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Sample records for ras signaling pathway

  1. Canonical RTK-Ras-ERK signaling and related alternative pathways

    PubMed Central

    Sundaram, Meera V.

    2013-01-01

    Receptor Tyrosine Kinase (RTK)-Ras-Extracellular signal-regulated kinase (ERK) signaling pathways control many aspects of C. elegans development and behavior. Studies in C. elegans helped elucidate the basic framework of the RTK-Ras-ERK pathway and continue to provide insights into its complex regulation, its biological roles, how it elicits cell-type appropriate responses, and how it interacts with other signaling pathways to do so. C. elegans studies have also revealed biological contexts in which alternative RTK- or Ras-dependent pathways are used instead of the canonical pathway. PMID:23908058

  2. Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway.

    PubMed

    Han, Chang Woo; Jeong, Mi Suk; Jang, Se Bok

    2017-09-16

    Ras proteins are small GTPases that serve as master moderators of a large number of signaling pathways involved in various cellular processes. Activating mutations in Ras are found in about one-third of cancers. H-REV107, a K-Ras binding protein, plays an important role in determining K-Ras function. H-REV107 is a member of the HREV107 family of class II tumor suppressor genes and a growth inhibitory Ras target gene that suppresses cellular growth, differentiation, and apoptosis. Expression of H-REV107 was strongly reduced in about 50% of human carcinoma cell lines. However, the specific molecular mechanism by which H-REV107 inhibits Ras is still unknown. In the present study, we suggest that H-REV107 forms a strong complex with activating oncogenic mutation Q61H K-Ras from various biochemical binding assays and modeled structures. In addition, the interaction sites between K-Ras and H-REV107 were predicted based on homology modeling. Here, we found that some structure-based mutants of the K-Ras disrupted the complex formation with H-REV107. Finally, a novel molecular mechanism describing K-Ras and H-REV107 binding is suggested and insights into new K-Ras effector target drugs are provided. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. A misexpression screen identifies genes that can modulate RAS1 pathway signaling in Drosophila melanogaster.

    PubMed Central

    Huang, A M; Rubin, G M

    2000-01-01

    Differentiation of the R7 photoreceptor cell is dependent on the Sevenless receptor tyrosine kinase, which activates the RAS1/mitogen-activated protein kinase signaling cascade. Kinase suppressor of Ras (KSR) functions genetically downstream of RAS1 in this signal transduction cascade. Expression of dominant-negative KSR (KDN) in the developing eye blocks RAS pathway signaling, prevents R7 cell differentiation, and causes a rough eye phenotype. To identify genes that modulate RAS signaling, we screened for genes that alter RAS1/KSR signaling efficiency when misexpressed. In this screen, we recovered three known genes, Lk6, misshapen, and Akap200. We also identified seven previously undescribed genes; one encodes a novel rel domain member of the NFAT family, and six encode novel proteins. These genes may represent new components of the RAS pathway or components of other signaling pathways that can modulate signaling by RAS. We discuss the utility of gain-of-function screens in identifying new components of signaling pathways in Drosophila. PMID:11063696

  4. Post-transcriptional regulation of the Ras-ERK/MAPK signaling pathway.

    PubMed

    Whelan, Jarrett T; Hollis, Sarah E; Cha, Dong Seok; Asch, Adam S; Lee, Myon-Hee

    2012-03-01

    The Ras-ERK/MAP (Mitogen-Activated Protein) kinase signaling pathway governs many cellular processes such as proliferation, differentiation, cell fate, homeostasis, and survival in all eukaryotes. Constitutive activation of the Ras-ERK/MAPK signaling pathway often leads to promotion of abnormal cell growth and tumorigenesis. Although the regulation of the Ras-ERK/MAPK signaling pathway by post-translational modification has been well elucidated, post-transcriptional regulations of this pathway are beginning to emerge in invertebrates and this work is extended to humans. In this review, we describe the conserved regulation of Ras-ERK/MAPK signaling by RNA-binding proteins (PUF, KH-domain, HuR, and LARP) and microRNAs (let-7 family miRNAs) and important implications for human diseases including cancers. Copyright © 2011 Wiley Periodicals, Inc.

  5. Genetic analysis of Ras signalling pathways in cell proliferation, migration and survival.

    PubMed

    Drosten, Matthias; Dhawahir, Alma; Sum, Eleanor Y M; Urosevic, Jelena; Lechuga, Carmen G; Esteban, Luis M; Castellano, Esther; Guerra, Carmen; Santos, Eugenio; Barbacid, Mariano

    2010-03-17

    We have used mouse embryonic fibroblasts (MEFs) devoid of Ras proteins to illustrate that they are essential for proliferation and migration, but not for survival, at least in these cells. These properties are unique to the Ras subfamily of proteins because ectopic expression of other Ras-like small GTPases, even when constitutively active, could not compensate for the absence of Ras proteins. Only constitutive activation of components of the Raf/Mek/Erk pathway was sufficient to sustain normal proliferation and migration of MEFs devoid of Ras proteins. Activation of the phosphatidylinositol 3-kinase (PI3K)/PTEN/Akt and Ral guanine exchange factor (RalGEF)/Ral pathways, either alone or in combination, failed to induce proliferation or migration of Rasless cells, although they cooperated with Raf/Mek/Erk signalling to reproduce the full response mediated by Ras signalling. In contrast to current hypotheses, Ras signalling did not induce proliferation by inducing expression of D-type Cyclins. Rasless MEFs had normal levels of Cyclin D1/Cdk4 and Cyclin E/Cdk2. However, these complexes were inactive. Inactivation of the pocket proteins or knock down of pRb relieved MEFs from their dependence on Ras signalling to proliferate.

  6. Mutant K-RAS Promotes Invasion and Metastasis in Pancreatic Cancer Through GTPase Signaling Pathways

    PubMed Central

    Padavano, Julianna; Henkhaus, Rebecca S; Chen, Hwudaurw; Skovan, Bethany A; Cui, Haiyan; Ignatenko, Natalia A

    2015-01-01

    Pancreatic ductal adenocarcinoma is one of the most aggressive malignancies, characterized by the local invasion into surrounding tissues and early metastasis to distant organs. Oncogenic mutations of the K-RAS gene occur in more than 90% of human pancreatic cancers. The goal of this study was to investigate the functional significance and downstream effectors of mutant K-RAS oncogene in the pancreatic cancer invasion and metastasis. We applied the homologous recombination technique to stably disrupt K-RAS oncogene in the human pancreatic cell line MiaPaCa-2, which carries the mutant K-RASG12C oncogene in both alleles. Using in vitro assays, we found that clones with disrupted mutant K-RAS gene exhibited low RAS activity, reduced growth rates, increased sensitivity to the apoptosis inducing agents, and suppressed motility and invasiveness. In vivo assays showed that clones with decreased RAS activity had reduced tumor formation ability in mouse xenograft model and increased survival rates in the mouse orthotopic pancreatic cancer model. We further examined molecular pathways downstream of mutant K-RAS and identified RhoA GTP activating protein 5, caveolin-1, and RAS-like small GTPase A (RalA) as key effector molecules, which control mutant K-RAS-dependent migration and invasion in MiaPaCa-2 cells. Our study provides rational for targeting RhoA and RalA GTPase signaling pathways for inhibition of pancreatic cancer metastasis. PMID:26512205

  7. The Ras-Erk-ETS-Signaling Pathway Is a Drug Target for Longevity

    PubMed Central

    Slack, Cathy; Alic, Nazif; Foley, Andrea; Cabecinha, Melissa; Hoddinott, Matthew P.; Partridge, Linda

    2015-01-01

    Summary Identifying the molecular mechanisms that underlie aging and their pharmacological manipulation are key aims for improving lifelong human health. Here, we identify a critical role for Ras-Erk-ETS signaling in aging in Drosophila. We show that inhibition of Ras is sufficient for lifespan extension downstream of reduced insulin/IGF-1 (IIS) signaling. Moreover, direct reduction of Ras or Erk activity leads to increased lifespan. We identify the E-twenty six (ETS) transcriptional repressor, Anterior open (Aop), as central to lifespan extension caused by reduced IIS or Ras attenuation. Importantly, we demonstrate that adult-onset administration of the drug trametinib, a highly specific inhibitor of Ras-Erk-ETS signaling, can extend lifespan. This discovery of the Ras-Erk-ETS pathway as a pharmacological target for animal aging, together with the high degree of evolutionary conservation of the pathway, suggests that inhibition of Ras-Erk-ETS signaling may provide an effective target for anti-aging interventions in mammals. Video Abstract PMID:26119340

  8. Disorders of dysregulated signal traffic through the RAS-MAPK pathway: phenotypic spectrum and molecular mechanisms

    PubMed Central

    Tartaglia, Marco; Gelb, Bruce D.

    2010-01-01

    RAS GTPases control a major signaling network implicated in several cellular functions, including cell fate determination, proliferation, survival, differentiation, migration, and senescence. Within this network, signal flow through the RAF-MEK-ERK pathway, the first identified mitogen-associated protein kinase (MAPK) cascade, mediates early and late developmental processes controlling morphology determination, organogenesis, synaptic plasticity and growth. Signaling through the RAS-MAPK cascade is tightly controlled, and its enhanced activation represents a well-known event in oncogenesis. Unexpectedly, in the past few years, inherited dysregulation of this pathway has been recognized as the cause underlying a group of clinically related disorders sharing facial dysmorphism, cardiac defects, reduced postnatal growth, ectodermal anomalies, variable cognitive deficits and susceptibility to certain malignancies as major features. These disorders are caused by heterozygosity for mutations in genes encoding RAS proteins, regulators of RAS function, modulators of RAS interaction with effectors or downstream signal transducers. Here, we provide an overview of the phenotypic spectrum associated with germline mutations perturbing RAS-MAPK signaling, the unpredicted molecular mechanisms converging towards the dysregulation of this signaling cascade, and major genotype-phenotype correlations. PMID:20958325

  9. Reciprocal regulatory interactions between the Notch and Ras signaling pathways in the Drosophila embryonic mesoderm.

    PubMed

    Carmena, Ana; Buff, Eugene; Halfon, Marc S; Gisselbrecht, Stephen; Jiménez, Fernando; Baylies, Mary K; Michelson, Alan M

    2002-04-15

    Convergent intercellular signals must be precisely integrated in order to elicit specific biological responses. During specification of muscle and cardiac progenitors from clusters of equivalent cells in the Drosophila embryonic mesoderm, the Ras/MAPK pathway--activated by both epidermal and fibroblast growth factor receptors--functions as an inductive cellular determination signal, while lateral inhibition mediated by Notch antagonizes this activity. A critical balance between these signals must be achieved to enable one cell of an equivalence group to segregate as a progenitor while its neighbors assume a nonprogenitor identity. We have investigated whether these opposing signals directly interact with each other, and we have examined how they are integrated by the responding cells to specify their unique fates. Our findings reveal that Ras and Notch do not function independently; rather, we have uncovered several modes of cross-talk between these pathways. Ras induces Notch, its ligand Delta, and the epidermal growth factor receptor antagonist, Argos. We show that Delta and Argos then synergize to nonautonomously block a positive autoregulatory feedback loop that amplifies a fate-inducing Ras signal. This feedback loop is characterized by Ras-mediated upregulation of proximal components of both the epidermal and fibroblast growth factor receptor pathways. In turn, Notch activation in nonprogenitors induces its own expression and simultaneously suppresses both Delta and Argos levels, thereby reinforcing a unidirectional inhibitory response. These reciprocal interactions combine to generate the signal thresholds that are essential for proper specification of progenitors and nonprogenitors from groups of initially equivalent cells.

  10. An Interdisciplinary Approach for Designing Kinetic Models of the Ras/MAPK Signaling Pathway.

    PubMed

    Reis, Marcelo S; Noël, Vincent; Dias, Matheus H; Albuquerque, Layra L; Guimarães, Amanda S; Wu, Lulu; Barrera, Junior; Armelin, Hugo A

    2017-01-01

    We present in this article a methodology for designing kinetic models of molecular signaling networks, which was exemplarily applied for modeling one of the Ras/MAPK signaling pathways in the mouse Y1 adrenocortical cell line. The methodology is interdisciplinary, that is, it was developed in a way that both dry and wet lab teams worked together along the whole modeling process.

  11. The PDZ Protein Canoe/AF-6 Links Ras-MAPK, Notch and Wingless/Wnt Signaling Pathways by Directly Interacting with Ras, Notch and Dishevelled

    PubMed Central

    Carmena, Ana; Speicher, Stephan; Baylies, Mary

    2006-01-01

    Over the past few years, it has become increasingly apparent that signal transduction pathways are not merely linear cascades; they are organized into complex signaling networks that require high levels of regulation to generate precise and unique cell responses. However, the underlying regulatory mechanisms by which signaling pathways cross-communicate remain poorly understood. Here we show that the Ras-binding protein Canoe (Cno)/AF-6, a PDZ protein normally associated with cellular junctions, is a key modulator of Wingless (Wg)/Wnt, Ras-Mitogen Activated Protein Kinase (MAPK) and Notch (N) signaling pathways cross-communication. Our data show a repressive effect of Cno/AF-6 on these three signaling pathways through physical interactions with Ras, N and the cytoplasmic protein Dishevelled (Dsh), a key Wg effector. We propose a model in which Cno, through those interactions, actively coordinates, at the membrane level, Ras-MAPK, N and Wg signaling pathways during progenitor specification. PMID:17183697

  12. Spatial Segregation of Ras Signaling

    PubMed Central

    Chang, Eric C.; Philips, Mark R.

    2010-01-01

    The Ras GTPases act as binary switches for signal transduction pathways that are important for growth regulation and tumorigenesis. Despite the biochemical simplicity of this switch, Ras proteins control multiple pathways, and the functions of the four mammalian Ras proteins are not overlapping. This raises an important question—how does a Ras protein selectively regulate a particular activity? One recently emerging model suggests that a single Ras protein can control different functions by acting in distinct cellular compartments. A critical test of this model is to identify pathways that are selectively controlled by Ras when it is localized to a particular compartment. A recent study has examined Ras signaling in the fission yeast Schizosaccharomyces pombe, which expresses only one Ras protein that controls two separate evolutionarily conserved pathways. This study demonstrates that whereas Ras localized to the plasma membrane selectively regulates a MAP kinase pathway to mediate mating pheromone signaling, Ras localized to the endomembrane activates a Cdc42 pathway to mediate cell polarity and protein trafficking. This study has provided unambiguous evidence for compartmentalized signaling of Ras. PMID:16931912

  13. Ral-GTPases mediate a distinct downstream signaling pathway from Ras that facilitates cellular transformation.

    PubMed Central

    Urano, T; Emkey, R; Feig, L A

    1996-01-01

    Ral proteins (RalA and RalB) comprise a distinct family of Ras-related GTPases (Feig and Emkey, 1993). Recently, Ral-GDS, the exchange factor that activates Ral proteins, has been shown to bind specifically to the activated forms of RasH, R-Ras and Rap1A, in the yeast two-hybrid system. Here we demonstrate that although all three GTPases have the capacity to bind Ral-GDS in mammalian cells, only RasH activates Ral-GDS. Furthermore, although constitutively activated Ra1A does not induce oncogenic transformation on its own, its expression enhances the transforming activities of both RasH and Raf. Finally, a dominant inhibitory form of RalA suppresses the transforming activities of both RasH and Raf. These results demonstrate that activation of Ral-GDS and thus its target, Ral, constitutes a distinct downstream signaling pathway from RasH that potentiates oncogenic transformation. Images PMID:8631302

  14. In TCR-Stimulated T-cells, N-ras Regulates Specific Genes and Signal Transduction Pathways

    PubMed Central

    Lynch, Stephen J.; Zavadil, Jiri; Pellicer, Angel

    2013-01-01

    It has been recently shown that N-ras plays a preferential role in immune cell development and function; specifically: N-ras, but not H-ras or K-ras, could be activated at and signal from the Golgi membrane of immune cells following a low level T-cell receptor stimulus. The goal of our studies was to test the hypothesis that N-ras and H-ras played distinct roles in immune cells at the level of the transcriptome. First, we showed via mRNA expression profiling that there were over four hundred genes that were uniquely differentially regulated either by N-ras or H-ras, which provided strong evidence in favor of the hypothesis that N-ras and H-ras have distinct functions in immune cells. We next characterized the genes that were differentially regulated by N-ras in T cells following a low-level T-cell receptor stimulus. Of the large pool of candidate genes that were differentially regulated by N-ras downstream of TCR ligation, four genes were verified in qRT-PCR-based validation experiments (Dntt, Slc9a6, Chst1, and Lars2). Finally, although there was little overlap between individual genes that were regulated by N-ras in unstimulated thymocytes and stimulated CD4+ T-cells, there was a nearly complete correspondence between the signaling pathways that were regulated by N-ras in these two immune cell types. PMID:23755101

  15. Peroxiredoxin II promotes hepatic tumorigenesis through cooperation with Ras/Forkhead box M1 signaling pathway.

    PubMed

    Park, Y-H; Kim, S-U; Kwon, T-H; Kim, J-M; Song, I-S; Shin, H-J; Lee, B-K; Bang, D-H; Lee, S-J; Lee, D-S; Chang, K-T; Kim, B-Y; Yu, D-Y

    2016-07-07

    The current study was carried out to define the involvement of Peroxiredoxin (Prx) II in progression of hepatocellular carcinoma (HCC) and the underlying molecular mechanism(s). Expression and function of Prx II in HCC was determined using H-ras(G12V)-transformed HCC cells (H-ras(G12V)-HCC cells) and the tumor livers from H-ras(G12V)-transgenic (Tg) mice and HCC patients. Prx II was upregulated in H-ras(G12V)-HCC cells and H-ras(G12V)-Tg mouse tumor livers, the expression pattern of which highly similar to that of forkhead Box M1 (FoxM1). Moreover, either knockdown of FoxM1 or site-directed mutagenesis of FoxM1-binding site of Prx II promoter significantly reduced Prx II levels in H-ras(G12V)-HCC cells, indicating FoxM1 as a direct transcription factor of Prx II in HCC. Interestingly, the null mutation of Prx II markedly decreased the number and size of tumors in H-ras(G12V)-Tg livers. Consistent with this, knockdown of Prx II in H-ras(G12V)-HCC cells reduced the expression of cyclin D1, cell proliferation, anchorage-independent growth and tumor formation in athymic nude mice, whereas overexpression of Prx II increased or aggravated the tumor phenotypes. Importantly, the expression of Prx II was correlated with that of FoxM1 in HCC patients. The activation of extracellular signal-related kinase (ERK) pathway and the expression of FoxM1 and cyclin D1 were highly dependent on Prx II in H-ras(G12V)-HCC cells and H-ras(G12V)-Tg livers. Prx II is FoxM1-dependently-expressed antioxidant in HCC and function as an enhancer of Ras(G12V) oncogenic potential in hepatic tumorigenesis through activation of ERK/FoxM1/cyclin D1 cascade.

