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1.
Sequence analysis of 27 alleles of each of the three Ras-related genes in Drosophila melanogaster indicates that they all have low levels of polymorphism but may experience slightly different evolutionary pressures. No
amino acid replacement substitutions were indicated in any of the sequences, or in the sibling species D. simulans and D. mauritiana. The Dras1 gene, which is the major ras homologue in Drosophila, has less within-species variation in D. melanogaster relative to the amount of divergence from the sibling species than does Dras2, although the contrast was not significant by the HKA test. Dras2 appears to be maintaining two classes of haplotype in D. melanogaster, one of which is closer to the alleles observed in the sibling species, suggesting that this is not likely to be a pseudogene
despite the absence of a mutant phenotype. Although differences in level of expression may affect the function of the genes,
it is concluded that genetic variation in the Ras signal transduction pathways cannot be attributed to catalytic variation
in the Ras proteins.
Received: 5 November 1998 / Accepted: 26 March 1999 相似文献
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Marsha J. Miller Lise Rioux Gregory V. Prendergast Sarah Cannon Michael A. White Judy L. Meinkoth 《Molecular and cellular biology》1998,18(7):3718-3726
Ras mutants with the ability to interact with different effectors have played a critical role in the identification of Ras-dependent signaling pathways. We used two mutants, RasS35 and RasG37, which differ in their ability to bind Raf-1, to examine Ras-dependent signaling in thyroid epithelial cells. Wistar rat thyroid cells are dependent upon thyrotropin (TSH) for growth. Although TSH-stimulated mitogenesis requires Ras, TSH activates protein kinase A (PKA) and downregulates signaling through Raf and the mitogen-activated protein kinase (MAPK) cascade. Cells expressing RasS35, a mutant which binds Raf, or RasG37, a mutant which binds RalGDS, exhibited TSH-independent proliferation. RasS35 stimulated morphological transformation and anchorage-independent growth. RasG37 stimulated proliferation but not transformation as measured by these indices. TSH exerted markedly different effects on the Ras mutants and transiently repressed MAPK phosphorylation in RasS35-expressing cells. In contrast, TSH stimulated MAPK phosphorylation and growth in cells expressing RasG37. The Ras mutants, in turn, exerted differential effects on TSH signaling. RasS35 abolished TSH-stimulated changes in cell morphology and thyroglobulin expression, while RasG37 had no effect on these activities. Together, the data indicate that cross talk between Ras and PKA discriminates between distinct Ras effector pathways. 相似文献
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Isabel Fuentes-Calvo Piero Crespo Eugenio Santos José M. López-Novoa Carlos Martínez-Salgado 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2013,1833(12):2734-2744
In addition to their role as oncogenes, Ras GTPases are key regulators of cell function. There is a proven relationship between the signaling pathways of transforming growth factor-β1 (TGF- β1) and Ras GTPases. Each of the Ras isoforms (H, N and K) exhibits specific modulatory activity on different cellular pathways. Our purpose has been to study some of the mechanisms involved in the development of renal fibrosis, assessing the individual role of N-Ras in basal and TGF-β1-mediated extracellular matrix (ECM) synthesis, proliferation, and migration in immortalized N-Ras deficient fibroblasts (N-ras?/?). Compared to normal counterparts, fibroblasts deficient for N-Ras exhibited higher basal activity levels of phosphatidylinositol-3-kinase (PI3K)/Akt and MEK/Erk, accompanied by upregulated collagen synthesis and diminished proliferation and migration rates. We found that the absence of N-Ras did not affect TGF-β1-induced proliferation and migration, which required PI3K/Akt but not Erk1/2 activation. Similar effector pathway dependence was found for fibronectin and collagen type I expression.Our results indicate that N-Ras might contribute to renal fibrosis through the down-regulation of ECM synthesis and up-regulation proliferation and migration modulating Akt activation. N-Ras also regulates TGF-β1-induced collagen I and fibronectin expression through Erk-independent pathways. 相似文献
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Sara S. Tolsma Justin D. Cohen Lorna S. Ehrlich Noel P. Bouck 《Experimental cell research》1993,205(2)
