Brain; Cell Aggregation; Cell Differentiation; Cell Transformation, Neoplastic; Colchicine; Cytoplasm; Depression, Chemical; Electrophoresis, Polyacrylamide Gel; Female; Guanosine Triphosphate; In Vitro Techniques; Microtubules; Nerve Tissue Proteins; Nitrosourea Compounds; Nucleotides; Polymers; Protein Binding; Radiation Effects; Rats; Rats, Inbred Strains; Swine; Temperature; Time Factors; Ultraviolet Rays; Uterine Cervical Neoplasms