  16. BRAF vs RAS oncogenes: are mutations of the same pathway equal? differential signalling and therapeutic implications

    PubMed Central

    Oikonomou, Eftychia; Koustas, Evangelos; Goulielmaki, Maria; Pintzas, Alexander

    2014-01-01

    As the increased knowledge of tumour heterogeneity and genetic alterations progresses, it exemplifies the need for further personalized medicine in modern cancer management. Here, the similarities but also the differential effects of RAS and BRAF oncogenic signalling are examined and further implications in personalized cancer diagnosis and therapy are discussed. Redundant mechanisms mediated by the two oncogenes as well as differential regulation of signalling pathways and gene expression by RAS as compared to BRAF are addressed. The implications of RAS vs BRAF differential functions, in relevant tumour types including colorectal cancer, melanoma, lung cancer are discussed. Current therapeutic findings and future viewpoints concerning the exploitation of RAS-BRAF-pathway alterations for the development of novel therapeutics and efficient rational combinations, as well as companion tests for relevant markers of response will be evaluated. The concept that drug-resistant cells may also display drug dependency, such that altered dosing may prevent the emergence of lethal drug resistance posed a major therapy hindrance. PMID:25361007

  17. The effect of aquaporin 5 overexpression on the Ras signaling pathway

    SciTech Connect

    Woo, Janghee; Lee, Juna; Kim, Myoung Sook; Jang, Se Jin; Sidransky, David; Moon, Chulso

    2008-03-07

    Human aquaporin 5 (AQP5) has been shown to be overexpressed in multiple cancers, such as pancreatic cancer and colon cancer. Furthermore, it has been reported that ectopic expression of AQP5 leads to many phenotypic changes characteristic of transformation. However, the biochemical mechanism leading to transformation in AQP5-overexpressing cells has not been clearly elucidated. In this report, the overexpression of AQP5 in NIH3T3 cells demonstrated a significant effect on Ras activity and, thus, cell proliferation. Furthermore, this influence was shown to be mediated by phosphorylation of the PKA consensus site of AQP5. This is the first evidence demonstrating an association between AQP5 and a signaling pathway, namely the Ras signal transduction pathway, which may be the basis of the oncogenic properties seen in AQP-overexpressing cells.

  18. Epac activation induces histone deacetylase nuclear export via a Ras-dependent signalling pathway.

    PubMed

    Métrich, Mélanie; Laurent, Anne-Coline; Breckler, Magali; Duquesnes, Nicolas; Hmitou, Isabelle; Courillau, Delphine; Blondeau, Jean-Paul; Crozatier, Bertrand; Lezoualc'h, Frank; Morel, Eric

    2010-10-01

    Epac (Exchange protein directly activated by cAMP) is a sensor for cAMP and represents a novel mechanism for governing cAMP signalling. Epac is a guanine nucleotide exchange factor (GEF) for the Ras family of small GTPases, Rap. Previous studies demonstrated that, in response to a prolonged beta-adrenergic stimulation Epac induced cardiac myocyte hypertrophy. The aim of our study was to further characterize Epac downstream effectors involved in cardiac myocyte growth. Here, we found that Epac led to the activation of the small G protein H-Ras in primary neonatal cardiac myocytes. A Rap GTPase activating protein (RapGAP) partially inhibited Epac-induced H-Ras activation. Interestingly, we found that H-Ras activation involved the GEF domain of Epac. However, Epac did not directly induce exchange activity on this small GTPase protein. Instead, the effect of Epac on H-Ras activation was dependent on a signalling cascade involving phospholipase C (PLC)/inositol 1,3,5 triphosphate receptor (IP3R) and an increase intracellular calcium. In addition, we found that Epac activation induced histone deacetylase type 4 (HDAC4) translocation. Whereas HDAC5 alone was unresponsive to Epac, it became responsive to Epac in the presence of HDAC4 in COS cells. Consistent with its effect on HDAC cytoplasmic shuttle, Epac activation also increased the prohypertrophic transcription factor MEF2 in a CaMKII dependent manner in primary cardiac myocytes. Thus, our data show that Epac activates a prohypertrophic signalling pathway which involves PLC, H-Ras, CaMKII and HDAC nuclear export. Copyright 2010 Elsevier Inc. All rights reserved.

  19. c-Ki-ras oncogene amplification and FGF2 signaling pathways in the mouse Y1 adrenocortical cell line.

    PubMed

    Forti, Fábio L; Costa, Erico T; Rocha, Kátia M; Moraes, Miriam S; Armelin, Hugo A

    2006-06-01

    The mouse Y1 adrenocortical tumor cell line is highly responsive to FGF2-(Fibroblast Growth Factor 2) and possesses amplified and over-expressed c-Ki-ras proto-oncogene. We previously reported that this genetic lesion leads to high constitutive levels of activation of the c-Ki-Ras-GTP-->PI3K-->Akt signaling pathway (Forti et al. 2002). On the other hand, activation levels of another important pathway downstream of c-Ki-Ras-GTP, namely, Raf-->MEK-->ERK, remain strictly dependent on FGF2 stimulation (Rocha et al. 2003). Here we show that, first, FGF2 transiently up-regulates the c-Ki-Ras-GTP-->PI3K-->Akt pathway, in spite of its high basal levels. Second, c-Ki-Ras-GTP transient up-regulation likely underlies activation of the ERK1/2 pathway by FGF2. Third, c-Ki-Ras-GTP high basal levels suppress activation of the c-H-Ras onco-protein. But, Y1 cells, expressing dominant negative mutant RasN17, display a rapid and transient up-regulation of c-H-Ras-GTP upon FGF2 treatment. Elucidation of FGF2-signaling pathways in Y1 tumor cells can uncover new targets for drug development of interest in cancer therapy.

  20. The molecular basis of viral oncolysis: usurpation of the Ras signaling pathway by reovirus.

    PubMed Central

    Strong, J E; Coffey, M C; Tang, D; Sabinin, P; Lee, P W

    1998-01-01

    NIH-3T3 cells, which are resistant to reovirus infection, became susceptible when transformed with activated Sos or Ras. Restriction of reovirus proliferation in untransformed NIH-3T3 cells was not at the level of viral gene transcription, but rather at the level of viral protein synthesis. An analysis of cell lysates revealed that a 65 kDa protein was phosphorylated in untransformed NIH-3T3 cells, but only after infection with reovirus. This protein was not phosphorylated in infected or uninfected transformed cells. The 65 kDa protein was determined to be the double-stranded RNA-activated protein kinase (PKR), whose phosphorylation leads to translation inhibition. Inhibition of PKR phosphorylation by 2-aminopurine, or deletion of the Pkr gene, led to drastic enhancement of reovirus protein synthesis in untransformed cells. The emerging picture is one in which early viral transcripts trigger PKR phosphorylation in untransformed cells, which in turn leads to inhibition of translation of viral genes; this phosphorylation event is blocked by an element(s) in the Ras pathway in the transformed cells, allowing viral protein synthesis to ensue. The usurpation of the Ras signaling pathway therefore constitutes the basis of reovirus oncolysis. PMID:9628872

  1. siRNA blocking the RAS signalling pathway and inhibits the growth of oesophageal squamous cell carcinoma in nude mice.

    PubMed

    Wang, Xinjie; Zheng, Yuling; Fan, Qingxia; Zhang, Xudong; Shi, Yonggang

    2014-12-01

    The aim of this study was to study RAS-siRNA blocking RAS pathway and suppressing cell growth in human oesophageal squamous cell carcinoma in nude mice. The methods in this study was to construct RAS-siRNA expression vector, establish 40 oesophageal squamous cell carcinoma xenograft animal models and divided them into five groups: control group, siRNA control group, RAS-siRNA group, paclitaxel group and RAS-siRNA and paclitaxel group. We observed tumour growth in nude mice, studied histology by HE staining, tumour growth inhibition by TUNEL assay and detected the RAS, MAPK and cyclin D1 protein expression by immunohistochemistry and western blot. We have obtained the following results: (i) successfully established animal models; (ii) nude mice in each group after treatment inhibited tumour volume was significantly reduced compared with the control group (p < 0.05); (iii) compared with the control group, the number of apoptotic cells were significantly increased in the siRNA control group and the RAS-siRNA group, and the number of apoptosis cells in the paclitaxel and RAS-siRNA group is significantly most than the paclitaxel group and RAS-siRNA group (p < 0.05); and (iv) after treatment, RAS, MAPK and cyclin D1 expression in five groups was decreasing gradually. After adding paclitaxel, the protein expression in the paclitaxel and RAS-siRNA group was significantly lower than that of paclitaxel group, negative control and paclitaxel group (p < 0.05). We therefore conclude that RAS-siRNA can block the RAS signal transduction pathway, reduce the activity of tumour cells, arrest tumour cell cycle, promote apoptosis, inhibit cell proliferation and increase tumour cell sensitivity to chemotherapeutic drugs.

  2. Plasma membrane regulates Ras signaling networks

    PubMed Central

    Chavan, Tanmay Sanjeev; Muratcioglu, Serena; Marszalek, Richard; Jang, Hyunbum; Keskin, Ozlem; Gursoy, Attila; Nussinov, Ruth; Gaponenko, Vadim

    2015-01-01

    Ras GTPases activate more than 20 signaling pathways, regulating such essential cellular functions as proliferation, survival, and migration. How Ras proteins control their signaling diversity is still a mystery. Several pieces of evidence suggest that the plasma membrane plays a critical role. Among these are: (1) selective recruitment of Ras and its effectors to particular localities allowing access to Ras regulators and effectors; (2) specific membrane-induced conformational changes promoting Ras functional diversity; and (3) oligomerization of membrane-anchored Ras to recruit and activate Raf. Taken together, the membrane does not only attract and retain Ras but also is a key regulator of Ras signaling. This can already be gleaned from the large variability in the sequences of Ras membrane targeting domains, suggesting that localization, environment and orientation are important factors in optimizing the function of Ras isoforms. PMID:27054048

  3. Plasma membrane regulates Ras signaling networks.

    PubMed

    Chavan, Tanmay Sanjeev; Muratcioglu, Serena; Marszalek, Richard; Jang, Hyunbum; Keskin, Ozlem; Gursoy, Attila; Nussinov, Ruth; Gaponenko, Vadim

    2015-01-01

    Ras GTPases activate more than 20 signaling pathways, regulating such essential cellular functions as proliferation, survival, and migration. How Ras proteins control their signaling diversity is still a mystery. Several pieces of evidence suggest that the plasma membrane plays a critical role. Among these are: (1) selective recruitment of Ras and its effectors to particular localities allowing access to Ras regulators and effectors; (2) specific membrane-induced conformational changes promoting Ras functional diversity; and (3) oligomerization of membrane-anchored Ras to recruit and activate Raf. Taken together, the membrane does not only attract and retain Ras but also is a key regulator of Ras signaling. This can already be gleaned from the large variability in the sequences of Ras membrane targeting domains, suggesting that localization, environment and orientation are important factors in optimizing the function of Ras isoforms.

  4. Actin-induced hyperactivation of the Ras signaling pathway leads to apoptosis in Saccharomyces cerevisiae.

    PubMed

    Gourlay, C W; Ayscough, K R

    2006-09-01

    Recent research has revealed a conserved role for the actin cytoskeleton in the regulation of aging and apoptosis among eukaryotes. Here we show that the stabilization of the actin cytoskeleton caused by deletion of Sla1p or End3p leads to hyperactivation of the Ras signaling pathway. The consequent rise in cyclic AMP (cAMP) levels leads to the loss of mitochondrial membrane potential, accumulation of reactive oxygen species (ROS), and cell death. We have established a mechanistic link between Ras signaling and actin by demonstrating that ROS production in actin-stabilized cells is dependent on the G-actin binding region of the cyclase-associated protein Srv2p/CAP. Furthermore, the artificial elevation of cAMP directly mimics the apoptotic phenotypes displayed by actin-stabilized cells. The effect of cAMP elevation in inducing actin-mediated apoptosis functions primarily through the Tpk3p subunit of protein kinase A. This pathway represents the first defined link between environmental sensing, actin remodeling, and apoptosis in Saccharomyces cerevisiae.

  5. The Drosophila secreted protein Argos regulates signal transduction in the Ras/MAPK pathway.

    PubMed

    Sawamoto, K; Okabe, M; Tanimura, T; Mikoshiba, K; Nishida, Y; Okano, H

    1996-08-25

    The Drosophila argos gene encodes a secreted protein with an EGF motif which acts as an inhibitor of cellular differentiation in multiple developmental processes. To investigate the cellular pathways regulated by Argos, we screened for mutations which could modify the phenotype caused by overexpression of argos. We show that the effects of argos overexpression on the eye and wing vein development are suppressed by gain-of-function mutations of the MAPKK/D-MEK gene (Dsor1/D-mek) and the MAPK/ERK-A gene (rolled) and were enhanced by loss-of-function mutations of Star. Loss-of-function mutations in components of the Ras/MAPK signaling cascade act as dominant suppressors of the phenotype caused by the argos null mutations. A loss-of-function argos mutation enhanced the overproduction of R7 neurons caused by gain-of-function alleles of Son of sevenless and Dsor1. Conversely, overexpression of argos inhibited formation of the extra R7 cells that was caused by high-level MAPK/ERK-A activity. A phenotype of the sev; argos double mutants revealed that sev is epistatic to argos. These results provide evidence that Argos negatively regulates signal transduction events in the Ras/MAPK cascade.

  6. Effects of acrylonitrile on lymphocyte lipid rafts and RAS/RAF/MAPK/ERK signaling pathways.

    PubMed

    Li, X J; Li, B; Huang, J S; Shi, J M; Wang, P; Fan, W; Zhou, Y L

    2014-09-26

    Acrylonitrile (ACN) is a widely used chemical in the production of plastics, resins, nitriles, acrylic fibers, and synthetic rubber. Previous epidemiological investigations and animal studies have confirmed that ACN affects the lymphocytes and spleen. However, the immune toxicity mechanism is unknown. Lipid rafts are cell membrane structures that are rich in cholesterol and involved in cell signal transduction. The B cell lymophoma-10 (Bcl10) protein is a joint protein that is important in lymphocyte development and signal pathways. This study was conducted to examine the in vitro effects of ACN. We separated lipid rafts, and analyzed Bcl10 protein and caveolin. Western blotting was used to detect mitogen-activated protein kinase (MAPK) and phosphorylated MAPK levels. The results indicated that with increasing ACN concentration, the total amount of Bcl10 remained stable, but was concentrated mainly in part 4 to part 11 in electrophoretic band district which is high density in gradient centrifugation. Caveolin-1 was evaluated as a lipid raft marker protein; caveolin-1 content and position were relatively unchanged. Western blotting showed that in a certain range, MAPK protein was secreted at a higher level. At some ACN exposure levels, MAPK protein secretion was significantly decreased compared to the control group (P < 0.05). These results indicate that ACN can cause immune toxicity by damaging lipid raft structures, causing Bcl10 protein and lipid raft separation and restraining Ras-Raf-MAPK-extracellular signal-regulated kinase signaling pathways.

  7. Plumbagin inhibits tumour angiogenesis and tumour growth through the Ras signalling pathway following activation of the VEGF receptor-2

    PubMed Central

    Lai, Li; Liu, Junchen; Zhai, Dong; Lin, Qingxiang; He, Lijun; Dong, Yanmin; Zhang, Jing; Lu, Binbin; Chen, Yihua; Yi, Zhengfang; Liu, Mingyao

    2012-01-01

    BACKGROUND AND PURPOSE Angiogenesis-based therapy is an effective anti-tumour strategy and previous reports have shown some beneficial effects of a naturally occurring bioactive compound plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone). Here, we sought to determine the biological effects of plumbagin on signalling mechanisms during tumour angiogenesis. EXPERIMENTAL APPROACH The effects of plumbagin were evaluated in various in vitro assays which utilised human umbilical vein endothelial cells (HUVEC) proliferation, migration and tube formation. Plumbagin was also evaluated in vivo using chicken embryo chorioallantoic membrane (CAM) and mouse corneal micropocket models., Human colon carcinoma and prostate cancer xenograft mouse models were used to evaluate the effects of plumbagin on angiogenesis. Immunofluorescence, GST pull-down and Western blotting were employed to explore the underlying mechanisms of VEGF receptor (VEGFR)2-mediated Ras signalling pathways. KEY RESULTS Plumbagin not only inhibited endothelial cell proliferation, migration and tube formation but also suppressed chicken chorioallantoic membrane neovascularzation and VEGF-induced mouse corneal angiogenesis. Moreover, plumbagin suppressed tumour angiogenesis and tumour growth in human colon carcinoma and prostate cancer xenograft mouse models. At a molecular level, plumbagin blocked the Ras/Rac/cofilin and Ras/MEK signalling pathways mediated by VEGFR2 in HUVECs. CONCLUSIONS AND IMPLICATIONS Plumbagin inhibited tumour angiogenesis and tumour growth by interference with the VEGFR2-mediated Ras signalling pathway in endothelial cells. Our findings demonstrate a molecular basis for the effects of plumbagin and suggest that this compound might have therapeutic ant-tumour effects. PMID:21658027

  8. Overactivation of Ras signaling pathway in CD133+ MPNST cells.

    PubMed

    Borrego-Diaz, Emma; Terai, Kaoru; Lialyte, Kristina; Wise, Amanda L; Esfandyari, Tuba; Behbod, Fariba; Mautner, Victor F; Spyra, Melanie; Taylor, Sarah; Parada, Luis F; Upadhyaya, Meena; Farassati, Faris

    2012-07-01

    Cancer stem cells (CSCs) are believed to be the regenerative pool of cells responsible for repopulating tumors. Gaining knowledge about the signaling characteristics of CSCs is important for understanding the biology of tumors and developing novel anti-cancer therapies. We have identified a subpopulation of cells positive for CD133 (a CSC marker) from human primary malignant peripheral nerve sheath tumor (MPNST) cells which were absent in non-malignant Schwann cells. CD133 was also found to be expressed in human tissue samples and mouse MPNST cells. CD133+ cells were capable of forming spheres in non-adherent/serum-free conditions. The activation levels of Ras and its downstream effectors such as ERK, JNK, PI3K, p38K, and RalA were significantly increased in this population. Moreover, the CD133+ cells showed enhanced invasiveness which was linked to the increased expression of β-Catenin and Snail, two important proteins involved in the epithelial to mesenchymal transition, and Paxilin, a focal adhesion protein. Among other important characteristics of the CD133+ population, endoplasmic reticulum stress marker IRE1α was decreased, implying the potential sensitivity of CD133+ to the accumulation of unfolded proteins. Apoptotic indicators seemed to be unchanged in CD133+ cells when compared to the wild (unsorted) cells. Finally, in order to test the possibility of targeting CD133+ MPNST cells with Ras pathway pharmacological inhibitors, we exposed these cells to an ERK inhibitor. The wild population was more sensitive to inhibition of proliferation by this inhibitor as compared with the CD133+ cells supporting previous studies observing enhanced chemoresistance of these cells.

  9. Blocking downstream signaling pathways in the context of HDAC inhibition promotes apoptosis preferentially in cells harboring mutant Ras

    PubMed Central

    Luchenko, Victoria; Basseville, Agnes; Chakraborty, Arup R.; Kozlowski, Hanna; Pauly, Gary T.; Patel, Paresma; Schneider, Joel P.; Gottesman, Michael M.; Bates, Susan E.