Despite their familiar sensitivity to transformation by dominant-acting ras oncogenes, NIH/3T3 cells carry a ras suppressor. When tested by cell fusion they were able to suppress the anchorage-independent phenotype of both mouse and human cells transformed by activated H-ras or N-ras. This suppression occurred without a decrease in expression of the activated ras oncogene. Ras-transformed NIH/3T3 clones cured of their oncogene by benzamide treatment reverted to a non-transformed phenotype, but had lost the ability to suppress other ras transformants, indicating that their initial transformation was accompanied by suppressor loss. In hamster cells an active ras oncogene increased the rate of chromosome segregation by >100-fold. These results suggest that in vitro transformation of NIH/3T3 cells by ras may be more similar to multistep in vivo tumor development than previously suspected, involving not only expression of an active oncogene but also loss of a suppressor activity, perhaps induced by the clastogenic oncogene. 相似文献
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Two lung and two colon carcinoma cell lines of human origin, which contained the same activated rasK transforming gene, expressed abnormal species of p21 that were distinct from the p21 proteins expressed in normal human cells and other human carcinomas. The abnormal species of p21 expressed by three of these cell lines were indistinguishable from each other, but differed from the abnormal p21 expressed by one lung carcinoma cell line. NIH cells transformed by DNAs of these carcinomas expressed the same abnormal p21 species, indicating that these abnormal proteins were encoded by the activated rasK genes detected by transfection. These results indicate that transforming activity of rasK genes in human lung and colon carcinoma cell lines is activated by mutations which alter the structure of their gene products, and that activation of rasK genes can result from different molecular alterations in different individual neoplasms. 相似文献
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Toshiko Yamada-Okabe Rikuo Doi Hisafumi Yamada-Okabe 《Journal of cellular biochemistry》1996,61(2):172-181
Point mutation of the c-H-ras gene significantly increases cellular transforming activities of Ras. Since posttranslational modification and subsequent membrane localization are essential for the biological activities of Ras, we examined whether or not the mutation also affects these two factors. The normal (Gly12) or the transforming (Val12) c-H-ras gene was expressed in NIH3T3 cells using a metallothionein promoter. Expression of either type of Ras was efficiently induced by the cadmium treatment of these cells, and immunoprecipitation of metabolically labeled cell extracts revealed that both normal and transforming Ras were expressed as four differently migrating forms on SDS-polyacrylamide gels, two of which were slower migrating cytosolic precursors and the other two were faster migrating membrane-bound forms. There was no significant difference in half lives between normal and transforming Ras; however, posttranslational modification was quite different between the two types of Ras. Transforming Ras was processed and became membrane-bound forms much more efficiently than normal Ras. Interestingly, posttranslational modification and membrane localization of Ras was significantly inhibited when the c-myc oncogene was co-expressed with Ras. In contrast to the c-myc oncogene, expression of either wild type or mutant p53 did not affect the posttranslational modification of Ras, suggesting that the c-myc oncogene specifically impairs the posttranslational modification of Ras. © 1996 Wiley-Liss, Inc. 相似文献
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G. M. Truesdell C. Jones T. Holt G. Henderson M. B. Dickman 《Molecular & general genetics : MGG》1999,262(1):46-54
Ras is a low-molecular-weight guanine nucleotide (GDP/GTP)-binding protein that transduces signals for growth and differentiation
in eukaryotes. In mammals, the importance of Ras in regulating growth is underscored by the observation that activating mutations
in ras genes are found in many animal tumors. Colletotrichum trifolii is a filamentous fungal pathogen of alfalfa which causes anthracnose disease. To investigate signaling pathways that regulate
growth and development in this fungus, a gene encoding a Ras homolog (CT-Ras) was cloned from C. trifolii. CT-Ras exhibited extensive amino acid similarity to Ras proteins from higher and lower eukaryotes. A single amino acid change
resulting in mutationally activated CT-Ras induced cellular transformation of mouse (NIH 3T3) fibroblasts and tumor formation
in nu/nu mice. In Colletotrichum, mutationally activated CT-Ras induced abnormal hyphal proliferation and defects in polarized growth, and significantly reduced
differentiation in a nutrient-dependent manner. These results show that C. trifolii Ras is a functional growth regulator in both mammals and fungi, and demonstrate that proper regulation of Ras is required
for normal fungal growth and development.
