共查询到20条相似文献,搜索用时 31 毫秒
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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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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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Protein kinase C δ (PKCδ) modulates cell survival and apoptosis in diverse cellular systems. We recently reported that PKCδ functions as a critical anti-apoptotic signal transducer in cells containing activated p21Ras and results in the activation of AKT, thereby promoting cell survival. How PKCδ is regulated by p21Ras, however, remains incompletely understood. In this study, we show that PKCδ, as a transducer of anti-apoptotic signals, is activated by phosphotidylinositol 3′ kinase/phosphoinositide-dependent kinase 1 (PI3K–PDK1) to deliver the survival signal to Akt in the environment of activated p21Ras. PDK1 is upregulated in cells containing an activated p21Ras. Knock-down of PDK1, PKCδ, or AKT forces cells containing activated p21Ras to undergo apoptosis. PDK1 regulates PKCδ activity, and constitutive expression of PDK1 increases PKCδ activity in different cell types. Conversely, expression of a kinase-dead (dominant-negative) PDK1 significantly suppresses PKCδ activity. p21Ras-mediated survival signaling is therefore regulated by via a PI3K–AKT pathway, which is dependent upon both PDK1 and PKCδ, and PDK1 activates and regulates PKCδ to determine the fate of cells containing a mutated, activated p21Ras. 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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Lee JS Gil JE Kim JH Kim TK Jin X Oh SY Sohn YW Jeon HM Park HJ Park JW Shin YJ Chung YG Lee JB You S Kim H 《Biochemical and biophysical research communications》2008,365(3):496-502
Here, we show that H-rasV12 causes the p53-knockout mouse astrocytes (p53−/− astrocytes) to be transformed into brain cancer stem-like cells. H-rasV12 triggers the p53−/− astrocytes to express a Nestin and a Cd133, which are expressed in normal and cancer neural stem cells. H-rasV12 also induces the formation of a single cell-derived neurosphere under neural stem cell culture conditions. Furthermore, H-rasV12-overexpressing p53−/− astrocytes (p53−/−ast-H-rasV12) possess an in vitro self-renewal capacity, and are aberrantly differentiated into Tuj1-positve neurons both in vitro and in vivo. Amongst a variety of Ras-mediated canonical signaling pathways, we demonstrated that the MEK/ERK signaling pathway is responsible for neurosphere formation in p53-deficient astrocytes, whereas the PI3K/AKT signaling pathway is involved in oncogenic transformation in these cells. These findings suggest that the activation of Ras signaling pathways promotes the generation of brain cancer stem-like cells from p53-deficient mouse astrocytes by changing cell fate and transforming cell properties. 相似文献
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Loss of the cell polarity gene could cooperate with oncogenic Ras to drive tumor growth and invasion, which critically depends on the c-Jun N-terminal Kinase (JNK) signaling pathway in Drosophila. By performing a genetic screen, we have identified Src42A, the ortholog of mammalian Src, as a key modulator of both RasV12/lgl−/− triggered tumor invasion and loss of cell polarity gene-induced cell migration. Our genetic study further demonstrated that the Bendless (Ben)/dUev1a ubiquitin E2 complex is an essential regulator of Src42A-induced, JNK-mediated cell migration. Furthermore, we showed that ectopic Ben/dUev1a expression induced invasive cell migration along with increased MMP1 production in wing disc epithelia. Moreover, Ben/dUev1a could cooperate with RasV12 to promote tumor overgrowth and invasion. In addition, we found that the Ben/dUev1a complex is required for ectopic Src42A-triggered cell death and endogenous Src42A-dependent thorax closure. Our data not only provide a mechanistic insight into the role of Src in development and disease but also propose a potential oncogenic function for Ubc13 and Uev1a, the mammalian homologs of Ben and dUev1a. 相似文献