    2016-01-01

    We previously demonstrated activation of the mitogen-activated protein kinase (MAPK) pathway in a series of romidepsin-selected T-cell lymphoma cell lines as a mechanism of resistance to the histone deacetylase inhibitor (HDI), romidepsin. As Ras mutation leads to activation of both the MAPK and the phosphoinositide 3-kinase (PI3K) pathway, we examined whether combining romidepsin with small molecule pathway inhibitors would lead to increased apoptosis in cancers harboring Ras mutations. We treated 18 Ras mutant or wild-type cell lines with romidepsin in the presence of a MEK inhibitor (PD-0325901) and/or an AKT inhibitor (MK-2206) and examined apoptosis by flow cytometry. A short-term treatment schedule of romidepsin (25 ng/ml for 6 h) was used to more closely model clinical administration. Romidepsin in combination with a MEK and an AKT inhibitor induced apoptosis preferentially in cells harboring mutant versus wild-type Ras (69.1% vs. 21.1%, p < 0.0001). Similar results were found in a subset of cell lines when belinostat was combined with the MEK and AKT inhibitors and when romidepsin was combined with the dual extracellular signaling-related kinase (ERK)/PI3K inhibitor, D-87503, which inhibited both the MAPK and PI3K pathways at 5–10 μM. The observed apoptosis was caspase-dependent and required Bak and Bax expression. Cells with wild-type or mutant Ras treated with romidepsin alone or in combination with the MEK inhibitor displayed increased expression of proapoptotic Bim. We thus conclude that cancers bearing Ras mutations, such as pancreatic cancer, can be targeted by the combination of an HDI and a dual inhibitor of the MAPK and PI3K pathways. PMID:27634878

  10. CREBBP knockdown enhances RAS/RAF/MEK/ERK signaling in Ras pathway mutated acute lymphoblastic leukemia but does not modulate chemotherapeutic response.

    PubMed

    Dixon, Zach A; Nicholson, Lindsay; Zeppetzauer, Martin; Matheson, Elizabeth; Sinclair, Paul; Harrison, Christine J; Irving, Julie A E

    2017-04-01

    Relapsed acute lymphoblastic leukemia is the most common cause of cancer-related mortality in young people and new therapeutic strategies are needed to improve outcome. Recent studies have shown that heterozygous inactivating mutations in the histone acetyl transferase, CREBBP, are particularly frequent in relapsed childhood acute lymphoblastic leukemia and associated with a hyperdiploid karyotype and KRAS mutations. To study the functional impact of CREBBP haploinsufficiency in acute lymphoblastic leukemia, RNA interference was used to knock down expression of CREBBP in acute lymphoblastic leukemia cell lines and various primagraft acute lymphoblastic leukemia cells. We demonstrate that attenuation of CREBBP results in reduced acetylation of histone 3 lysine 18, but has no significant impact on cAMP-dependent target gene expression. Impaired induction of glucocorticoid receptor targets was only seen in 1 of 4 CREBBP knockdown models, and there was no significant difference in glucocorticoid-induced apoptosis, sensitivity to other acute lymphoblastic leukemia chemotherapeutics or histone deacetylase inhibitors. Importantly, we show that CREBBP directly acetylates KRAS and that CREBBP knockdown enhances signaling of the RAS/RAF/MEK/ERK pathway in Ras pathway mutated acute lymphoblastic leukemia cells, which are still sensitive to MEK inhibitors. Thus, CREBBP mutations might assist in enhancing oncogenic RAS signaling in acute lymphoblastic leukemia but do not alter response to MEK inhibitors. Copyright© Ferrata Storti Foundation.

  11. Epiregulin expression by Ets-1 and ERK signaling pathway in Ki-ras-transformed cells.

    PubMed

    Cho, Min-Chul; Choi, Hee-Sook; Lee, Sojung; Kim, Bo Yeon; Jung, Mira; Park, Sue Nie; Yoon, Do-Young

    2008-12-19

    Epiregulin belongs to the epidermal growth factor family, binds to the epidermal growth factor receptor, and its expression is upregulated in various cancer cells, but the regulatory mechanism is unclear. We investigated the regulatory mechanism of epiregulin expression in Ki-ras-transformed cancer cells. In 267B1/Ki-ras cells, the RAF/MEK/ERK pathway was constitutively activated, epiregulin was up-regulated, and the expression and phosphorylation of Ets-1 were augmented. The inhibition of ERK by PD98059 decreased epiregulin and Ets-1 expression and suppressed the growth of 267B1/Ki-ras cells. A chromatin immunoprecipitation assay demonstrated that Ets-1 was bound to human epiregulin promoter, and this binding was abolished by PD98059. Silencing of Ets-1 by RNA interference decreased cellular epiregulin transcript expression. We suggest that the Ki-ras mutation in 267B1 prostate cells constitutively activates the RAF/MEK/ERK pathway and induces the activation of the Ets-1 transcription factor, ultimately leading to the increased expression of epiregulin.

  12. Manumycin inhibits ras signal transduction pathway and induces apoptosis in COLO320-DM human colon tumourcells

    PubMed Central

    Paolo, A Di; Danesi, R; Nardini, D; Bocci, G; Innocenti, F; Fogli, S; Barachini, S; Marchetti, A; Bevilacqua, G; Tacca, M Del

    2000-01-01

    The aim of the present study was to assess the cytotoxicity of manumycin, a specific inhibitor of farnesyl:protein transferase, as well as its effects on protein isoprenylation and kinase-dependent signal transduction in COLO320-DM human colon adenocarcinoma which harbours a wild-type K- ras gene. Immunoblot analysis of isolated cell membranes and total cellular lysates of COLO320-DM cells demonstrated that manumycin dose-dependently reduced p21 ras farnesylation with a 50% inhibitory concentration (IC50) of 2.51 ± 0.11 μM and 2.68 ± 0.20 μM, respectively, while the geranylgeranylation of p21 rhoA and p21 rap1 was not affected. Manumycin dose-dependently inhibited (IC50= 2.40 ± 0.67 μM) the phosphorylation of the mitogen-activated protein kinase/extracellular-regulated kinase 2 (p42MAPK/ERK2), the main cytoplasmic effector of p21 ras, as well as COLO320-DM cell growth (IC50= 3.58 ± 0.27 μM) without affecting the biosynthesis of cholesterol. Mevalonic acid (MVA, 100 μM), a substrate of the isoprenoid synthesis, was unable to protect COLO320-DM cells from manumycin cytotoxicity. Finally, manumycin 1–25 μM for 24–72 h induced oligonucleosomal fragmentation in a dose- and time-dependent manner and MVA did not protect COLO320-DM cells from undergoing DNA cleavage. The present findings indicate that the inhibition of p21 ras processing and signal transduction by manumycin is associated with marked inhibition of cell proliferation and apoptosis in colon cancer cells and the effect on cell growth does not require the presence of a mutated ras gene for maximal expression of chemotherapeutic activity. © 2000 Cancer Research Campaign PMID:10732765

  13. Noncanonical control of C. elegans germline apoptosis by the insulin/IGF-1 and Ras/MAPK signaling pathways

    PubMed Central

    Perrin, A J; Gunda, M; Yu, B; Yen, K; Ito, S; Forster, S; Tissenbaum, H A; Derry, W B

    2013-01-01

    The insulin/IGF-1 pathway controls a number of physiological processes in the nematode worm Caenorhabditis elegans, including development, aging and stress response. We previously found that the Akt/PKB ortholog AKT-1 dampens the apoptotic response to genotoxic stress in the germline by negatively regulating the p53-like transcription factor CEP-1. Here, we report unexpected rearrangements to the insulin/IGF-1 pathway, whereby the insulin-like receptor DAF-2 and 3-phosphoinositide-dependent protein kinase PDK-1 oppose AKT-1 to promote DNA damage-induced apoptosis. While DNA damage does not affect phosphorylation at the PDK-1 site Thr350/Thr308 of AKT-1, it increased phosphorylation at Ser517/Ser473. Although ablation of daf-2 or pdk-1 completely suppressed akt-1-dependent apoptosis, the transcriptional activation of CEP-1 was unaffected, suggesting that daf-2 and pdk-1 act independently or downstream of cep-1 and akt-1. Ablation of the akt-1 paralog akt-2 or the downstream target of the insulin/IGF-1 pathway daf-16 (a FOXO transcription factor) restored sensitivity to damage-induced apoptosis in daf-2 and pdk-1 mutants. In addition, daf-2 and pdk-1 mutants have reduced levels of phospho-MPK-1/ERK in their germ cells, indicating that the insulin/IGF-1 pathway promotes Ras signaling in the germline. Ablation of the Ras effector gla-3, a negative regulator of mpk-1, restored sensitivity to apoptosis in daf-2 mutants, suggesting that gla-3 acts downstream of daf-2. In addition, the hypersensitivity of let-60/Ras gain-of-function mutants to damage-induced apoptosis was suppressed to wild-type levels by ablation of daf-2. Thus, insulin/IGF-1 signaling selectively engages AKT-2/DAF-16 to promote DNA damage-induced germ cell apoptosis downstream of CEP-1 through the Ras pathway. PMID:22935616

  14. Noncanonical control of C. elegans germline apoptosis by the insulin/IGF-1 and Ras/MAPK signaling pathways.

    PubMed

    Perrin, A J; Gunda, M; Yu, B; Yen, K; Ito, S; Forster, S; Tissenbaum, H A; Derry, W B

    2013-01-01

    The insulin/IGF-1 pathway controls a number of physiological processes in the nematode worm Caenorhabditis elegans, including development, aging and stress response. We previously found that the Akt/PKB ortholog AKT-1 dampens the apoptotic response to genotoxic stress in the germline by negatively regulating the p53-like transcription factor CEP-1. Here, we report unexpected rearrangements to the insulin/IGF-1 pathway, whereby the insulin-like receptor DAF-2 and 3-phosphoinositide-dependent protein kinase PDK-1 oppose AKT-1 to promote DNA damage-induced apoptosis. While DNA damage does not affect phosphorylation at the PDK-1 site Thr350/Thr308 of AKT-1, it increased phosphorylation at Ser517/Ser473. Although ablation of daf-2 or pdk-1 completely suppressed akt-1-dependent apoptosis, the transcriptional activation of CEP-1 was unaffected, suggesting that daf-2 and pdk-1 act independently or downstream of cep-1 and akt-1. Ablation of the akt-1 paralog akt-2 or the downstream target of the insulin/IGF-1 pathway daf-16 (a FOXO transcription factor) restored sensitivity to damage-induced apoptosis in daf-2 and pdk-1 mutants. In addition, daf-2 and pdk-1 mutants have reduced levels of phospho-MPK-1/ERK in their germ cells, indicating that the insulin/IGF-1 pathway promotes Ras signaling in the germline. Ablation of the Ras effector gla-3, a negative regulator of mpk-1, restored sensitivity to apoptosis in daf-2 mutants, suggesting that gla-3 acts downstream of daf-2. In addition, the hypersensitivity of let-60/Ras gain-of-function mutants to damage-induced apoptosis was suppressed to wild-type levels by ablation of daf-2. Thus, insulin/IGF-1 signaling selectively engages AKT-2/DAF-16 to promote DNA damage-induced germ cell apoptosis downstream of CEP-1 through the Ras pathway.

  15. Aurora kinase A interacts with H-Ras and potentiates Ras-MAPK signaling.

    PubMed

    Umstead, MaKendra; Xiong, Jinglin; Qi, Qi; Du, Yuhong; Fu, Haian

    2017-02-03

    In cancer, upregulated Ras promotes cellular transformation and proliferation in part through activation of oncogenic Ras-MAPK signaling. While directly inhibiting Ras has proven challenging, new insights into Ras regulation through protein-protein interactions may offer unique opportunities for therapeutic intervention. Here we report the identification and validation of Aurora kinase A (Aurora A) as a novel Ras binding protein. We demonstrate that the kinase domain of Aurora A mediates the interaction with the N-terminal domain of H-Ras. Further more, the interaction of Aurora A and H-Ras exists in a protein complex with Raf-1. We show that binding of H-Ras to Raf-1 and subsequent MAPK signaling is enhanced by Aurora A, and requires active H-Ras. Thus, the functional linkage between Aurora A and the H-Ras/Raf-1 protein complex may provide a mechanism for Aurora A's oncogenic activity through direct activation of the Ras/MAPK pathway.

  16. Expression of oncogenic K-ras from its endogenous promoter leads to a partial block of erythroid differentiation and hyperactivation of cytokine-dependent signaling pathways.

    PubMed

    Zhang, Jing; Liu, Yangang; Beard, Caroline; Tuveson, David A; Jaenisch, Rudolf; Jacks, Tyler E; Lodish, Harvey F

    2007-06-15

    When overexpressed in primary erythroid progenitors, oncogenic Ras leads to the constitutive activation of its downstream signaling pathways, severe block of terminal erythroid differentiation, and cytokine-independent growth of primary erythroid progenitors. However, whether high-level expression of oncogenic Ras is required for these phenotypes is unknown. To address this issue, we expressed oncogenic K-ras (K-ras(G12D)) from its endogenous promoter using a tetracycline-inducible system. We show that endogenous K-ras(G12D) leads to a partial block of terminal erythroid differentiation in vivo. In contrast to results obtained when oncogenic Ras was overexpressed from retroviral vectors, endogenous levels of K-ras(G12D) fail to constitutively activate but rather hyperactivate cytokine-dependent signaling pathways, including Stat5, Akt, and p44/42 MAPK, in primary erythroid progenitors. This explains previous observations that hematopoietic progenitors expressing endogenous K-ras(G12D) display hypersensitivity to cytokine stimulation in various colony assays. Our results support efforts to modulate Ras signaling for treating hematopoietic malignancies.

  17. A novel SOS1 mutation in Costello/CFC syndrome affects signaling in both RAS and PI3K pathways.

    PubMed

    Tumurkhuu, Munkhtuya; Saitoh, Makiko; Takita, Junko; Mizuno, Yoko; Mizuguchi, Masashi

    2013-04-01

    Pathological upregulation of the RAS/MAPK pathway causes Costello, Noonan and cardio-facio-cutaneous (CFC) syndrome; however, little is known about PI3K/AKT signal transduction in these syndromes. Previously, we found a novel mutation of the SOS1 gene (T158A) in a patient with Costello/CFC overlapping phenotype. The aim of this study was to investigate how this mutation affects RAS/MAPK as well as PI3K/AKT pathway signal transduction. Wild-type and mutant (T158A) Son of Sevenless 1 (SOS1) were transfected into 293T cells. The levels of phospho- and total ERK1/2, AKT, p70S6K and pS6 were examined under epidermal growth factor (EGF) stimulation. After EGF stimulation, the ratio of phospho-ERK1/2 to total ERK1/2 was highest at 5 min in mutant (T158A) SOS1 cells, and at 15 min in wild-type SOS1 cells. Phospho-AKT was less abundant at 60 min in mutant than in wild-type SOS1 cells. Phosphorylation at various sites in p70S6K differed between wild-type and mutant cells. Eighteen hours after activation by EGF, the ratio of phospho-ERK1/2 to total ERK1/2 remained significantly higher in mutant than in wild-type SOS1 cells, but that of phospho-AKT to total AKT was unchanged. T158A is located in the histone-like domain, which may have a role in auto-inhibition of RAS exchanger activity of SOS1. T158A may disrupt auto-inhibition and enhance RAS signaling. T158A also affects PI3K/AKT signaling, probably via negative feedback via phospho-p70S6K. The SOS1 T158A mutation altered the phosphorylation of gene products involved in both RAS/MAPK and PI3K/AKT pathways.

  18. Dissection of Ras-Dependent Signaling Pathways Controlling Aggressive Tumor Growth of Human Fibrosarcoma Cells: Evidence for a Potential Novel Pathway

    PubMed Central

    Gupta, Swati; Plattner, Rina; Der, Channing J.; Stanbridge, Eric J.

    2000-01-01

    Activation of multiple signaling pathways is required to trigger the full spectrum of in vitro and in vivo phenotypic traits associated with neoplastic transformation by oncogenic Ras. To determine which of these pathways are important for N-ras tumorigenesis in human cancer cells and also to investigate the possibility of cross talk among the pathways, we have utilized a human fibrosarcoma cell line (HT1080), which contains an endogenous mutated allele of the N-ras gene, and its derivative (MCH603c8), which lacks the mutant N-ras allele. We have stably transfected MCH603c8 and HT1080 cells with activating or dominant-negative mutant cDNAs, respectively, of various components of the Raf, Rac, and RhoA pathways. In previous studies with these cell lines we showed that loss of mutant Ras function results in dramatic changes in the in vitro phenotypic traits and conversion to a weakly tumorigenic phenotype in vivo. We report here that only overexpression of activated MEK contributed significantly to the conversion of MCH603c8 cells to an aggressive tumorigenic phenotype. Furthermore, we have demonstrated that blocking the constitutive activation of the Raf-MEK, Rac, or RhoA pathway alone is not sufficient to block the aggressive tumorigenic phenotype of HT1080, despite affecting a number of in vitro-transformed phenotypic traits. We have also demonstrated the possibility of bidirectional cross talk between the Raf-MEK-ERK pathway and the Rac-JNK or RhoA pathway. Finally, overexpression of activated MEK in MCH603c8 cells appears to result in the activation of an as-yet-unidentified target(s) that is critical for the aggressive tumorigenic phenotype. PMID:11094080

  19. Severe craniosynostosis with Noonan syndrome phenotype associated with SHOC2 mutation: clinical evidence of crosslink between FGFR and RAS signaling pathways.

    PubMed

    Takenouchi, Toshiki; Sakamoto, Yoshiaki; Miwa, Tomoru; Torii, Chiharu; Kosaki, Rika; Kishi, Kazuo; Takahashi, Takao; Kosaki, Kenjiro

    2014-11-01

    Dysregulation in the RAS signaling cascade results in a family of malformation syndromes called RASopathies. Meanwhile, alterations in FGFR signaling cascade are responsible for various syndromic forms of craniosynostosis. In general, the phenotypic spectra of RASopathies and craniosynostosis syndromes do not overlap. Recently, however, mutations in ERF, a downstream molecule of the RAS signaling cascade, have been identified as a cause of complex craniosynostosis, suggesting that the RAS and FGFR signaling pathways can interact in the pathogenesis of malformation syndromes. Here, we document a boy with short stature, developmental delay, and severe craniosynostosis involving right coronal, bilateral lambdoid, and sagittal sutures with a de novo mutation in exon1 of SHOC2 (c.4A>G p.Ser2Gly). This observation further supports the existence of a crosslink between the RAS signaling cascade and craniosynostosis. In retrospect, the propositus had physical features suggestive of a dysregulated RAS signaling cascade, such as fetal pleural effusion, fetal hydrops, and atrial tachycardia. In addition to an abnormal cranial shape, which has been reported for this specific mutation, craniosynostosis might be a novel associated phenotype. In conclusion, the phenotypic combination of severe craniosynostosis and RASopathy features observed in the propositus suggests an interaction between the RAS and FGFR signaling cascades. Patients with craniosynostosis in combination with any RASopathy feature may require mutation screening for molecules in the FGFR-RAS signaling cascade.

  20. Effects of Germline Mutations in the Ras/MAPK Signaling Pathway on Adaptive Behavior: Cardiofaciocutaneous Syndrome and Noonan Syndrome

    PubMed Central

    Pierpont, Elizabeth I.; Pierpont, Mary Ella; Mendelsohn, Nancy J.; Roberts, Amy E.; Tworog-Dube, Erica; Rauen, Katherine A.; Seidenberg, Mark S.