Received: 20 October 1998 / Accepted: 23 April 1999 相似文献
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Naumov I Kazanov D Lisiansky V Starr A Aroch I Shapira S Kraus S Arber N 《Experimental cell research》2012,318(2):160-168
BackgroundFunctional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35–40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras.ResultsA recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our “gene therapy” approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce ~ 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by ~ 35% tumor progression in vivo in already established tumors.ConclusionsSelective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC. 相似文献
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The ras genes from yeast and mammalian cells were fused to plant expression promoters, and introduced into plant cells via Agrobacterium, to study their effect on cell growth and development. All introduced ras genes had a strong inhibitory effect on callus and shoot regeneration from plant tissues. This is consistent with earlier findings that heterologous ras genes were highly lethal to protoplasts following direct DNA uptake. These effects could not be reversed by increasing exogenous or endogenous cytokinin levels. These effects were also independent of the v-Ha-ras mutations in functionally important regions of Ras proteins such as effector-binding and membrane-binding sites. Similarly, co-transformation with the genes encoding the Ras-negative regulators, GTPase-activating protein and neurofibromin did not affect the ras inhibitory effect, indicating that the mechanism of ras inhibition of plant cells is not related to normal ras cellular functions. This conclusion was supported by further studies in which ras gene expression was modified using various promoters and antisense constructs. The introduced ras sequences remained fully inhibitory regardless of which promoters (inducible or tissue-specific) or which orientations (sense or antisense) were tested. This strongly suggests that the ras DNA sequence itself, rather than the Ras protein or ras mRNA, is directly involved in the inhibitory effect. The mechanism underlying this novel phenomenon remains unknown. Introduced ras genes may inhibit plant cell growth by inducing co-suppression of unknown endogenous ras or ras-related genes, thereby leading to the arrest of cell growth. 相似文献
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Réda Hadj-Slimane Perayot Pamonsinlapatham Jean-Philippe Herbeuval Christiane Garbay Yves Lepelletier Françoise Raynaud 《Cellular signalling》2010,22(8):1214-1221
Several cancers are treated by interferons α and γ in association with conventional chemotherapy due to the resistance observed with interferon treatment alone. The frequency of un-sensitive cancer depends on tumor origin and oncogenic genes. Preclinical studies have highlighted interferon resistance in many cancers such as colon carcinoma due to oncogenic Ras. However, the resistance mechanism remains elusive. Apoptosis and proliferation of Raswt and mutated RasV12 transformed colon carcinoma cells treated with several recombinant interferon combinations were analyzed by flow cytometer and immunoblot. Apoptotic pathways of resistant RasV12 cells were investigated using siRNA strategy to determine key proteins involved in this process. We show that interferons α and γ synergized to induce human Raswt colon carcinoma cell (HT29) apoptosis by caspases and PARP-1 cleavages in contrast to RasV12 mutated colon carcinoma cells (SW480, HT29 clone). However, RasV12 siRNA restored interferon sensitivity of RasV12-HT29 clone to apoptosis. Survivin siRNA increased interferon apoptosis in Raswt cells demonstrating the key role of this protein in cell survival. RasV12 mutation in HT29 clone neutralized the interferon effect on Survivin suppression and maintained high level of phospho-Aurora-B/Histone H3, which protected cells from apoptosis. SiRNA strategy against both Aurora-B and Survivin in RasV12 cells synergized to restore interferon -induced apoptosis. RasV12 cells are less sensitive than Raswt cells to interferon induced cell apoptosis due to a Survivin/Aurora-B survival alternative pathway. Taken together, these results may provide interest in siRNA-therapeutic strategy and diagnostic relevance for therapy. 相似文献
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Daniel Filchtinski Alma Rüppel Ingrid R. Vetter Julia M. Shifman 《Journal of molecular biology》2010,399(3):422-435