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Chronic inflammation is associated with epithelial to mesenchymal transition (EMT) and cancer progression however the relationship between inflammation and EMT remains unclear. Here, we have exploited zebrafish to visualize and quantify the earliest events during epithelial cell transformation induced by oncogenic HRasV12. Live imaging revealed that expression of HRasV12 in the epidermis results in EMT and chronic neutrophil and macrophage infiltration. We have developed an in vivo system to probe and quantify gene expression changes specifically in transformed cells from chimeric zebrafish expressing oncogenic HRasV12 using translating ribosomal affinity purification (TRAP). We found that the expression of genes associated with EMT, including slug, vimentin and mmp9, are enriched in HRasV12 transformed epithelial cells and that this enrichment requires the presence of neutrophils. An early signal induced by HRasV12 in epithelial cells is the expression of il-8 (cxcl8) and we found that the chemokine receptor, Cxcr2, mediates neutrophil but not macrophage recruitment to the transformed cells. Surprisingly, we also found a cell autonomous role for Cxcr2 signaling in transformed cells for both neutrophil recruitment and EMT related gene expression associated with Ras transformation. Taken together, these findings implicate both autocrine and paracrine signaling through Cxcr2 in the regulation of inflammation and gene expression in transformed epithelial cells. 相似文献
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Cancer genomes accumulate numerous genetic and epigenetic modifications. Yet, human cellular transformation can be accomplished by a few genetically defined elements. These elements activate key pathways required to support replicative immortality and anchorage independent growth, a predictor of tumorigenesis in vivo. Here, we provide evidence that the Hippo tumor suppressor pathway is a key barrier to Ras‐mediated cellular transformation. The Hippo pathway targets YAP1 for degradation via the βTrCP‐SCF ubiquitin ligase complex. In contrast, the Ras pathway acts oppositely, to promote YAP1 stability through downregulation of the ubiquitin ligase complex substrate recognition factors SOCS5/6. Depletion of SOCS5/6 or upregulation of YAP1 can bypass the requirement for oncogenic Ras in anchorage independent growth in vitro and tumor formation in vivo. Through the YAP1 target, Amphiregulin, Ras activates the endogenous EGFR pathway, which is required for transformation. Thus, the oncogenic activity of RasV12 depends on its ability to counteract Hippo pathway activity, creating a positive feedback loop, which depends on stabilization of YAP1. 相似文献
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Caveolin‐1 down‐regulation is required for Wnt5a‐Frizzled 2 signalling in Ha‐RasV12‐induced cell transformation
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Hsiu‐Kuan Lin Hsi‐Hui Lin Yu‐Wei Chiou Ching‐Lung Wu Wen‐Tai Chiu Ming‐Jer Tang 《Journal of cellular and molecular medicine》2018,22(5):2631-2643
Caveolin‐1 (Cav1) is down‐regulated during MK4 (MDCK cells harbouring inducible Ha‐RasV12 gene) transformation by Ha‐RasV12. Cav1 overexpression abrogates the Ha‐RasV12‐driven transformation of MK4 cells; however, the targeted down‐regulation of Cav1 is not sufficient to mimic this transformation. Cav1‐silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction‐related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I‐CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha‐RasV12‐inducing MK4 cells increased exosome‐like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I‐CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I‐CM (MK4+I‐EXs). Wnt5a, a downstream product of Ha‐RasV12, was markedly secreted by MK4+I‐CM and MK4+I‐EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha‐RasV12‐ and MK4+I‐CM‐induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down‐regulation, either by Ha‐RasV12 or targeted shRNA, increased frizzled‐2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I‐EXs in MDCK cells. These data suggest that Cav1‐dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha‐RasV12‐Wnt5a‐Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha‐RasV12‐driven cell transformation. 相似文献