    2011-01-01

    Cardiofaciocutaneous syndrome (CFC) and Noonan syndrome (NS) are two phenotypically overlapping genetic disorders whose underlying molecular etiologies affect a common signaling pathway. Mutations in the BRAF, MEK1 and MEK2 genes cause most cases of CFC and mutations in PTPN11, SOS1, KRAS and RAF1 typically cause NS. Although both syndromes are associated with developmental delays of varying severity, the extent to which the behavioral profiles differ may shed light on the different roles these respective genes play in development of skills necessary for everyday functioning. In this study, profiles of adaptive behavior of individuals with CFC and NS who had confirmed pathogenic mutations in Ras/MAPK pathway genes were investigated. Patterns of strengths and weaknesses, age-related differences, and risk factors for difficulties in adaptive skills were assessed. Although genes acting more downstream in the Ras/MAPK pathway were associated with more difficulties in adaptive functioning than genes more upstream in the pathway, several inconsistencies highlight the wide spectrum of possible developmental courses in CFC and NS. Along with clinical and genetic factors, variables such as chronological age, gestational age at birth and parental education levels accounted for significant variance in adaptive skills. Results indicate that there is wide heterogeneity in adaptive ability in CFC and NS, but that these abilities are correlated to some extent with the specific disease-causing genes. PMID:20186801

  1. DA-Raf-dependent inhibition of the Ras-ERK signaling pathway in type 2 alveolar epithelial cells controls alveolar formation.

    PubMed

    Watanabe-Takano, Haruko; Takano, Kazunori; Sakamoto, Akemi; Matsumoto, Kenji; Tokuhisa, Takeshi; Endo, Takeshi; Hatano, Masahiko

    2014-06-03

    Alveolar formation is coupled to the spatiotemporally regulated differentiation of alveolar myofibroblasts (AMYFs), which contribute to the morphological changes of interalveolar walls. Although the Ras-ERK signaling pathway is one of the key regulators for alveolar formation in developing lungs, the intrinsic molecular and cellular mechanisms underlying its role remain largely unknown. By analyzing the Ras-ERK signaling pathway during postnatal development of lungs, we have identified a critical role of DA-Raf1 (DA-Raf)-a dominant-negative antagonist for the Ras-ERK signaling pathway-in alveolar formation. DA-Raf-deficient mice displayed alveolar dysgenesis as a result of the blockade of AMYF differentiation. DA-Raf is predominantly expressed in type 2 alveolar epithelial cells (AEC2s) in developing lungs, and DA-Raf-dependent MEK1/2 inhibition in AEC2s suppresses expression of tissue inhibitor of matalloprotienase 4 (TIMP4), which prevents a subsequent proteolytic cascade matrix metalloproteinase (MMP)14-MMP2. Furthermore, MMP14-MMP2 proteolytic cascade regulates AMYF differentiation and alveolar formation. Therefore, DA-Raf-dependent inhibition of the Ras-ERK signaling pathway in AEC2s is required for alveolar formation via triggering MMP2 activation followed by AMYF differentiation. These findings reveal a pivotal role of the Ras-ERK signaling pathway in the dynamic regulation of alveolar development.

  2. Ocular albinism type 1-induced melanoma cell migration is mediated through the RAS/RAF/MEK/ERK signaling pathway.

    PubMed

    Bai, Jun; Xie, Xin; Lei, Yun; An, Gaili; He, Li; Lv, Xiaopeng

    2014-07-01

    Malignant melanoma has the highest risk of mortality among all types of skin cancer due to its highly metastatic potential. The ocular albinism type 1 (OA1) protein is a pigment cell‑specific glycoprotein, which shares significant structural and functional features with G protein‑coupled receptors. However, the role of OA1 in melanoma has yet to be elucidated. The present study aimed to investigate whether OA1 is involved in melanoma cell migration. OA1 was found to stimulate cell migration in a dose‑dependent manner in cultured human melanoma cells. Furthermore, knockdown of OA1 using small interfering RNA was observed to significantly inhibit melanoma cell migration. In addition, the mechanism underlying OA1‑induced melanoma cell migration was investigated. Stimulation of the RAS/RAF/mitogen activated protein kinase kinase (MEK)/extracellular signal‑regulated kinase (ERK) pathway using growth factors enhanced OA1 expression and melanoma cell migration, whereas inhibition of this pathway using U0126 was observed to markedly decrease OA1 expression and the number of migrated cells. These findings indicate that OA1 is involved in melanoma cell migration and that OA1‑induced melanoma cell migration is mediated through the RAS/RAF/MEK/ERK signaling pathway. Therefore, OA1 may serve as a novel therapeutic target for melanoma.

  3. Inhibition of the Ras/Raf/ERK1/2 Signaling Pathway Restores Cultured Spinal Cord-Injured Neuronal Migration, Adhesion, and Dendritic Spine Development.

    PubMed

    Xu, Dongdong; Cao, Fujiang; Sun, Shiwei; Liu, Tao; Feng, Shiqing

    2016-08-01

    The Ras/Raf/ERK1/2 signaling pathway plays an important role in central and peripheral neurons in functions such as dendritic arborization, neuronal polarity, and axon assembly. However, emerging evidence also shows that up-regulation of this signaling pathway may lead to the development of spinal cord injury. The present study aimed to determine the effects of Ras/Raf/ERK1/2 signaling pathway inhibition on properties of spinal cord-injured neurons. First, neurons from spinal cord-injured C57BL/6 J mouse pups and sham-operated C57BL/6 J mouse pups were harvested. Then, immunofluorescence, western blotting, cell adhesion and cell migration assays, and DiI labeling were employed to investigate the effect of Ras/Raf/ERK1/2 signaling pathway inhibition on spinal cord-injured neurons. Immunofluorescence results of synapse formation indicated that the experimental spinal cord injury model was successfully established. Western blot results identified upregulated Erk phosphorylation in the spinal cord-injured neurons, and also showed that U0126 inhibited phosphorylation of Erk, which is a downstream kinase in the Ras/Raf signaling pathway. Additionally, cell migration and adhesion was significantly increased in the spinal cord-injured neurons. DiI labeling results also showed an increased formation of mature spines after inhibition of Ras/Raf/ERK1/2 signaling. Taken together, these results suggested that the Ras/Raf/ERK1/2 signaling pathway could serve as an effective treatment target for spinal cord injury.

  4. The Ras/PKA signaling pathway may control RNA polymerase II elongation via the Spt4p/Spt5p complex in Saccharomyces cerevisiae.

    PubMed Central

    Howard, Susie C; Hester, Arelis; Herman, Paul K

    2003-01-01

    The Ras signaling pathway in Saccharomyces cerevisiae controls cell growth via the cAMP-dependent protein kinase, PKA. Recent work has indicated that these effects on growth are due, in part, to the regulation of activities associated with the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. However, the precise target of these Ras effects has remained unknown. This study suggests that Ras/PKA activity regulates the elongation step of the RNA polymerase II transcription process. Several lines of evidence indicate that Spt5p in the Spt4p/Spt5p elongation factor is the likely target of this control. First, the growth of spt4 and spt5 mutants was found to be very sensitive to changes in Ras/PKA signaling activity. Second, mutants with elevated levels of Ras activity shared a number of specific phenotypes with spt5 mutants and vice versa. Finally, Spt5p was efficiently phosphorylated by PKA in vitro. Altogether, the data suggest that the Ras/PKA pathway might be directly targeting a component of the elongating polymerase complex and that this regulation is important for the normal control of yeast cell growth. These data point out the interesting possibility that signal transduction pathways might directly influence the elongation step of RNA polymerase II transcription. PMID:14668364

  5. G-CSF improves CUMS-induced depressive behaviors through downregulating Ras/ERK/MAPK signaling pathway.

    PubMed

    Li, Hui; Linjuan-Li; Wang, Yaping

    2016-10-28

    Neuronal plasticity in hippocampal neurons is closely related to memory, mood and behavior as well as in the development of depression. Granulocyte colony-stimulating factor (G-CSF) can promote neuronal plasticity and enhance motor skills. However, the function of G-CSF in depression remains poorly understood. In this study, we explored the biological role and potential molecular mechanism of G-CSF on depression-like behaviors. Our results showed that G-CSF was significantly downregulated in the hippocampus of chronic unexpected mild stress (CUMS) rats. Administration of G-CSF significantly reversed CUMS-induced depression-like behaviors in the open field test (OFT), sucrose preference test (SPT) and forced swimming test (FST). Moreover, G-CSF upregulated the expression of synaptic-associated proteins including polysialylated form of neural cell adhesion molecule (PSA-NCAM), synaptophysin (SYN), and postsynaptic density protein 95 (PSD-95) in the hippocampus and G-CSF significantly increased cell viability rate of hippocampal neurons in vitro. Further studies indicated that the renin-angiotensin system (Ras)/extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (MAPK) signaling pathways was involved in the regulation of G-CSF on depressive-like behaviors and neuronal plasticity in CUMS rats. Taken together, our results showed that G-CSF improves depression-like behaviors via inhibiting Ras/ERK/MAPK signaling pathways. Our study suggests that G-CSF may be a promising therapeutic strategy for the treatment of depression.

  6. Lupeol, a fruit and vegetable based triterpene, induces apoptotic death of human pancreatic adenocarcinoma cells via inhibition of Ras signaling pathway.

    PubMed

    Saleem, Mohammad; Kaur, Satwinderjeet; Kweon, Mee-Hyang; Adhami, Vaqar Mustafa; Afaq, Farrukh; Mukhtar, Hasan

    2005-11-01

    Pancreatic cancer is an exceptionally aggressive disease, the treatment of which has largely been unsuccessful due to higher resistance offered by pancreatic cancer cells to conventional approaches such as surgery, radiation and/or chemotherapy. The aberration of Ras oncoprotein has been linked to the induction of multiple signaling pathways and to the resistance offered by pancreatic cancer cells to apoptosis. Therefore, there is a need for development of new and effective chemotherapeutic agents which can target multiple pathways to induce responsiveness of pancreatic cancer cells to death signals. In this study, human pancreatic adenocarcinoma cells AsPC-1 were used to investigate the effect of Lupeol on cell growth and its effects on the modulation of multiple Ras-induced signaling pathways. Lupeol caused a dose-dependent inhibition of cell growth as assessed by MTT assay and induction of apoptosis as assessed by flow cytometry, fluorescence microscopy and western blotting. Lupeol treatment to cells was found to significantly reduce the expression of Ras oncoprotein and modulate the protein expression of various signaling molecules involved in PKCalpha/ODC, PI3K/Akt and MAPKs pathways along with a significant reduction in the activation of NFkappaB signaling pathway. Our data suggest that Lupeol can adopt a multi-prong strategy to target multiple signaling pathways leading to induction of apoptosis and inhibition of growth of pancreatic cancer cells. Lupeol could be a potential agent against pancreatic cancer, however, further in-depth in vivo studies are warranted.

  7. Insulin-like growth factor-1 induces hyperproliferation of PKD1 cystic cells via a Ras/Raf dependent signalling pathway

    PubMed Central

    Parker, Emma; Newby, Linda J; Sharpe, Claire C; Rossetti, Sandro; Streets, Andrew J; Harris, Peter C; O’Hare, Michael J; Ong, Albert CM

    2008-01-01

    Tubular cell proliferation occurs early and precedes cyst formation in autosomal dominant polycystic kidney disease (ADPKD). To identify key alterations in cell signalling which regulate cell proliferation in ADPKD, we examined the potential role of insulin-like growth factor-1 (IGF-1) mediated signalling pathways. Conditionally immortalised tubular epithelial cells were generated from ADPKD patients with characterised germline PKD1 mutations and normal individuals. Germline and somatic PKD1 (but not PKD2) mutations were identified in PKD1 cystic cells by DHPLC. All lines showed a reduction or absence of polycystin-1 but normal polycystin-2 expression. Polycystin-1 deficiency was associated with increased sensitivity to IGF-1 as well as a permissive effect of cAMP on cell growth. The increase in cell proliferation to both agents was dependent on PI3 kinase and ERK activity. Inhibition of Ras or Raf activity also abolished stimulated cell proliferation. Ras activation assays revealed significantly higher IGF-1 stimulated levels of GTP-Ras in cystic cells compared to control cells but cAMP alone had no effect on GTP-Ras levels. These results suggest that haploinsufficiency of PC1 may lower the threshold for activation of a Ras-Raf mediated signalling cascade leading to growth-factor induced hyperproliferation. Inhibition of Ras or Raf activation could be a useful therapeutic approach to reducing tubular cell proliferation in ADPKD. PMID:17396115

  8. Involvement of the Ras/extracellular signal-regulated kinase signalling pathway in the regulation of ERCC-1 mRNA levels by insulin.

    PubMed Central

    Lee-Kwon, W; Park, D; Bernier, M

    1998-01-01

    Expression of DNA repair enzymes, which includes ERCC-1, might be under the control of hormonal and growth factor stimulation. In the present study it was observed that insulin increased ERCC-1 mRNA levels both in Chinese hamster ovary cells overexpressing human insulin receptors (HIRc cells) and in fully differentiated 3T3-L1 adipocytes. To investigate the mechanisms underlying the increase in ERCC-1 gene expression in HIRc cells, we used a variety of pharmacological tools known to inhibit distinct signalling pathways. None of these inhibitors affected the amount of ERCC-1 mRNA in unstimulated cells. The pretreatment of cells with two chemically unrelated phosphatidylinositol 3'-kinase inhibitors, wortmannin and LY294002, failed to block the doubling of ERCC-1 mRNA content by insulin. Similarly, inhibition of pp70 S6 kinase by rapamycin had no apparent effects on this insulin response. In contrast, altering the p21(ras)-dependent pathway with either manumycin, an inhibitor of Ras farnesylation, or PD98059, an inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) kinase, suppressed the induction of ERCC-1 mRNA by insulin (P<0.001). Furthermore inhibition of RNA and protein synthesis negatively regulated the expression of this insulin-regulated gene (P<0.005). These results suggest that insulin enhances ERCC-1 mRNA levels by the activation of the Ras-ERK-dependent pathway without the involvement of the phosphatidylinositol 3'-kinase/pp70 S6 kinase. PMID:9531502

  9. Hepatitis B virus X protein induces RNA polymerase III-dependent gene transcription and increases cellular TATA-binding protein by activating the Ras signaling pathway.

    PubMed

    Wang, H D; Trivedi, A; Johnson, D L

    1997-12-01

    Our previous studies have shown that the hepatitis B virus protein, X, activates all three classes of RNA polymerase III (pol III)-dependent promoters by increasing the cellular level of TATA-binding protein (TBP) (H.-D. Wang et al., Mol. Cell. Biol. 15:6720-6728, 1995), a limiting transcription component (A. Trivedi et al., Mol. Cell. Biol. 16:6909-6916, 1996). We have investigated whether these X-mediated events are dependent on the activation of the Ras/Raf-1 signaling pathway. Transient expression of a dominant-negative mutant Ras gene (Ras-ala15) in a Drosophila S-2 stable cell line expressing X (X-S2), or incubation of the cells with a Ras farnesylation inhibitor, specifically blocked both the X-dependent activation of a cotransfected tRNA gene and the increase in cellular TBP levels. Transient expression of a constitutively activated form of Ras (Ras-val12) in control S2 cells produced both an increase in tRNA gene transcription and an increase in cellular TBP levels. These events are not cell type specific since X-mediated gene induction was also shown to be dependent on Ras activation in a stable rat 1A cell line expressing X. Furthermore, increases in RNA pol III-dependent gene activity and TBP levels could be restored in X-S2 cells expressing Ras-ala15 by coexpressing a constitutively activated form of Raf-1. These events are serum dependent, and when the cells are serum deprived, the X-mediated effects are augmented. Together, these results demonstrate that the X-mediated induction of RNA pol III-dependent genes and increase in TBP are both dependent on the activation of the Ras/Raf-1 signaling cascade. In addition, these studies define two new and important consequences mediated by the activation of the Ras signal transduction pathway: an increase in the central transcription factor, TBP, and the induction of RNA pol III-dependent gene activity.

  10. Regulation of SREBPs by Sphingomyelin in Adipocytes via a Caveolin and Ras-ERK-MAPK-CREB Signaling Pathway

    PubMed Central

    Makdissy, Nehman; Popa, Iuliana; Younsi, Mohamed; Valet, Philippe; Brunaud, Laurent; Ziegler, Olivier; Quilliot, Didier

    2015-01-01

    Sterol response element binding protein (SREBP) is a key transcription factor in insulin and glucose metabolism. We previously demonstrated that elevated levels of membrane sphingomyelin (SM) were related to peroxisome proliferator–activated receptor-γ (PPARγ), which is a known target gene of SREBP-1 in adipocytes. However, the role of SM in SREBP expression in adipocytes remains unknown. In human abdominal adipose tissue from obese women with various concentrations of fasting plasma insulin, SREBP-1 proteins decreased in parallel with increases in membrane SM levels. An inverse correlation was found between the membrane SM content and the levels of SREBP-1c/ERK/Ras/PPARγ/CREB proteins. For the first time, we demonstrate the effects of SM and its signaling pathway in 3T3-F442A adipocytes. These cells were enriched or unenriched with SM in a range of concentrations similar to those observed in obese subjects by adding exogenous natural SMs (having different acyl chain lengths) or by inhibiting neutral sphingomyelinase. SM accumulated in caveolae of the plasma membrane within 24 h and then in the intracellular space. SM enrichment decreased SREBP-1 through the inhibition of extracellular signal-regulated protein kinase (ERK) but not JNK or p38 mitogen-activated protein kinase (MAPK). Ras/Raf-1/MEK1/2 and KSR proteins, which are upstream mediators of ERK, were down-regulated, whereas SREBP-2/caveolin and cholesterol were up-regulated. In SM-unmodulated adipocytes treated with DL-1-Phenyl-2-Palmitoylamino-3-morpholino-1-propanol (PPMP), where the ceramide level increased, the expression levels of SREBPs and ERK were modulated in an opposite direction relative to the SM-enriched cells. SM inhibited the insulin-induced expression of SREBP-1. Rosiglitazone, which is an anti-diabetic agent and potent activator of PPARγ, reversed the effects of SM on SREBP-1, PPARγ and CREB. Taken together, these findings provide novel insights indicating that excess membrane SM

  11. Sprouty, an intracellular inhibitor of Ras signaling.

    PubMed

    Casci, T; Vinós, J; Freeman, M

    1999-03-05

    Sprouty was identified in a genetic screen as an inhibitor of Drosophila EGF receptor signaling. The Egfr triggers cell recruitment in the eye, and sprouty- eyes have excess photoreceptors, cone cells, and pigment cells. Sprouty's function is, however, more widespread. We show that it also interacts genetically with the receptor tyrosine kinases Torso and Sevenless, and it was first discovered through its effect on FGF receptor signaling. In contrast to an earlier proposal that Sprouty is extracellular, we show by biochemical analysis that Sprouty is an intracellular protein, associated with the inner surface of the plasma membrane. Sprouty binds to two intracellular components of the Ras pathway, Drk and Gap1. Our results indicate that Sprouty is a widespread inhibitor of Ras pathway signal transduction.