Ras is a small GTP-binding protein that is an essential molecular switch for a wide variety of signaling pathways including the control of cell proliferation, cell cycle progression and apoptosis. In the GTP-bound state, Ras can interact with its effectors, triggering various signaling cascades in the cell. In the GDP-bound state, Ras looses its ability to bind to known effectors. The interaction of the GTP-bound Ras (RasGTP) with its effectors has been studied intensively. However, very little is known about the much weaker interaction between the GDP-bound Ras (RasGDP) and Ras effectors. We investigated the factors underlying the nucleotide-dependent differences in Ras interactions with one of its effectors, Raf kinase. Using computational protein design, we generated mutants of the Ras-binding domain of Raf kinase (Raf) that stabilize the complex with RasGDP. Most of our designed mutations narrow the gap between the affinity of Raf for RasGTP and RasGDP, producing the desired shift in binding specificity towards RasGDP. A combination of our best designed mutation, N71R, with another mutation, A85K, yielded a Raf mutant with a 100-fold improvement in affinity towards RasGDP. The Raf A85K and Raf N71R/A85K mutants were used to obtain the first high-resolution structures of RasGDP bound to its effector. Surprisingly, these structures reveal that the loop on Ras previously termed the switch I region in the RasGDP·Raf mutant complex is found in a conformation similar to that of RasGTP and not RasGDP. Moreover, the structures indicate an increased mobility of the switch I region. This greater flexibility compared to the same loop in RasGTP is likely to explain the natural low affinity of Raf and other Ras effectors to RasGDP. Our findings demonstrate that an accurate balance between a rigid, high-affinity conformation and conformational flexibility is required to create an efficient and stringent molecular switch. 相似文献
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F. Shira Neuman-Silberberg Eyal Schejter F. Michael Hoffmann Ben-Zion Shilo 《Cell》1984,37(3):1027-1033
Three Drosophila genes homologous to the Ha-ras probe were isolated and mapped to positions 85D, 64B, and 62B on chromosome 3. Two of these genes (termed Dras1 and Dras2) were sequenced. In the case of Dras1, which contains multiple introns, a cDNA clone was isolated and sequenced. In the case of Dras2, the nucleotide sequence of the genomic clone was determined. Each gene codes for a protein with a predicted molecular weight of 21.6 kd. Alignment of the amino acid sequence of Dras1 with the vertebrate Ha-ras protein shows that at the amino terminus and central portion (residues 1–121 and 137–164) the two proteins are remarkably similar, and have an overall homology of 75%. The Dras2 gene lacks significant homology to the vertebrate counterpart at the extreme amino terminus and is homologous only between positions 28–120 and 139–161 (overall homology of 50%). This result suggests that the N terminus of p21 forms a distinct regulatory or functional domain. At the carboxy terminus, the major region of variability among the vertebrate ras proteins, the two Drosophila sequences also display considerable variability. However, both appear to be more similar to exon 4B of the Ki-ras gene. 相似文献
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《Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism》1996,1299(1):146-154
The effect of ras transformation (rasB fibroblasts) on basal and serum-stimulated diacylglcerol (DAG) composition and mass was examined over time with respect to changes in membrane phospholipid composition and ceramide mass. RasB cells vs. Nontransformed control cells (rasD and NR6) had chronically elevated DAG levels (up to 240 min) following serum stimulation, indicating a defect in the recovery phase of the intracellular DAG pulse. Ras transformation also had a dramatic effect on DAG composition. Molecular species analysis revealed that DAG from unstimulated rasB cells was enriched inthe Δ9 desaturase fatty acyl species (monoenoate 18:1 (n − 7) and 18:1 (n−9)), and depleted in arachidonic acid (20:4(n−6)). With the exception of glycerophosphoinositol (GPI), DAG remodeling paralleled the compositional alterations in individual phospholipid classes. Importantly, ras transformation altered the fatty acyl composition of sphingomyelin, a precursor to the ceramide second messenger. With the addition of serum, control cells (rasD) had a progressive increase in ceramide mass with levels approximately 5-fold higher by 240 min. In contrast, ceramide levels did not increase in rasB cells at either 4 or 240 min. These results demonstrate that ras-oncogene, in addition to its effects on DAG metabolism, can also abolish the cellular increase in ceramide mass in response to serum stimulation. Since DAG and ceramide may have opposing biological functions, the prolonged elevation of DAG and the suppression of ceramide levels would be consistent with an enhanced proliferative capacity. 相似文献