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Zhiwei Wang Shadan Ali Sanjeev Banerjee Bin Bao Yiwei Li Asfar S. Azmi Murray Korc Fazlul H. Sarkar 《Journal of cellular physiology》2013,228(3):556-562
Pancreatic ductal adenocarcinoma (PDAC) is one of the most frequently diagnosed cancers and the fourth leading cause of cancer‐related death in the United States, suggesting that there is an urgent need to design novel strategies for achieving better treatment outcome of patients diagnosed with PDAC. Our previous study has shown that activation of Notch and NF‐κB play a critical role in the development of PDAC in the compound K‐RasG12D and Ink4a/Arf deficient transgenic mice. However, the exact molecular mechanism by which mutated K‐Ras and Ink4a/Arf deficiency contribute to progression of PDAC remains largely elusive. In the present study, we used multiple methods, such as real‐time RT‐PCR, Western blotting assay, and immunohistochemistry to gain further mechanistic insight. We found that the deletion of Ink4a/Arf in K‐RasG12D expressing mice led to high expression of PDGF‐D signaling pathway in the tumor and tumor‐derived cell line (RInk‐1 cells). Furthermore, PDGF‐D knock‐down in RInk‐1 cells resulted in the inhibition of pancreatosphere formation and down‐regulation of EZH2, CD44, EpCAM, and vimentin. Moreover, we demonstrated that epithelial–mesenchymal transition (EMT) was induced in the compound mice, which is linked with aggressiveness of PDAC. In addition, we demonstrated that tumors from compound transgenic mice have higher expression of cancer stem cell (CSC) markers. These results suggest that the acquisition of EMT phenotype and induction of CSC characteristics could be linked with the aggressiveness of PDAC mediated in part through the activation of PDGF‐D, signaling. J. Cell. Physiol. 228: 556–562, 2013. © 2012 Wiley Periodicals, Inc. 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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Mohammad Zafrullah Adriana Haimovitz-Friedman Richard Kolesnick 《Biochemical and biophysical research communications》2009,390(3):434-463
Whether kinase suppressor of Ras1 (KSR1) is an active kinase that phosphorylates c-Raf-1 or a scaffold that coordinates signaling along the Ras/ERK1 signaling module is actively debated. In this study, we generated a monoclonal antibody against a c-Raf-1 peptide containing phosphorylated Thr269, the putative target for KSR1 kinase activity. We show that this antibody detects Thr269-phosphorylated c-Raf-1 in A431 cells upon epidermal growth factor (EGF) stimulation, preceding MEK1 activation. Furthermore, this antibody detects in vitro phosphorylation of FLAG-c-Raf-1 and kinase-dead FLAG-c-Raf-1(K375M) by immunopurified KSR1, but fails to detect phosphorylation of FLAG-c-Raf-1(K375M/T269V), engineered with a Thr269 to valine substitution. To provide unequivocal evidence that KSR1 is a legitimate kinase, we purified KSR1 to homogeneity, confirmed by mass spectrometry, renatured it in-gel, and demonstrated that it phosphorylates BSA-conjugated c-Raf-1 peptide at Thr269. These studies add to emerging data validating KSR1 as a kinase that phosphorylates c-Raf-1. 相似文献
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Jessica K. Sawyer Zahra Kabiri Ruth A. Montague Scott R. Allen Rebeccah Stewart Sarah V. Paramore Erez Cohen Hamed Zaribafzadeh Christopher M. Counter Donald T. Fox 《PLoS genetics》2020,16(12)
Signal transduction pathways are intricately fine-tuned to accomplish diverse biological processes. An example is the conserved Ras/mitogen-activated-protein-kinase (MAPK) pathway, which exhibits context-dependent signaling output dynamics and regulation. Here, by altering codon usage as a novel platform to control signaling output, we screened the Drosophila genome for modifiers specific to either weak or strong Ras-driven eye phenotypes. Our screen enriched for regions of the genome not previously connected with Ras phenotypic modification. We mapped the underlying gene from one modifier to the ribosomal gene RpS21. In multiple contexts, we show that RpS21 preferentially influences weak Ras/MAPK signaling outputs. These data show that codon usage manipulation can identify new, output-specific signaling regulators, and identify RpS21 as an in vivo Ras/MAPK phenotypic regulator. 相似文献