  12. Control of Mammary Differentiation by Ras-Dependent Signal Transduction Pathways

    DTIC Science & Technology

    2005-05-01

    differentiation and apoptosis during pregnancy and lactation. These processes are initiated by a complex series of signals that include mammotrophic hormones...differentiation and apoptosis during pregnancy and lactation. These processes are initiated by a complex series of signals which include mammotrophic...differentiation will provide critical insight into control of this process in HCI I cells. Because inhibition of differentiation in HCI 1 cells

  13. SPRED1 Interferes with K-ras but Not H-ras Membrane Anchorage and Signaling

    PubMed Central

    Siljamäki, Elina

    2016-01-01

    The Ras/mitogen-activated protein kinase (MAPK) signaling pathway is tightly controlled by negative feedback regulators, such as the tumor suppressor SPRED1. The SPRED1 gene also carries loss-of-function mutations in the RASopathy Legius syndrome. Growth factor stimulation translocates SPRED1 to the plasma membrane, triggering its inhibitory activity. However, it remains unclear whether SPRED1 there acts at the level of Ras or Raf. We show that pharmacological or galectin-1 (Gal-1)-mediated induction of B- and C-Raf-containing dimers translocates SPRED1 to the plasma membrane. This is facilitated in particular by SPRED1 interaction with B-Raf and, via its N terminus, with Gal-1. The physiological significance of these novel interactions is supported by two Legius syndrome-associated mutations that show diminished binding to both Gal-1 and B-Raf. On the plasma membrane, SPRED1 becomes enriched in acidic membrane domains to specifically perturb membrane organization and extracellular signal-regulated kinase (ERK) signaling of active K-ras4B (here, K-ras) but not H-ras. However, SPRED1 also blocks on the nanoscale the positive effects of Gal-1 on H-ras. Therefore, a combinatorial expression of SPRED1 and Gal-1 potentially regulates specific patterns of K-ras- and H-ras-dependent signaling output. More broadly, our results open up the possibility that related SPRED and Sprouty proteins act in a similar Ras and Raf isoform-specific manner. PMID:27503857

  14. SPRED1 Interferes with K-ras but Not H-ras Membrane Anchorage and Signaling.

    PubMed

    Siljamäki, Elina; Abankwa, Daniel

    2016-10-15

    The Ras/mitogen-activated protein kinase (MAPK) signaling pathway is tightly controlled by negative feedback regulators, such as the tumor suppressor SPRED1. The SPRED1 gene also carries loss-of-function mutations in the RASopathy Legius syndrome. Growth factor stimulation translocates SPRED1 to the plasma membrane, triggering its inhibitory activity. However, it remains unclear whether SPRED1 there acts at the level of Ras or Raf. We show that pharmacological or galectin-1 (Gal-1)-mediated induction of B- and C-Raf-containing dimers translocates SPRED1 to the plasma membrane. This is facilitated in particular by SPRED1 interaction with B-Raf and, via its N terminus, with Gal-1. The physiological significance of these novel interactions is supported by two Legius syndrome-associated mutations that show diminished binding to both Gal-1 and B-Raf. On the plasma membrane, SPRED1 becomes enriched in acidic membrane domains to specifically perturb membrane organization and extracellular signal-regulated kinase (ERK) signaling of active K-ras4B (here, K-ras) but not H-ras. However, SPRED1 also blocks on the nanoscale the positive effects of Gal-1 on H-ras. Therefore, a combinatorial expression of SPRED1 and Gal-1 potentially regulates specific patterns of K-ras- and H-ras-dependent signaling output. More broadly, our results open up the possibility that related SPRED and Sprouty proteins act in a similar Ras and Raf isoform-specific manner.

  15. Acquisition of anoikis resistance promotes alterations in the Ras/ERK and PI3K/Akt signaling pathways and matrix remodeling in endothelial cells.

    PubMed

    de Sousa Mesquita, Ana Paula; de Araújo Lopes, Silvana; Pernambuco Filho, Paulo Castanho A; Nader, Helena B; Lopes, Carla Cristina

    2017-06-26

    Anoikis is a programmed cell death induced upon cell detachment from extracellular matrix. Anoikis resistance is a critical mechanism in tumor metastasis. Cancer cells deregulate and adapt their metabolism to survive in the absence of adhesion, spreading metastases to distant organs. These adaptations include abnormal regulation of growth factor receptors activating prosurvival signaling pathways, such as the Ras/ERK and PI3K/Akt pathways, and extracellular matrix remodeling, leading to metastasis by an increase of invasiveness and inhibiting anoikis. This study investigates the possible involvement of ECM components and signaling pathways in the regulation of resistance to anoikis in endothelial cells (EC). Endothelial cells submitted to stressful conditions by blocking adhesion to substrate (anoikis resistance) display an up-regulation of Ras/ERK and PI3k/Akt pathways by high expression of Ras, ERK, PI3K (p110α) and Akt (Thr 308). After ERK and PI3K inhibiting, all EC-derived cell lines studied showed lower growth, a decrease in invasive potential and a higher rate of apoptosis. Furthermore, anoikis-resistant cell lines display a decrease in the expression of fibronectin, collagen IV and hyaluronic acid and an increase in the expression of laminin, perlecan, αv, β3, α5 and β1 integrins subunits, hyaluronidades 1, 2 and 3 and metalloproteinases 2 and 9. These results indicate that the acquisition of anoikis resistance induced remodeling of the extracellular matrix and overexpression of the PI3K/Akt and Ras/ERK pathway components. Acquisition of resistance to anoikis is a potentially crucial step in endothelial cell transformation.

  16. Gene expression studies demonstrate that the K-ras/Erk MAP kinase signal transduction pathway and other novel pathways contribute to the pathogenesis of cumene-induced lung tumors.

    PubMed

    Wakamatsu, Nobuko; Collins, Jennifer B; Parker, Joel S; Tessema, Mathewos; Clayton, Natasha P; Ton, Thai-Vu T; Hong, Hue-Hua L; Belinsky, Steven; Devereux, Theodora R; Sills, Robert C; Lahousse, Stephanie A

    2008-07-01

    National Toxicology Program (NTP) inhalation studies demonstrated that cumene significantly increased the incidence of alveolar/bronchiolar adenomas and carcinomas in B6C3F1 mice. Cumene or isopropylbenzene is a component of crude oil used primarily in the production of phenol and acetone. The authors performed global gene expression analysis to distinguish patterns of gene regulation between cumene-induced tumors and normal lung tissue and to look for patterns based on the presence or absence of K-ras and p53 mutations in the tumors. Principal component analysis segregated the carcinomas into groups with and without K-ras mutations, but failed to separate the tumors based on p53 mutation status. Expression of genes associated with the Erk MAP kinase signaling pathway was significantly altered in carcinomas with K-ras mutations compared to tumors without K-ras mutations or normal lung. Gene expression analysis also suggested that cumene-induced carcinomas with K-ras mutations have greater malignant potential than those without mutations. In addition, significance analysis of function and expression (SAFE) demonstrated expression changes of genes regulated by histone modification in carcinomas with K-ras mutations. The gene expression analysis suggested the formation of alveolar/bronchiolar carcinomas in cumene-exposed mice typically involves mutation of K-ras, which results in increased Erk MAP kinase signaling and modification of histones.

  17. The Ras/PKA signaling pathway of Saccharomyces cerevisiae exhibits a functional interaction with the Sin4p complex of the RNA polymerase II holoenzyme.

    PubMed Central

    Howard, S C; Chang, Y W; Budovskaya, Y V; Herman, P K

    2001-01-01

    Saccharomyces cerevisiae cells enter into the G(0)-like resting state, stationary phase, in response to specific types of nutrient limitation. We have initiated a genetic analysis of this resting state and have identified a collection of rye mutants that exhibit a defective transcriptional response to nutrient deprivation. These transcriptional defects appear to disrupt the control of normal growth because the rye mutants are unable to enter into a normal stationary phase upon nutrient deprivation. In this study, we examined the mutants in the rye1 complementation group and found that rye1 mutants were also defective for stationary phase entry. Interestingly, the RYE1 gene was found to be identical to SIN4, a gene that encodes a component of the yeast Mediator complex within the RNA polymerase II holoenzyme. Moreover, mutations that affected proteins within the Sin4p module of the Mediator exhibited specific genetic interactions with the Ras protein signaling pathway. For example, mutations that elevated the levels of Ras signaling, like RAS2(val19), were synthetic lethal with sin4. In all, our data suggest that specific proteins within the RNA polymerase II holoenzyme might be targets of signal transduction pathways that are responsible for coordinating gene expression with cell growth. PMID:11560888

  18. Mechanisms of Ras membrane organization and signaling

    PubMed Central

    Abankwa, Daniel; Gorfe, Alemayehu A.; Hancock, John F.

    2009-01-01

    Understanding the signalling function of Ras GTPases has been the focus of much research for over 20 years. Both the catalytic domain and the membrane anchoring C terminal hypervariable region (HVR) of Ras are necessary for its cellular function. However, while the highly conserved catalytic domain has been characterized in atomic detail, the structure of the full-length membrane-bound Ras has remained elusive. Lack of structural knowledge on the full-length protein limited our understanding of Ras signalling. For example, structures of the Ras catalytic domain solved in complex with effectors do not provide a basis for the functional specificity of different Ras isoforms. Recent molecular dynamics simulations in combination with biophysical and cell biological experiments have shown that the HVR and parts of the G domain cofunction with the lipid tails to anchor H-ras to the plasma membrane. In the GTP-bound state, H-ras adopts an orientation that allows read out by Ras effectors and translation into corresponding MAPK signalling. Here we discuss details of an analysis that suggests a novel balance model for Ras functioning. The balance model rationalizes Ras membrane orientation and may help explain isoform specific interactions of Ras with its effectors and modulators. PMID:18758236

  19. Endogenous K-ras signaling in erythroid differentiation.

    PubMed

    Zhang, Jing; Lodish, Harvey F

    2007-08-15

    K-ras is one of the most frequently mutated genes in virtually all types of human cancers. Using mouse fetal liver erythroid progenitors as a model system, we studied the role of endogenous K-ras signaling in erythroid differentiation. When oncogenic K-ras is expressed from its endogenous promoter, it hyperactivates cytokine-dependent signaling pathways and results in a partial block in erythroid differentiation. In erythroid progenitors deficient in K-ras, cytokine-dependent Akt activation is greatly reduced, leading to delays in erythroid differentiation. Thus, both loss- and gain-of-Kras functions affect erythroid differentiation through modulation of cytokine signaling. These results support the notion that in human cancer patients oncogenic Ras signaling might be controlled by antagonizing essential cytokines.

  20. Ras trafficking, localization and compartmentalized signalling.

    PubMed

    Prior, Ian A; Hancock, John F

    2012-04-01

    Ras proteins are proto-oncogenes that are frequently mutated in human cancers. Three closely related isoforms, HRAS, KRAS and NRAS, are expressed in all cells and have overlapping but distinctive functions. Recent work has revealed how differences between the Ras isoforms in their trafficking, localization and protein-membrane orientation enable signalling specificity to be determined. We review the various strategies used to characterize compartmentalized Ras localization and signalling. Localization is an important contextual modifier of signalling networks and insights from the Ras system are of widespread relevance for researchers interested in signalling initiated from membranes.

  1. KSR: a MAPK scaffold of the Ras pathway?

    PubMed

    Morrison, D K

    2001-05-01

    Kinase Suppressor of Ras (KSR) is an intriguing component of the Ras pathway that was first identified by genetic studies performed in Drosophila melanogaster and Caenorhabditis elegans. In both organisms, inactivating mutations in KSR suppress the phenotypic effects induced by activated Ras. These findings together with the fact that KSR contains many structural features characteristic of a protein kinase led to early speculation that KSR is a kinase functioning upstream of the Ras pathway component Raf-1 or in a parallel Ras-dependent pathway. However, in the six years since its discovery, KSR has been found to lack several key properties of known protein kinases, which has cast doubt on whether KSR is indeed a functional enzyme. A major breakthrough in our understanding of the role of KSR in signal transduction has come from recent findings that KSR interacts with several components of the MAP kinase cascade, including Raf-1, MEK1/2 and ERK1/2. The model now emerging is that KSR acts as a scaffolding protein that coordinates the assembly of a membrane-localized, multiprotein MAP kinase complex, a vital step in Ras-mediated signal transduction. Thus, while Kinase Suppressor of Ras may be its name, phosphorylation may not be its game.

  2. Digital signaling and hysteresis characterize Ras activation in lymphoid cells

    PubMed Central

    Das, Jayajit; Ho, Mary; Zikherman, Julie; Govern, Christopher; Yang, Ming; Weiss, Arthur; Chakraborty, Arup K.; Roose, Jeroen P.

    2009-01-01

    Activation of Ras proteins underlies functional decisions in diverse cell types. Two molecules, RasGRP and SOS, catalyze Ras activation in lymphocytes. Binding of active Ras to SOS′ allosteric pocket markedly increases SOS′ activity establishing a positive feedback loop for SOS-mediated Ras activation. Integrating in silico and in vitro studies, we demonstrate that digital signaling in lymphocytes (cells are “on” or “off”) is predicated upon feedback regulation of SOS. SOS′ feedback loop leads to hysteresis in the dose-response curve, which can enable a capacity to sustain Ras activation as stimuli are withdrawn and exhibit “memory” of past encounters with antigen. Ras activation via RasGRP alone is analog (graded increase in amplitude with stimulus). We describe how complementary analog (RasGRP) and digital (SOS) pathways act on Ras to efficiently convert analog input to digital output. Numerous predictions regarding the impact of our findings on lymphocyte function and development are noted. PMID:19167334

  3. Sodium phenylacetate inhibits the Ras/MAPK signaling pathway to induce reduction of the c-Raf-1 protein in human and canine breast cancer cells.

    PubMed

    Watanabe, Manabu; Miyajima, Nozomi; Igarashi, Maki; Endo, Yoshifumi; Watanabe, Natsuko; Sugano, Sumio

    2009-11-01

    An aromatic fatty acid, phenylacetate (PA), has been shown to have cytostatic, antitumor and cell differentiation-inducing effects on various kinds of tumors. Previously, we have demonstrated cell growth inhibition, malignant phenotype reduction and cell differentiation effects of sodium phenylacetate (NaPA) treatment in a canine mammary tumor cell line. To clarify the molecular mechanism of these effects, we examined the expression of Ras/MAPK signaling pathway-related molecules in human and canine breast cancer cell lines, and found that the level of c-Raf-1 protein was reduced by 5, 10 and 20 mM of NaPA treatments, though Ras activation was maintained. Dephosphorylation of c-Raf-1 at Serine (Ser) 259, Ser 338, and Ser 621 were also seen in NaPA-treated cells. Downstream factors in the pathway, such as mitogen-activated protein kinase/ERK kinase (MEK)1/2 and ERK1/2, showed decreased activity, and accordingly, expressions of cyclinD1, c-myc, and inactivation of p90 ribosomal S6 kinase (RSK), which are MAPK targets, were reduced. We also observed the reduction of cell-cycle-promoted molecules, such as cdc1/cdk2, cdk4, PCNA cyclin A, and cyclin B, and the increased expression of p27kip1. Furthermore, expression of an epithelial marker, E-cadherin, was increased by NaPA treatment. These results suggest that one of the molecular targets of NaPA treatment was the reduction of c-Raf-1 protein, and that its reduction results in the decrease of malignant characteristics of tumor cells through blockage of the Ras/MAPK signaling pathway.

  4. Melatonin Represses Oxidative Stress-Induced Activation of the MAP Kinase and mTOR Signaling Pathways in H4IIE Hepatoma Cells Through Inhibition of Ras

    PubMed Central

    Kimball, Scot R.; Abbas, Ahmed; Jefferson, Leonard S.

    2009-01-01

    SUMMARY Reactive oxygen species (ROS) have been implicated in the pathogenesis of a variety of diseases, and antioxidant treatment is currently being investigated as a potential therapy to attenuate the detrimental effects of ROS-mediated oxidative stress. Melatonin is a potent naturally produced antioxidant, which acts through various mechanisms to ameliorate the toxic effects of ROS. However, little is known about the mechanisms or signaling pathways through which melatonin acts to reverse the effects of ROS. In the present study, the effect of melatonin treatment on the hydrogen peroxide (H2O2)-induced activation of the mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) signaling pathways was assessed in H4IIE hepatoma cells. It was found that melatonin strongly attenuated H2O2-induced activation of the ERK1/2 and p38 MAP kinases, as well as several of their downstream targets. Melatonin also attenuated the H2O2-induced phosphorylation of Akt and the Akt substrate mTOR, as well as a downstream target of mTOR action, 4E-BP1. Upregulation of ERK1/2, p38, and Akt signaling by H2O2 was accompanied by activation of Ras, an effect that was blocked by melatonin. Overall, the results suggest that melatonin acts to prevent many of the H2O2-induced alterations in the MAPK and mTOR signaling pathways through inhibition of Ras, at least in H4IIE hepatoma cells. PMID:18410586

  5. Effect of angiotensin II and small GTPase Ras signaling pathway inhibition on early renal changes in a murine model of obstructive nephropathy.

    PubMed

    Rodríguez-Peña, Ana B; Fuentes-Calvo, Isabel; Docherty, Neil G; Arévalo, Miguel; Grande, María T; Eleno, Nélida; Pérez-Barriocanal, Fernando; López-Novoa, José M

    2014-01-01

    Tubulointerstitial fibrosis is a major feature of chronic kidney disease. Unilateral ureteral obstruction (UUO) in rodents leads to the development of renal tubulointerstitial fibrosis consistent with histopathological changes observed in advanced chronic kidney disease in humans. The purpose of this study was to assess the effect of inhibiting angiotensin II receptors or Ras activation on early renal fibrotic changes induced by UUO. Animals either received angiotensin II or underwent UUO. UUO animals received either losartan, atorvastatin, and farnesyl transferase inhibitor (FTI) L-744,832, or chaetomellic acid A (ChA). Levels of activated Ras, phospho-ERK1/2, phospho-Akt, fibronectin, and α-smooth muscle actin were subsequently quantified in renal tissue by ELISA, Western blot, and/or immunohistochemistry. Our results demonstrate that administration of angiotensin II induces activation of the small GTPase Ras/Erk/Akt signaling system, suggesting an involvement of angiotensin II in the early obstruction-induced activation of renal Ras. Furthermore, upstream inhibition of Ras signalling by blocking either angiotensin AT1 type receptor or by inhibiting Ras prenylation (atorvastatin, FTI o ChA) reduced the activation of the Ras/Erk/Akt signaling system and decreased the early fibrotic response in the obstructed kidney. This study points out that pharmacological inhibition of Ras activation may hold promise as a future strategy in the prevention of renal fibrosis.

  6. Effect of Angiotensin II and Small GTPase Ras Signaling Pathway Inhibition on Early Renal Changes in a Murine Model of Obstructive Nephropathy

    PubMed Central

    Rodríguez-Peña, Ana B.; Fuentes-Calvo, Isabel; Docherty, Neil G.; Arévalo, Miguel; Grande, María T.; Eleno, Nélida; Pérez-Barriocanal, Fernando; López-Novoa, José M.