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p19ARF-independent induction of p53 and cell cycle arrest by Raf in murine keratinocytes 总被引:2,自引:0,他引:2
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In tumorigenesis of the skin, activated Ras co-operates with mutations that inactivate the tumour suppressor p53, but the molecular basis for this co-operation remains unresolved. Here we show that activation of the Raf/MAP kinase pathway in primary mouse keratinocytes leads to a p53 and p21Cip1-dependent cycle arrest and to terminal differentiation. Raf activation in keratinocytes lacking p53 or p21Cip1 genes leads to expression of differentiation markers, but the cells do not cease to proliferate. Thus, loss of p53 or p21Cip1 function is necessary to disable growth-inhibitory Raf/MAP kinase signalling. Activation of oncogenes, including Ras, has been reported to stabilize and activate p53 via induction of the tumour suppressor p19ARF. However, the response to Raf in p19ARF–/– keratinocytes was indistinguishable from wild-type controls. Thus, p19ARF is not essential for Raf-induced p53 induction and cell cycle arrest in keratinocytes, indicating that oncogenes engage p53 activity via multiple mechanisms. 相似文献
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V. G. Mitrofanov A. I. Chekunova E. S. Zelentsova L. N. Gauze G. N. Bakhtoyarov 《Cell and Tissue Biology》2011,5(5):440-443
The Dras1 gene was mapped by in situ hybridization to polytene chromosomes of several sibling species of the Drosophila virilis group and their hybrids. A 1037-bp fragment of Dras1 gene from the D. virilis genome was used as the probe. The gene sequence was localized in the region of a 25 A-B disk in chromosome 2 (in accordance
with the D. virilis polytene chromosome map (Gubenko and Evgen’ev, 1984). 相似文献
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Caenorhabditis elegans SUR-5, a Novel but Conserved Protein, Negatively Regulates LET-60 Ras Activity during Vulval Induction 总被引:1,自引:0,他引:1
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The let-60 ras gene acts in a signal transduction pathway to control vulval differentiation in Caenorhabditis elegans. By screening suppressors of a dominant negative let-60 ras allele, we isolated three loss-of-function mutations in the sur-5 gene which appear to act as negative regulators of let-60 ras during vulval induction. sur-5 mutations do not cause an obvious mutant phenotype of their own, and they appear to specifically suppress only one of the two groups of let-60 ras dominant negative mutations, suggesting that the gene may be involved in a specific aspect of Ras activation. Consistent with its negative function, overexpressing sur-5 from an extragenic array partially suppresses the Multivulva phenotype of an activated let-60 ras mutation and causes synergistic phenotypes with a lin-45 raf mutation. We have cloned sur-5 and shown that it encodes a novel protein. We have also identified a potential mammalian SUR-5 homolog that is about 35% identical to the worm protein. SUR-5 also has some sequence similarity to acetyl coenzyme A synthetases and is predicted to contain ATP/GTP and AMP binding sites. Our results suggest that sur-5 gene function may be conserved through evolution. 相似文献
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Ras proteins play important roles in development especially for cell proliferation and differentiation in various organisms. However, their functions in the most insect species are still not clear. We identified three ras cDNAs from the silk worm, Bombyx mori. These sequences corresponded to three Ras of Drosophila melanogaster, but not to three mammalian Ras (H-Ras, K-Ras, N-Ras). Subsequently, the expression profiles of ras were investigated by quantitative real-time PCR using whole body of individuals from the embryonic to adult stages, and various tissues of 4th and 5th instar larvae. Each of three Bombyx ras showed different expression patterns. We also showed membrane localization of their products. These results indicate that the three Bombyx Ras are functional and have different roles. 相似文献