    2014-01-01

    Tubulointerstitial fibrosis is a major feature of chronic kidney disease. Unilateral ureteral obstruction (UUO) in rodents leads to the development of renal tubulointerstitial fibrosis consistent with histopathological changes observed in advanced chronic kidney disease in humans. The purpose of this study was to assess the effect of inhibiting angiotensin II receptors or Ras activation on early renal fibrotic changes induced by UUO. Animals either received angiotensin II or underwent UUO. UUO animals received either losartan, atorvastatin, and farnesyl transferase inhibitor (FTI) L-744,832, or chaetomellic acid A (ChA). Levels of activated Ras, phospho-ERK1/2, phospho-Akt, fibronectin, and α-smooth muscle actin were subsequently quantified in renal tissue by ELISA, Western blot, and/or immunohistochemistry. Our results demonstrate that administration of angiotensin II induces activation of the small GTPase Ras/Erk/Akt signaling system, suggesting an involvement of angiotensin II in the early obstruction-induced activation of renal Ras. Furthermore, upstream inhibition of Ras signalling by blocking either angiotensin AT1 type receptor or by inhibiting Ras prenylation (atorvastatin, FTI o ChA) reduced the activation of the Ras/Erk/Akt signaling system and decreased the early fibrotic response in the obstructed kidney. This study points out that pharmacological inhibition of Ras activation may hold promise as a future strategy in the prevention of renal fibrosis. PMID:25101263

  7. HMGB1 promotes the secretion of multiple cytokines and potentiates the osteogenic differentiation of mesenchymal stem cells through the Ras/MAPK signaling pathway

    PubMed Central

    Feng, Lin; Xue, Deting; Chen, Erman; Zhang, Wei; Gao, Xiang; Yu, Jiawei; Feng, Yadong; Pan, Zhijun

    2016-01-01

    High mobility group box 1 (HMGB1) protein has been previously been detected in the inflammatory microenvironment of bone fractures. It is well known that HMGB1 acts as a chemoattractant to mesenchymal stem cells (MSCs). In the present study, the effects of HMGB1 on cytokine secretion from MSCs were determined, and the molecular mechanisms underlying these effects of HMGB1 on osteogenic differentiation were elucidated. To detect cytokine secretion, antibody array assays were performed, which demonstrated that HGMB1 induced the differential secretion of cytokines that are predominantly associated with cell development, regulation of growth and cell migration, stress responses, and immune system functions. Moreover, the secretion of epidermal growth factor receptor (EGFR) was significantly upregulated by HMGB1. The EGFR-activated Ras/MAPK pathway regulates the osteogenic differentiation of MSCs. These results suggested that HMGB1 enhances the secretion of various cytokines by MSCs and promotes osteogenic differentiation via the Ras/MAPK signaling pathway. The present study may provide a theoretical basis for the development of novel techniques for the treatment of bone fractures in the future. PMID:28105126

  8. Spred is a Sprouty-related suppressor of Ras signalling.

    PubMed

    Wakioka, T; Sasaki, A; Kato, R; Shouda, T; Matsumoto, A; Miyoshi, K; Tsuneoka, M; Komiya, S; Baron, R; Yoshimura, A

    2001-08-09

    Cellular proliferation, and differentiation of cells in response to extracellular signals, are controlled by the signal transduction pathway of Ras, Raf and MAP (mitogen-activated protein) kinase. The mechanisms that regulate this pathway are not well known. Here we describe two structurally similar tyrosine kinase substrates, Spred-1 and Spred-2. These two proteins contain a cysteine-rich domain related to Sprouty (the SPR domain) at the carboxy terminus. In Drosophila, Sprouty inhibits the signalling by receptors of fibroblast growth factor (FGF) and epidermal growth factor (EGF) by suppressing the MAP kinase pathway. Like Sprouty, Spred inhibited growth-factor-mediated activation of MAP kinase. The Ras-MAP kinase pathway is essential in the differentiation of neuronal cells and myocytes. Expression of a dominant negative form of Spred and Spred-antibody microinjection revealed that endogenous Spred regulates differentiation in these types of cells. Spred constitutively associated with Ras but did not prevent activation of Ras or membrane translocation of Raf. Instead, Spred inhibited the activation of MAP kinase by suppressing phosphorylation and activation of Raf. Spred may represent a class of proteins that modulate Ras-Raf interaction and MAP kinase signalling.

  9. Modulation of Presynaptic Plasticity and Learning by the H-ras/Extracellular Signal-Regulated Kinase/Synapsin I Signaling Pathway

    PubMed Central

    Kushner, Steven A.; Elgersma, Ype; Murphy, Geoffrey G.; Jaarsma, Dick; van Woerden, Geeske M.; Hojjati, Mohammad Reza; Cui, Yijun; LeBoutillier, Janelle C.; Marrone, Diano F.; Choi, Esther S.; De Zeeuw, Chris I.; Petit, Ted L.; Pozzo-Miller, Lucas; Silva, Alcino J.

    2009-01-01

    Molecular and cellular studies of the mechanisms underlying mammalian learning and memory have focused almost exclusively on postsynaptic function. We now reveal an experience-dependent presynaptic mechanism that modulates learning and synaptic plasticity in mice. Consistent with a presynaptic function for endogenous H-ras/extracellular signal-regulated kinase (ERK) signaling, we observed that, under normal physiologic conditions in wild-type mice, hippocampus-dependent learning stimulated the ERK-dependent phosphorylation of synapsin I, and MEK (MAP kinase kinase)/ERK inhibition selectively decreased the frequency of miniature EPSCs. By generating transgenic mice expressing a constitutively active form of H-ras (H-rasG12V), which is abundantly localized in axon terminals, we were able to increase the ERK-dependent phosphorylation of synapsin I. This resulted in several presynaptic changes, including a higher density of docked neurotransmitter vesicles in glutamatergic terminals, an increased frequency of miniature EPSCs, and increased paired-pulse facilitation. In addition, we observed facilitated neurotransmitter release selectively during high-frequency activity with consequent increases in long-term potentiation. Moreover, these mice showed dramatic enhancements in hippocampus-dependent learning. Importantly, deletion of synapsin I, an exclusively presynaptic protein, blocked the enhancements of learning, presynaptic plasticity, and long-term potentiation. Together with previous invertebrate studies, these results demonstrate that presynaptic plasticity represents an important evolutionarily conserved mechanism for modulating learning and memory. PMID:16237176

  10. Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

    Cancer.gov

    The NCI CPTAC program is applying its expertise in quantitative proteomics to develop assays for RAS pathway proteins. Targets include key phosphopeptides that should increase our understanding of how the RAS pathway is regulated.

  11. Regulation of Ras signaling and function by plasma membrane microdomains.

    PubMed

    Goldfinger, Lawrence E; Michael, James V

    2017-02-07

    Together H-, N- and KRAS mutations are major contributors to ~30% of all human cancers. Thus, Ras inhibition remains an important anti-cancer strategy. The molecular mechanisms of isotypic Ras oncogenesis are still not completely understood. Monopharmacological therapeutics have not been successful in the clinic. These disappointing outcomes have led to attempts to target elements downstream of Ras, mainly targeting either the Phosphatidylinositol 3-Kinase (PI3K) or Mitogen-Activated Protein Kinase (MAPK) pathways. While several such approaches are moderately effective, recent efforts have focused on preclinical evaluation of combination therapies to improve efficacies. This review will detail current understanding of the contributions of plasma membrane microdomain targeting of Ras to mitogenic and tumorigenic signaling and tumor progression. Moreover, this review will outline novel approaches to target Ras in cancers, including targeting schemes for new drug development, as well as putative re-purposing of drugs in current use to take advantage of blunting Ras signaling by interfering with Ras plasma membrane microdomain targeting and retention.

  12. Quadruple wild-type (WT) GIST: defining the subset of GIST that lacks abnormalities of KIT, PDGFRA, SDH, or RAS signaling pathways.

    PubMed

    Pantaleo, Maria A; Nannini, Margherita; Corless, Christopher L; Heinrich, Michael C

    2015-01-01

    A subset of GISTs lack mutations in the KIT/PDGFRA or RAS pathways and yet retain an intact succinate dehydrogensase (SDH) complex. We propose that these KIT/PDGFRA/SDH/RAS-P WT GIST tumors be designated as quadruple wild-type (WT) GIST. Further molecular and clinicophatological characterization of quadruple WT GIST will help to determine their prognosis as well as assist in the optimization of medical management, including clinical test of novel therapies.

  13. CCN2 is transiently expressed by keratinocytes during re-epithelialization and regulates keratinocyte migration in vitro by the ras-MEK-ERK signaling pathway.

    PubMed

    Kiwanuka, Elizabeth; Hackl, Florian; Caterson, Edward J; Nowinski, Daniel; Junker, Johan P E; Gerdin, Bengt; Eriksson, Elof

    2013-12-01

    CCN2 (previously known as connective tissue growth factor) is a multifunctional matricellular protein that has numerous effects on cell life and cell interactions with the connective tissue. Although the importance of CCN2 for the fibrotic process in wound healing has been well studied, the involvement of CCN2 in keratinocyte function has not yet been explored. Therefore, the aim of the present study was to investigate the role of CCN2 in the epidermis during wound healing. Immunohistochemistry was done on sections from full-thickness porcine wounds. The effect of CCN2 on the migration of cultured human keratinocytes exposed to scratch wounds, the effect on phosphorylation of extracellular signal-related kinases (ERK), and the effect of adding inhibitors to the ERK/mitogen-activated protein kinase pathway to human keratinocytes were studied. The CCN2 protein was transiently expressed in vivo at the leading keratinocyte edge during re-epithelialization of full-thickness porcine wounds. In vitro, exogenous addition of CCN2 to human keratinocyte cultures regulated keratinocyte migration and resulted in phosphorylation of ERK. The addition of inhibitors of ERK/mitogen-activated protein kinase counteracted the effect of CCN2 on migration. CCN2 was transiently expressed at the leading keratinocyte edge in vivo. The biologic importance of this was supported in vitro, because CCN2 regulated human keratinocyte migration through activation of the Ras-mitogen-activated protein kinase kinase-ERK signal transduction pathway. Published by Elsevier Inc.

  14. Ras Homolog Enriched in Brain (Rheb) Enhances Apoptotic Signaling*

    PubMed Central

    Karassek, Sascha; Berghaus, Carsten; Schwarten, Melanie; Goemans, Christoph G.; Ohse, Nadine; Kock, Gerd; Jockers, Katharina; Neumann, Sebastian; Gottfried, Sebastian; Herrmann, Christian; Heumann, Rolf; Stoll, Raphael

    2010-01-01

    Rheb is a homolog of Ras GTPase that regulates cell growth, proliferation, and regeneration via mammalian target of rapamycin (mTOR). Because of the well established potential of activated Ras to promote survival, we sought to investigate the ability of Rheb signaling to phenocopy Ras. We found that overexpression of lipid-anchored Rheb enhanced the apoptotic effects induced by UV light, TNFα, or tunicamycin in an mTOR complex 1 (mTORC1)-dependent manner. Knocking down endogenous Rheb or applying rapamycin led to partial protection, identifying Rheb as a mediator of cell death. Ras and c-Raf kinase opposed the apoptotic effects induced by UV light or TNFα but did not prevent Rheb-mediated apoptosis. To gain structural insight into the signaling mechanisms, we determined the structure of Rheb-GDP by NMR. The complex adopts the typical canonical fold of RasGTPases and displays the characteristic GDP-dependent picosecond to nanosecond backbone dynamics of the switch I and switch II regions. NMR revealed Ras effector-like binding of activated Rheb to the c-Raf-Ras-binding domain (RBD), but the affinity was 1000-fold lower than the Ras/RBD interaction, suggesting a lack of functional interaction. shRNA-mediated knockdown of apoptosis signal-regulating kinase 1 (ASK-1) strongly reduced UV or TNFα-induced apoptosis and suppressed enhancement by Rheb overexpression. In conclusion, Rheb-mTOR activation not only promotes normal cell growth but also enhances apoptosis in response to diverse toxic stimuli via an ASK-1-mediated mechanism. Pharmacological regulation of the Rheb/mTORC1 pathway using rapamycin should take the presence of cellular stress into consideration, as this may have clinical implications. PMID:20685651

  15. Sprouty-related Ena/Vasodilator-stimulated Phosphoprotein Homology 1-Domain-containing Protein (SPRED1), a Tyrosine-Protein Phosphatase Non-receptor Type 11 (SHP2) Substrate in the Ras/Extracellular Signal-regulated Kinase (ERK) Pathway*

    PubMed Central

    Quintanar-Audelo, Martina; Yusoff, Permeen; Sinniah, Saravanan; Chandramouli, Sumana; Guy, Graeme R.

    2011-01-01

    SHP2 is a tyrosine phosphatase involved in the activation of the Ras/ERK signaling pathway downstream of a number of receptor tyrosine kinases. One of the proposed mechanisms involving SHP2 in this context is to dephosphorylate and inactivate inhibitors of the Ras/ERK pathway. Two protein families bearing a unique, common domain, Sprouty and SPRED proteins, are possible candidates because they have been reported to inhibit the Ras/ERK pathway upon FGF activation. We tested whether any of these proteins are likely substrates of SHP2. Our findings indicate that Sprouty2 binds to the C-terminal tail of SHP2, which is an unlikely substrate binding site, whereas SPRED proteins bind to the tyrosine phosphatase domain that is known to be the binding site for its substrates. Overexpressed SHP2 was able to dephosphorylate SPREDs but not Sprouty2. Finally, we found two tyrosine residues on SPRED1 that are required, when phosphorylated, to inhibit Ras/ERK activation and identified Tyr-420 as a specific dephosphorylation target of SHP2. The evidence obtained indicates that SPRED1 is a likely substrate of SHP2, whose tyrosine dephosphorylation is required to attenuate the inhibitory action of SPRED1 in the Ras/ERK pathway. PMID:21531714

  16. Naringin-induced p21WAF1-mediated G(1)-phase cell cycle arrest via activation of the Ras/Raf/ERK signaling pathway in vascular smooth muscle cells.

    PubMed

    Lee, Eo-Jin; Moon, Gi-Seong; Choi, Won-Seok; Kim, Wun-Jae; Moon, Sung-Kwon

    2008-12-01

    The flavonoid naringin has been shown to play a role in preventing the development of cardiovascular disease. However, the exact molecular mechanisms underlying the roles of integrated cell cycle regulation and MAPK signaling pathways in the regulation of naringin-induced inhibition of cell proliferation in vascular smooth muscle cells (VSMCs) remain to be identified. Naringin treatment resulted in significant growth inhibition and G(1)-phase cell cycle arrest mediated by induction of p53-independent p21WAF1 expression; expression of cyclins and CDKs in VSMCs was also down-regulated. In addition, among the pathways examined, blockade of ERK function inhibited naringin-dependent p21WAF1 expression, reversed naringin-mediated inhibition of cell proliferation and decreased cell cycle proteins. Moreover, naringin treatment increased both Ras and Raf activations. Transfection of cells with dominant negative Ras (RasN17) and Raf (RafS621A) mutant genes suppressed naringin-induced ERK activity and p21WAF1 expression. Finally, naringin-induced reduction in cell proliferation and cell cycle protein was abolished in the presence of RasN17 and RafS621A mutant genes. The Ras/Raf/ERK pathway participates in p21WAF1 induction, leading to a decrease in cyclin D1/CDK4 and cyclin E/CDK2 complexes and in naringin-dependent inhibition of cell growth. These novel and unexpected findings provide a theoretical basis for preventive use of flavonoids to the atherosclerosis disease.

  17. Ras and Nox: linked signaling networks?

    PubMed Central

    Wu, Ru Feng; Terada, Lance S.

    2009-01-01

    Both Ras and Nox represent ancient gene families which control a broad range of cellular responses. Both families mediate signals governing motility, differentiation, and proliferation, and both inhabit overlapping subcellular microdomains. Yet little is known of the precise functional relationship between these two ubiquitous families. In this review, we examine the interface where these two large fields meet. PMID:19501154

  18. Acquisition of contextual discrimination involves the appearance of a RAS-GRF1/p38 mitogen-activated protein (MAP) kinase-mediated signaling pathway that promotes long term potentiation (LTP).

    PubMed

    Jin, Shan-Xue; Arai, Junko; Tian, Xuejun; Kumar-Singh, Rajendra; Feig, Larry A

    2013-07-26

    RAS-GRF1 is a guanine nucleotide exchange factor with the ability to activate RAS and RAC GTPases in response to elevated calcium levels. We previously showed that beginning at 1 month of age, RAS-GRF1 mediates NMDA-type glutamate receptor (NMDAR)-induction of long term depression in the CA1 region of the hippocampus of mice. Here we show that beginning at 2 months of age, when mice first acquire the ability to discriminate between closely related contexts, RAS-GRF1 begins to contribute to the induction of long term potentiation (LTP) in the CA1 hippocampus by mediating the action of calcium-permeable, AMPA-type glutamate receptors (CP-AMPARs). Surprisingly, LTP induction by CP-AMPARs through RAS-GRF1 occurs via activation of p38 MAP kinase rather than ERK MAP kinase, which has more frequently been linked to LTP. Moreover, contextual discrimination is blocked by knockdown of Ras-Grf1 expression specifically in the CA1 hippocampus, infusion of a p38 MAP kinase inhibitor into the CA1 hippocampus, or the injection of an inhibitor of CP-AMPARs. These findings implicate the CA1 hippocampus in the developmentally dependent capacity to distinguish closely related contexts through the appearance of a novel LTP-supporting signaling pathway.

  19. A new chemical inhibitor of angiogenesis and tumorigenesis that targets the VEGF signaling pathway upstream of Ras

    PubMed Central

    Desroches-Castan, Agnès; Quélard, Delphine; Demeunynck, Martine; Constant, Jean-François; Dong, Chongling; Keramidas, Michelle; Coll, Jean-Luc; Barette, Caroline; Lafanechère, Laurence; Feige, Jean-Jacques

    2015-01-01

    The efficacy of anti-angiogenic therapies on cancer patients is limited by the emergence of drug resistance, urging the search for second-generation drugs. In this study, we screened an academic chemical library (DCM, University of Grenoble-Alpes) and identified a leader molecule, COB223, that inhibits endothelial cell migration and proliferation. It inhibits also Lewis lung carcinoma (LLC/2) cell proliferation whereas it does not affect fibroblast proliferation. The anti-angiogenic activity of COB223 was confirmed using several in vitro and in vivo assays. In a mouse LLC/2 tumor model, ip administration of doses as low as 4 mg/kg COB223 efficiently reduced the tumor growth rate. We observed that COB223 inhibits endothelial cell ERK1/2 phosphorylation induced by VEGF, FGF-2 or serum and that it acts downstream of PKC and upstream of Ras. This molecule represents a novel anti-angiogenic and anti-tumorigenic agent with an original mechanism of action that deserves further development as an anti-cancer drug. PMID:25742784

  20. A new chemical inhibitor of angiogenesis and tumorigenesis that targets the VEGF signaling pathway upstream of Ras.

    PubMed

    Desroches-Castan, Agnès; Quélard, Delphine; Demeunynck, Martine; Constant, Jean-François; Dong, Chongling; Keramidas, Michelle; Coll, Jean-Luc; Barette, Caroline; Lafanechère, Laurence; Feige, Jean-Jacques

    2015-03-10

    The efficacy of anti-angiogenic therapies on cancer patients is limited by the emergence of drug resistance, urging the search for second-generation drugs. In this study, we screened an academic chemical library (DCM, University of Grenoble-Alpes) and identified a leader molecule, COB223, that inhibits endothelial cell migration and proliferation. It inhibits also Lewis lung carcinoma (LLC/2) cell proliferation whereas it does not affect fibroblast proliferation. The anti-angiogenic activity of COB223 was confirmed using several in vitro and in vivo assays. In a mouse LLC/2 tumor model, ip administration of doses as low as 4 mg/kg COB223 efficiently reduced the tumor growth rate. We observed that COB223 inhibits endothelial cell ERK1/2 phosphorylation induced by VEGF, FGF-2 or serum and that it acts downstream of PKC and upstream of Ras. This molecule represents a novel anti-angiogenic and anti-tumorigenic agent with an original mechanism of action that deserves further development as an anti-cancer drug.

  1. A genetic screen for novel components of the Ras/Mitogen-activated protein kinase signaling pathway that interact with the yan gene of Drosophila identifies split ends, a new RNA recognition motif-containing protein.

    PubMed Central

    Rebay, I; Chen, F; Hsiao, F; Kolodziej, P A; Kuang, B H; Laverty, T; Suh, C; Voas, M; Williams, A; Rubin, G M

    2000-01-01

    The receptor tyrosine kinase (RTK) signaling pathway is used reiteratively during the development of all multicellular organisms. While the core RTK/Ras/MAPK signaling cassette has been studied extensively, little is known about the nature of the downstream targets of the pathway or how these effectors regulate the specificity of cellular responses. Drosophila yan is one of a few downstream components identified to date, functioning as an antagonist of the RTK/Ras/MAPK pathway. Previously, we have shown that ectopic expression of a constitutively active protein (yan(ACT)) inhibits the differentiation of multiple cell types. In an effort to identify new genes functioning downstream in the Ras/MAPK/yan pathway, we have performed a genetic screen to isolate dominant modifiers of the rough eye phenotype associated with eye-specific expression of yan(ACT). Approximately 190,000 mutagenized flies were screened, and 260 enhancers and 90 suppressors were obtained. Among the previously known genes we recovered are four RTK pathway components, rolled (MAPK), son-of-sevenless, Star, and pointed, and two genes, eyes absent and string, that have not been implicated previously in RTK signaling events. We also isolated mutations in five previously uncharacterized genes, one of which, split ends, we have characterized molecularly and have shown to encode a member of the RRM family of RNA-binding proteins. PMID:10655223

  2. Lymphatic Vessel Abnormalities Arising from Disorders of Ras Signal Transduction

    PubMed Central

    Sevick-Muraca, Eva M.; King, Philip D.

    2013-01-01

    A number of genetic diseases in man have been described in which abnormalities in the development and function of the lymphatic vascular (LV) system are prominent features. The genes that are mutated in these diseases are varied and include genes that encode lymphatic endothelial cell (LEC) growth factor receptors and their ligands and transcription factors that control LEC fate and function. In addition, an increasing number of genes have been identified that encode components of the Ras signal transduction pathway that conveys signals from cell surface receptors to regulate cell growth, proliferation and differentiation. Gene targeting studies performed in mice have confirmed that the LV system is particularly susceptible to perturbations in the Ras pathway. PMID:24183794

  3. Rasputin, the Drosophila homologue of the RasGAP SH3 binding protein, functions in ras- and Rho-mediated signaling.

    PubMed

    Pazman, C; Mayes, C A; Fanto, M; Haynes, S R; Mlodzik, M

    2000-04-01

    The small GTPase Ras plays an important role in many cellular signaling processes. Ras activity is negatively regulated by GTPase activating proteins (GAPs). It has been proposed that RasGAP may also function as an effector of Ras activity. We have identified and characterized the Drosophila homologue of the RasGAP-binding protein G3BP encoded by rasputin (rin). rin mutants are viable and display defects in photoreceptor recruitment and ommatidial polarity in the eye. Mutations in rin/G3BP genetically interact with components of the Ras signaling pathway that function at the level of Ras and above, but not with Raf/MAPK pathway components. These interactions suggest that Rin is required as an effector in Ras signaling during eye development, supporting an effector role for RasGAP. The ommatidial polarity phenotypes of rin are similar to those of RhoA and the polarity genes, e.g. fz and dsh. Although rin/G3BP interacts genetically with RhoA, affecting both photoreceptor differentiation and polarity, it does not interact with the gain-of-function genotypes of fz and dsh. These data suggest that Rin is not a general component of polarity generation, but serves a function specific to Ras and RhoA signaling pathways.

  4. RAS signaling dysregulation in human embryonal Rhabdomyosarcoma.

    PubMed

    Martinelli, Simone; McDowell, Heather P; Vigne, Silvia Delle; Kokai, George; Uccini, Stefania; Tartaglia, Marco; Dominici, Carlo

    2009-11-01

    Rhabdomyosarcoma (RMS) is a common childhood solid tumor, resulting from dysregulation of the skeletal myogenesis program. Two major histological subtypes occur in childhood RMS, embryonal and alveolar. While chromosomal rearrangements account for the majority of alveolar tumors, the genetic defects underlying the pathogenesis of embryonal RMS remain largely undetermined. A few studies performed on small series of embryonal tumors suggest that dysregulation of RAS function may be relevant to disease pathogenesis. To explore further the biological and clinical relevance of mutations with perturbing consequences on RAS signaling in embryonal RMS, we investigated the prevalence of PTPN11, HRAS, KRAS, NRAS, BRAF, MEK1, and MEK2 mutations in a relatively large cohort of primary tumors. While HRAS and KRAS were found to be rarely mutated, we identified somatic NRAS lesions in 20% of cases. All mutations were missense and affected codon 61, with the introduction of a positive charged amino acid residue representing the most common event. PTPN11 was found mutated in one tumor specimen, confirming that somatic defects in this gene are relatively uncommon in RMS, while no mutation was observed in BRAF and MEK genes. Although no clear association of mutations with any clinical variable was observed, comparison of the outcome between mutation-positive and mutation-negative cases indicated a trend for a higher percentage of patients exhibiting a better outcome in the former. Our findings provide evidence that dysregulation of RAS signaling is a major event contributing to embryonal RMS pathogenesis. Copyright 2009 Wiley-Liss, Inc.

  5. Targeting the RAS pathway in melanoma.

    PubMed

    Ji, Zhenyu; Flaherty, Keith T; Tsao, Hensin

    2012-01-01

    Metastatic melanoma is a highly lethal type of skin cancer and is often refractory to all traditional chemotherapeutic agents. Key insights into the genetic makeup of melanoma tumors have led to the development of promising targeted agents. An activated RAS pathway, anchored by oncogenic BRAF, appears to be the central motor driving melanoma proliferation. Although recent clinical trials have brought enormous hope to patients with melanoma, adverse effects and novel escape mechanisms of these inhibitors have already emerged. Definition of the limits of the first successful targeted therapies will provide the basis for further advances in management of disseminated melanoma. In this review, the current state of targeted therapy for melanoma is discussed, including the potent BRAF(V600E) inhibitor vemurafenib. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Ras Family Small GTPase-mediated Neuroprotective Signaling in Stroke

    PubMed Central

    Shi, Geng-Xian; Andres, Douglas A.; Cai, Weikang

    2012-01-01

    Selective neuronal cell death is one of the major causes of neuronal damage following stroke, and cerebral cells naturally mobilize diverse survival signaling pathways to protect against ischemia. Importantly, therapeutic strategies designed to improve endogenous anti-apoptotic signaling appear to hold great promise in stroke treatment. While a variety of complex mechanisms have been implicated in the pathogenesis of stroke, the overall mechanisms governing the balance between cell survival and death are not well-defined. Ras family small GTPases are activated following ischemic insults, and in turn, serve as intrinsic switches to regulate neuronal survival and regeneration. Their ability to integrate diverse intracellular signal transduction pathways makes them critical regulators and potential therapeutic targets for neuronal recovery after stroke. This article highlights the contribution of Ras family GTPases to neuroprotective signaling cascades, including mitogen-activated protein kinase (MAPK) family protein kinase- and AKT/PKB-dependent signaling pathways as well as the regulation of cAMP response element binding (CREB), Forkhead box O (FoxO) and hypoxia-inducible factor 1(HIF1) transcription factors, in stroke. PMID:21521171

  7. Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

    DOE PAGES

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; ...

    2015-06-16

    Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referredmore » to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.« less

  8. Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

    SciTech Connect

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li -Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

    2015-06-16

    Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.

  9. Regulation of cell cycle progression and apoptosis by the Ras/Raf/MEK/ERK pathway (Review).

    PubMed

    Chang, Fumin; Steelman, Linda S; Shelton, John G; Lee, John T; Navolanic, Patrick M; Blalock, William L; Franklin, Richard; McCubrey, James A

    2003-03-01

    The Ras/Raf/MEK/ERK signal transduction pathway regulates cell cycle progression and apoptosis in diverse types of cells. Mutations in this pathway are often observed in transformed cell lines and frequently linked with human cancers. The Ras/Raf/MEK/ERK pathway can induce events both associated with cell proliferation and cell cycle arrest. The particular course chosen may depend on the strength and the particular Raf gene activated by Ras. This pathway also is involved in maintaining cell survival by modulating the activity of apoptotic molecules including Bad and Bcl-2. This review will discuss the regulation of the Ras/Raf/MEK/ERK pathway and how it modulates cell cycle progression and cell survival.

  10. Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module.

    PubMed

    Toettcher, Jared E; Weiner, Orion D; Lim, Wendell A

    2013-12-05

    The complex, interconnected architecture of cell-signaling networks makes it challenging to disentangle how cells process extracellular information to make decisions. We have developed an optogenetic approach to selectively activate isolated intracellular signaling nodes with light and use this method to follow the flow of information from the signaling protein Ras. By measuring dose and frequency responses in single cells, we characterize the precision, timing, and efficiency with which signals are transmitted from Ras to Erk. Moreover, we elucidate how a single pathway can specify distinct physiological outcomes: by combining distinct temporal patterns of stimulation with proteomic profiling, we identify signaling programs that differentially respond to Ras dynamics, including a paracrine circuit that activates STAT3 only after persistent (>1 hr) Ras activation. Optogenetic stimulation provides a powerful tool for analyzing the intrinsic transmission properties of pathway modules and identifying how they dynamically encode distinct outcomes.

  11. RasG signaling is important for optimal folate chemotaxis in Dictyostelium

    PubMed Central

    2014-01-01

    Background Signaling pathways linking receptor activation to actin reorganization and pseudopod dynamics during chemotaxis are arranged in complex networks. Dictyostelium discoideum has proven to be an excellent model system for studying these networks and a body of evidence has indicated that RasG and RasC, members of the Ras GTPase subfamily function as key chemotaxis regulators. However, recent evidence has been presented indicating that Ras signaling is not important for Dictyostelium chemotaxis. In this study, we have reexamined the role of Ras proteins in folate chemotaxis and then, having re-established the importance of Ras for this process, identified the parts of the RasG protein molecule that are involved. Results A direct comparison of folate chemotaxis methodologies revealed that rasG-C- cells grown in association with a bacterial food source were capable of positive chemotaxis, only when their initial position was comparatively close to the folate source. In contrast, cells grown in axenic medium orientate randomly regardless of their distance to the micropipette. Folate chemotaxis is restored in rasG-C- cells by exogenous expression of protein chimeras containing either N- or C- terminal halves of the RasG protein. Conclusions Conflicting data regarding the importance of Ras to Dictyostelium chemotaxis were the result of differing experimental methodologies. Both axenic and bacterially grown cells require RasG for optimal folate chemotaxis, particularly in weak gradients. In strong gradients, the requirement for RasG is relaxed, but only in bacterially grown cells. Both N- and C- terminal portions of the RasG protein are important for folate chemotaxis, suggesting that there are functionally important amino acids outside the well established switch I and switch II interaction surfaces. PMID:24742374

  12. Ras signaling pathway in the chemopreventive action of different ratios of fish oil and corn oil in experimentally induced colon carcinogenesis.

    PubMed

    Kansal, S; Negi, A K; Bhatnagar, A; Agnihotri, N

    2012-01-01

    Dietary factors play a significant role in colon cancer. The essential polyunsaturated fatty acids (PUFAs), n-3 PUFAs, and n-6 PUFAs exert inverse effect on cancer. This study was designed to understand the mechanism of chemopreventive action of different ratios of fish oil (FO) and corn oil (CO) in colon carcinoma. Wistar rats were divided into 3 groups: Group 1 received purified diet whereas Groups 2 and 3 received modified diet with FO:CO (1:1) and FO:CO (2.5:1), respectively. The groups were further subdivided into controls receiving ethylenediamine-tetra acetic-acid and treated groups received dimethylhydrazine-dihydrochloride (DMH)/wk for 4 wk. Animals sacrificed 48 h after last injection constituted initiation phase and that sacrificed after 16 wk constituted post-initiation phase. Differential effect of different ratios of FO and CO was analyzed in isolated colonocytes. In both phases, DMH treatment showed an increase in pan Ras, Raf, MEK1/2, extracellular signal regulated kinase (Erk)1/2, and c-fos levels. Akt levels were increased in post-initiation phase only. Treatment with FO + CO (1:1) + DMH decreased pan Ras, MEK1/2 and Erk1/2 levels in post-initiation phase whereas Raf and c-fos were decreased in both phases. Treatment with FO + CO (2.5:1) + DMH decreased Ras, Raf, MEK1/2, Erk1/2, and c-fos levels in both phases. Akt was decreased in post-initiation phase only. The chemo-preventive action of FO and CO may be mediated by time- and dose-dependent effect.

  13. Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade.

    PubMed Central

    Benn, J; Schneider, R J

    1994-01-01

    Hepatitis B virus produces a small (154-amino acid) transcriptional transactivating protein, HBx, which is required for viral infection and has been implicated in virus-mediated liver oncogenesis. However, the molecular mechanism for HBx activity and its possible influence on cell proliferation have remained obscure. A number of studies suggest that HBx may stimulate transcription by indirectly activating transcription factors, possibly by influencing cell signaling pathways. We now present biochemical evidence that HBx activates Ras and rapidly induces a cytoplasmic signaling cascade linking Ras, Raf, and mitogen-activated protein kinase (MAP kinase), leading to transcriptional transactivation. HBx strongly elevates levels of GTP-bound Ras, activated and phosphorylated Raf, and tyrosine-phosphorylated and activated MAP kinase. Transactivation of transcription factor AP-1 by HBx is blocked by inhibition of Ras or Raf activities but not by inhibition of Ca(2+)- and diacylglycerol-dependent protein kinase C. HBx was also found to stimulate DNA synthesis in serum-starved cells. The hepatitis B virus HBx protein therefore stimulates Ras-GTP complex formation and promotes downstream signaling through Raf and MAP kinases, and may influence cell proliferation. Images PMID:7937954

  14. SodC modulates ras and PKB signaling in Dictyostelium.

    PubMed

    Castillo, Boris; Kim, Seon-Hee; Sharief, Mujataba; Sun, Tong; Kim, Lou W

    2017-01-01

    We have previously reported that the basal RasG activity is aberrantly high in cells lacking Superoxide dismutase C (SodC). Here we report that other Ras proteins such as RasC and RasD activities are not affected in sodC(-) cells and mutagenesis studies showed that the presence of the Cys(118) in the Ras proteins is essential for the superoxide-mediated activation of Ras proteins in Dictyostelium. In addition to the loss of SodC, lack of extracellular magnesium ions increased the level of intracellular superoxide and active RasG proteins. Aberrantly active Ras proteins in sodC(-) cells persistently localized at the plasma membrane, but those in wild type cells under magnesium deficient medium exhibited intracellular vesicular localization. Interestingly, the aberrantly activated Ras proteins in wild type cells were largely insulated from their normal downstream events such as Phosphatidylinositol-3,4,5-P3 (PIP3) accumulation, Protein Kinase B (PKB) activation, and PKBs substrates phosphorylation. Intriguingly, however, aberrantly activated Ras proteins in sodC(-) cells were still engaged in signaling to their downstream targets, and thus excessive PKBs substrates phosphorylation persisted. In summary, we suggest that SodC and RasG proteins are essential part of a novel inhibitory mechanism that discourages oxidatively stressed cells from chemotaxis and thus inhibits the delivery of potentially damaged genome to the next generation.

  15. The transcription factor Gfi1 regulates G-CSF signaling and neutrophil development through the Ras activator RasGRP1

    PubMed Central

    de la Luz Sierra, Maria; Sakakibara, Shuhei; Gasperini, Paola; Salvucci, Ombretta; Jiang, Kan; McCormick, Peter J.; Segarra, Marta; Stone, Jim; Maric, Dragan; Zhu, Jinfang; Qian, Xiaolan; Lowy, Douglas R.

    2010-01-01

    The transcription factor growth factor independence 1 (Gfi1) and the growth factor granulocyte colony-stimulating factor (G-CSF) are individually essential for neutrophil differentiation from myeloid progenitors. Here, we provide evidence that the functions of Gfi1 and G-CSF are linked in the regulation of granulopoiesis. We report that Gfi1 promotes the expression of Ras guanine nucleotide releasing protein 1 (RasGRP1), an exchange factor that activates Ras, and that RasGRP1 is required for G-CSF signaling through the Ras/mitogen–activated protein/extracellular signal-regulated kinase (MEK/Erk) pathway. Gfi1-null mice have reduced levels of RasGRP1 mRNA and protein in thymus, spleen, and bone marrow, and Gfi1 transduction in myeloid cells promotes RasGRP1 expression. When stimulated with G-CSF, Gfi1-null myeloid cells are selectively defective at activating Erk1/2, but not signal transducer and activator of transcription 1 (STAT1) or STAT3, and fail to differentiate into neutrophils. Expression of RasGRP1 in Gfi1-deficient cells rescues Erk1/2 activation by G-CSF and allows neutrophil maturation by G-CSF. These results uncover a previously unknown function of Gfi1 as a regulator of RasGRP1 and link Gfi1 transcriptional control to G-CSF signaling and regulation of granulopoiesis. PMID:20203268

  16. Ras signaling is involved in the expression of Fas-L in glioma.

    PubMed

    Yang, B C; Wang, Y S; Liu, H S; Lin, S J

    2000-04-01

    Fas-L expresses on a variety of tumors and is suspected to modify the dialog between tumor and the immune system. However, the cellular abnormality in tumor cells leading to an aberrant expression of Fas-L is unclear. In this study, we demonstrate the involvement of Ras signaling in the Fas-L expression in several ways. First, the activated Ha-rasval12 gene enhanced the Fas-L expression of primary human glial cells. Second, blocking the Ras signal pathway in glioma cells by lovastatin or the Ha-rasAsn17 dominant-negative mutant gene resulted in reduced Fas-L expression. Transfection of the Ha-rasAsn17 into glioma cells also inhibited the activation of NFKB, which is a downstream component of Ras signaling. Accordingly, the membrane-permeable NFKB competitor suppressed the Fas-L expression. Furthermore, the Fas-L expression coincided with the Ras activity in the murine 212 cells, in which the Ras activity could be induced by isopropyl 3-D-thiogalactoside. In summary, these results suggest that the enhanced Ras signaling with consequential NFKB activation, which is a frequent defect found in tumors, could mediate the Fas-L expression of tumors.

  17. RAS-MAPK pathway epigenetic activation in cancer: miRNAs in action

    PubMed Central

    Masliah-Planchon, Julien; Garinet, Simon; Pasmant, Eric

    2016-01-01

    The highly conserved RAS-mitogen activated protein kinase (MAPK) signaling pathway is involved in a wide range of cellular processes including differentiation, proliferation, and survival. Somatic mutations in genes encoding RAS-MAPK components frequently occur in many tumors, making the RAS-MAPK a critical pathway in human cancer. Since the pioneering study reporting that let-7 miRNA acted as tumor suppressor by repressing the RAS oncogene, growing evidence has suggested the importance of miRNAs targeting the RAS-MAPK in oncogenesis. MiRNAs alterations in human cancers may act as a rheostat of the oncogenic RAS signal that is often amplified as cancers progress. However, specific mechanisms leading to miRNAs deregulation and their functional consequences in cancer are far from being fully elucidated. In this review, we provide an experimental-validated map of RAS-MAPK oncomiRs and tumor suppressor miRNAs from transmembrane receptor to downstream ERK proteins. MiRNAs could be further considered as potential genetic biomarkers for diagnosis, prognosis, or therapeutic purpose. PMID:26646588

  18. Role of Ras and Mapks in TGFbeta signaling.

    PubMed

    Mulder, K M

    2000-01-01

    Normal signaling by TGFbeta, in the absence of serum or exogenous factors, involves a rapid activation of Ras, Erks, and Sapks in proliferating cultures of TGFbeta-sensitive untransformed epithelial cells and human carcinoma cells. Expression of either RasN17 or dominant-negative (DN) MKK4, or addition of the MEK1 inhibitor PD98059, can block the ability of TGFbeta to induce AP-1 complex formation at the TGFbeta(1) promoter and to autoinduce its own production. The primary components present in this TGFbeta-stimulated AP-1 complex are JunD and Fra-2, although c-Jun, and possibly Fos B, may also be present. While there are two potential Smad binding elements (SBE's) in the TGFbeta(1) promoter, supershift assays suggest that at least one of these does not bind Smad4, and the other is unable to bind factors activated by TGFbeta. In contrast, TGFbeta autoinduction is Smad3-dependent, as DN Smad3 inhibits the ability of TGFbeta to stimulate TGFbeta(1) promoter activity. Our results indicate that TGFbeta can activate both the MKK4/Sapk and MEK/Erk pathways, through Ras and TGFbeta R(I) and R(II), to induce TGFbeta(1) production; Smad4 does not appear to be involved, and Smad3 appears to function independently of this Smad4. We also demonstrate that activation of the Ras/Mapk pathway by TGFbeta positively modulates Smad1-signaling-pathway activation by TGFbeta. In addition, Smad1 could enhance TGFbeta activation of the SBE reporter SBE-luc and this effect could be blocked by co-expression of a DN TGFbeta R(I) receptor or by the MEK1 inhibitor PD98059. This cross-talk between the MEK/Erk and Smad1 pathways was mediated through the four Erk consensus phosphorylation sites in the linker region of Smad1. Mutation of these sites resulted in a loss of the ligand-dependence of both Smad1-Smad4 interactions and nuclear accumulation of Smad1, as well as a loss of the ability of Smad1 to enhance TGFbeta-mediated SBE activation. Our results provide evidence that Erk

  19. Targeting of Ras-mediated FGF signaling suppresses Pten-deficient skin tumor.

    PubMed

    Mathew, Grinu; Hannan, Abdul; Hertzler-Schaefer, Kristina; Wang, Fen; Feng, Gen-Sheng; Zhong, Jian; Zhao, Jean J; Downward, Julian; Zhang, Xin

    2016-11-15

    Deficiency in PTEN (phosphatase and tensin homolog deleted on chromosome 10) is the underlying cause of PTEN hamartoma tumor syndrome and a wide variety of human cancers. In skin epidermis, we have previously identified an autocrine FGF signaling induced by loss of Pten in keratinocytes. In this study, we demonstrate that skin hyperplasia requires FGF receptor adaptor protein Frs2α and tyrosine phosphatase Shp2, two upstream regulators of Ras signaling. Although the PI3-kinase regulatory subunits p85α and p85β are dispensable, the PI3-kinase catalytic subunit p110α requires interaction with Ras to promote hyperplasia in Pten-deficient skin, thus demonstrating an important cross-talk between Ras and PI3K pathways. Furthermore, genetic and pharmacological inhibition of Ras-MAPK pathway impeded epidermal hyperplasia in Pten animals. These results reveal a positive feedback loop connecting Pten and Ras pathways and suggest that FGF-activated Ras-MAPK pathway is an effective therapeutic target for preventing skin tumor induced by aberrant Pten signaling.

  20. Ets Factors Regulate Neural Stem Cell Depletion and Gliogenesis in Ras Pathway Glioma.

    PubMed

    Breunig, Joshua J; Levy, Rachelle; Antonuk, C Danielle; Molina, Jessica; Dutra-Clarke, Marina; Park, Hannah; Akhtar, Aslam Abbasi; Kim, Gi Bum; Hu, Xin; Bannykh, Serguei I; Verhaak, Roel G W; Danielpour, Moise

    2015-07-14

    As the list of putative driver mutations in glioma grows, we are just beginning to elucidate the effects of dysregulated developmental signaling pathways on the transformation of neural cells. We have employed a postnatal, mosaic, autochthonous glioma model that captures the first hours and days of gliomagenesis in more resolution than conventional genetically engineered mouse models of cancer. We provide evidence that disruption of the Nf1-Ras pathway in the ventricular zone at multiple signaling nodes uniformly results in rapid neural stem cell depletion, progenitor hyperproliferation, and gliogenic lineage restriction. Abolishing Ets subfamily activity, which is upregulated downstream of Ras, rescues these phenotypes and blocks glioma initiation. Thus, the Nf1-Ras-Ets axis might be one of the select molecular pathways that are perturbed for initiation and maintenance in glioma. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  1. RALB provides critical survival signals downstream of Ras in acute myeloid leukemia

    PubMed Central

    Eckfeldt, Craig E.; Pomeroy, Emily J.; Lee, Robin D.W.; Hazen, Katherine S.; Lee, Lindsey A.; Moriarity, Branden S.; Largaespada, David A.

    2016-01-01

    Mutations that activate RAS proto-oncogenes and their effectors are common in acute myeloid leukemia (AML); however, efforts to therapeutically target Ras or its effectors have been unsuccessful, and have been hampered by an incomplete understanding of which effectors are required for AML proliferation and survival. We investigated the role of Ras effector pathways in AML using murine and human AML models. Whereas genetic disruption of NRAS(V12) expression in an NRAS(V12) and Mll-AF9-driven murine AML induced apoptosis of leukemic cells, inhibition of phosphatidylinositol-3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) signaling did not reproduce this effect. Conversely, genetic disruption of RALB signaling induced AML cell death and phenocopied the effects of suppressing oncogenic Ras directly – uncovering a novel role for RALB signaling in AML survival. Knockdown of RALB led to decreased phosphorylation of TBK1 and reduced BCL2 expression, providing mechanistic insight into RALB survival signaling in AML. Notably, we found that patient-derived AML blasts have higher levels of RALB-TBK1 signaling compared to normal blood leukocytes, supporting a pathophysiologic role for RALB signaling for AML patients. Overall, our work provides new insight into the specific roles of Ras effector pathways in AML and has identified RALB signaling as a key survival pathway. PMID:27556501

  2. The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis

    SciTech Connect

    Tsujita, Maristela; Batista, Wagner L.; Ogata, Fernando T.; Monteiro, Hugo P. Arai, Roberto J.

    2008-05-16

    p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras{sup C118S}) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG.

  3. The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis.

    PubMed

    Tsujita, Maristela; Batista, Wagner L; Ogata, Fernando T; Stern, Arnold; Monteiro, Hugo P; Arai, Roberto J

    2008-05-16

    p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras(C118S)) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG.

  4. MicroRNA-122 confers sorafenib resistance to hepatocellular carcinoma cells by targeting IGF-1R to regulate RAS/RAF/ERK signaling pathways.

    PubMed

    Xu, Yanmin; Huang, Ji; Ma, Leina; Shan, Juanjuan; Shen, Junjie; Yang, Zhi; Liu, Limei; Luo, Yongli; Yao, Chao; Qian, Cheng

    2016-02-28

    Sorafenib is the first-line treatment for advanced hepatocellular carcinoma (HCC), but the clinical response to sorafenib is seriously limited by drug resistance. In this study, we investigated the molecular mechanisms of sorafenib resistance in HCC cells. Our miRNA microarray data indicate that liver-specific miR-122 expression was significantly reduced in sorafenib-resistant cells. Overexpression of miR-122 made drug-tolerant cells sensitive to sorafenib and induced apoptosis. Insulin-like growth factor 1 receptor (IGF-1R) was validated as a target of miR-122 and was repressed by this miRNA. miR-122-induced apoptosis was repressed by the IGF-1R activator IGFI or IGFII. Conversely, the IGF-1R inhibitor PPP or NVP-AEW541 in combination with sorafenib significantly induced cell apoptosis and disrupted tolerance to drugs in vitro. These results indicated that activation of IGF-1R by ectopic down-regulation of miR-122 counteracted the effects of sorafenib-induced apoptosis, thus conferring sorafenib resistance. Further study revealed that activation of IGF-1R by miR-122 down-regulation contributed to activation of RAS/RAF/ERK signaling, which was associated with drug resistance. Our data imply that an intimate correlation between miR-122 and IGF-1R abnormal expression is a critical determinant of sorafenib tolerance. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. Abnormal Ras signaling in Costello syndrome (CS) negatively regulates enamel formation

    PubMed Central

    Goodwin, Alice F.; Tidyman, William E.; Jheon, Andrew H.; Sharir, Amnon; Zheng, Xu; Charles, Cyril; Fagin, James A.; McMahon, Martin; Diekwisch, Thomas G.H.; Ganss, Bernhard; Rauen, Katherine A.; Klein, Ophir D.

    2014-01-01

    RASopathies are syndromes caused by gain-of-function mutations in the Ras signaling pathway. One of these conditions, Costello syndrome (CS), is typically caused by an activating de novo germline mutation in HRAS and is characterized by a wide range of cardiac, musculoskeletal, dermatological and developmental abnormalities. We report that a majority of individuals with CS have hypo-mineralization of enamel, the outer covering of teeth, and that similar defects are present in a CS mouse model. Comprehensive analysis of the mouse model revealed that ameloblasts, the cells that generate enamel, lacked polarity, and the ameloblast progenitor cells were hyperproliferative. Ras signals through two main effector cascades, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways. To determine through which pathway Ras affects enamel formation, inhibitors targeting either PI3K or MEK 1 and 2 (MEK 1/2), kinases in the MAPK pathway, were utilized. MEK1/2 inhibition rescued the hypo-mineralized enamel, normalized the ameloblast polarity defect and restored normal progenitor cell proliferation. In contrast, PI3K inhibition only corrected the progenitor cell proliferation phenotype. We demonstrate for the first time the central role of Ras signaling in enamel formation in CS individuals and present the mouse incisor as a model system to dissect the roles of the Ras effector pathways in vivo. PMID:24057668

  6. Abnormal Ras signaling in Costello syndrome (CS) negatively regulates enamel formation.

    PubMed

    Goodwin, Alice F; Tidyman, William E; Jheon, Andrew H; Sharir, Amnon; Zheng, Xu; Charles, Cyril; Fagin, James A; McMahon, Martin; Diekwisch, Thomas G H; Ganss, Bernhard; Rauen, Katherine A; Klein, Ophir D

    2014-02-01

    RASopathies are syndromes caused by gain-of-function mutations in the Ras signaling pathway. One of these conditions, Costello syndrome (CS), is typically caused by an activating de novo germline mutation in HRAS and is characterized by a wide range of cardiac, musculoskeletal, dermatological and developmental abnormalities. We report that a majority of individuals with CS have hypo-mineralization of enamel, the outer covering of teeth, and that similar defects are present in a CS mouse model. Comprehensive analysis of the mouse model revealed that ameloblasts, the cells that generate enamel, lacked polarity, and the ameloblast progenitor cells were hyperproliferative. Ras signals through two main effector cascades, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways. To determine through which pathway Ras affects enamel formation, inhibitors targeting either PI3K or MEK 1 and 2 (MEK 1/2), kinases in the MAPK pathway, were utilized. MEK1/2 inhibition rescued the hypo-mineralized enamel, normalized the ameloblast polarity defect and restored normal progenitor cell proliferation. In contrast, PI3K inhibition only corrected the progenitor cell proliferation phenotype. We demonstrate for the first time the central role of Ras signaling in enamel formation in CS individuals and present the mouse incisor as a model system to dissect the roles of the Ras effector pathways in vivo.

  7. Reduced signaling of PI3K-Akt and RAS-MAPK pathways are the key targets for weight loss-induced cancer prevention by dietary calorie restriction and/or physical activity

    PubMed Central

    Standard, Joseph; Jiang, Yu; Yu, Miao; Su, Xiaoyu; Zhao, Zhihui; Xu, Jianteng; Chen, Jie; King, Brenee; Lu, Lizhi; Tomich, John; Baybutt, Richard; Wang, Weiqun

    2014-01-01

    Weight control through either dietary calorie restriction (DCR) or exercise has been associated with cancer prevention in animal models. However, the underlying mechanisms are not fully defined. Bioinformatics using genomics, proteomics, and lipidomics were employed to elucidate the molecular targets of weight control in a mouse skin cancer model. SENCAR mice were randomly assigned into 4 groups for 10 weeks: ad lib-fed sedentary control, ad lib-fed exercise (AE), exercise but pair-fed isocaloric amount of control (PE), and 20% DCR. Two hours after topical TPA treatment, skin epidermis was analyzed by Affymetrix for gene expression, DIGE for proteomics, and lipidomics for phospholipids. Body weights were significantly reduced in both DCR and PE but not AE mice versus the control. Among 39,000 transcripts, 411, 67, and 110 genes were significantly changed in DCR, PE, and AE, respectively. The expression of genes relevant to PI3K-Akt and Ras-MAPK signaling was effectively reduced by DCR and PE but not AE as measured through GenMAPP software. Proteomics analysis identified ~120 proteins, with 27 proteins significantly changed by DCR, including upregulated apolipoprotein A-1, a key antioxidant protein that decreases Ras-MAPK activity. Of the total 338 phospholipids analyzed by lipidomics, 57 decreased by PE including 5 phophatidylinositol species that serve as PI3K substrates. Although a full impact has not been determined yet, it appears the reduction of both Ras-MAPK and PI3K-Akt signaling pathways are cancer preventive targets that have been consistently demonstrated by three bioinformatics approaches. PMID:25283328

  8. Reduced signaling of PI3K-Akt and RAS-MAPK pathways is the key target for weight-loss-induced cancer prevention by dietary calorie restriction and/or physical activity.

    PubMed

    Standard, Joseph; Jiang, Yu; Yu, Miao; Su, Xiaoyu; Zhao, Zhihui; Xu, Jianteng; Chen, Jie; King, Brenee; Lu, Lizhi; Tomich, John; Baybutt, Richard; Wang, Weiqun

    2014-12-01

    Weight control through either dietary calorie restriction (DCR) or exercise has been associated with cancer prevention in animal models. However, the underlying mechanisms are not fully defined. Bioinformatics using genomics, proteomics and lipidomics was employed to elucidate the molecular targets of weight control in a mouse skin cancer model. SENCAR mice were randomly assigned into four groups for 10 weeks: ad-libitum-fed sedentary control, ad-libitum-fed exercise (AE), exercise but pair-fed isocaloric amount of control (PE) and 20% DCR. Two hours after topical TPA treatment, skin epidermis was analyzed by Affymetrix for gene expression, DIGE for proteomics and lipidomics for phospholipids. Body weights were significantly reduced in both DCR and PE but not AE mice versus the control. Among 39,000 transcripts, 411, 67 and 110 genes were significantly changed in DCR, PE and AE, respectively. The expression of genes relevant to PI3K-Akt and Ras-MAPK signaling was effectively reduced by DCR and PE but not AE as measured through GenMAPP software. Proteomics analysis identified ~120 proteins, with 27 proteins significantly changed by DCR, including up-regulated apolipoprotein A-1, a key antioxidant protein that decreases Ras-MAPK activity. Of the total 338 phospholipids analyzed by lipidomics, 57 decreased by PE including 5 phophatidylinositol species that serve as PI3K substrates. Although a full impact has not been determined yet, it appears that the reduction of both Ras-MAPK and PI3K-Akt signaling pathways is a cancer preventive target that has been consistently demonstrated by three bioinformatics approaches.

  9. Platelet-rich plasma stimulates human dermal fibroblast proliferation via a Ras-dependent extracellular signal-regulated kinase 1/2 pathway.

    PubMed

    Hara, Tomoya; Kakudo, Natsuko; Morimoto, Naoki; Ogawa, Takeshi; Lai, Fangyuan; Kusumoto, Kenji

    2016-12-01

    Platelet-rich plasma (PRP) contains a high concentration of several growth factors and contributes to soft-tissue engineering and wound healing. However, the effect of PRP on human dermal fibroblast proliferation and responses is unknown. This was investigated in the present study using PRP prepared from the whole human blood using the double-spin method. Human dermal fibroblast cultures were established from skin samples collected during plastic surgery. Platelet concentration and growth factor levels in PRP were estimated, and a cell proliferation assay was carried out after PRP treatment. The role of Ras-dependent extracellular signal-regulated kinase (ERK)1/2 in the effects of PRP was investigated in human dermal fibroblasts by suppressing ERK1/2 expression with an inhibitor or by short interfering (si)RNA-mediated knockdown, and assessing ERK1/2 phosphorylation by western blotting as well as proliferation in PRP-treated cells. We found that PRP stimulated human dermal fibroblast proliferation, which was suppressed by ERK1/2 inhibitor treatment (P < 0.01). ERK1/2 phosphorylation was increased in the presence of PRP, while siRNA-mediated knockdown of ERK1/2 blocked cell proliferation normally induced by PRP treatment (P < 0.01). These results demonstrate that PRP induces human dermal fibroblast proliferation via activation of ERK1/2 signaling. Our findings provide a basis for the development of agents that can promote wound healing and can be applied to soft-tissue engineering.

  10. Ras and Rap signaling in synaptic plasticity and mental disorders.

    PubMed

    Stornetta, Ruth L; Zhu, J Julius

    2011-02-01

    The Ras family GTPases (Ras, Rap1, and Rap2) and their downstream mitogen-activated protein kinases (ERK, JNK, and p38MAPK) and PI3K signaling cascades control various physiological processes. In neuronal cells, recent studies have shown that these parallel cascades signal distinct forms of AMPA-sensitive glutamate receptor trafficking during experience-dependent synaptic plasticity and adaptive behavior. Interestingly, both hypo- and hyperactivation of Ras/ Rap signaling impair the capacity of synaptic plasticity, underscoring the importance of a "happy-medium" dynamic regulation of the signaling. Moreover, accumulating reports have linked various genetic defects that either up- or down-regulate Ras/Rap signaling with several mental disorders associated with learning disability (e.g., Alzheimer's disease, Angelman syndrome, autism, cardio-facio-cutaneous syndrome, Coffin-Lowry syndrome, Costello syndrome, Cowden and Bannayan-Riley-Ruvalcaba syndromes, fragile X syndrome, neurofibromatosis type 1, Noonan syndrome, schizophrenia, tuberous sclerosis, and X-linked mental retardation), highlighting the necessity of happy-medium dynamic regulation of Ras/Rap signaling in learning behavior. Thus, the recent advances in understanding of neuronal Ras/Rap signaling provide a useful guide for developing novel treatments for mental diseases.

  11. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions.

    PubMed Central

    Kolch, W

    2000-01-01

    The Ras/Raf/MEK (mitogen-activated protein kinase/ERK kinase)/ERK (extracellular-signal-regulated kinase) pathway is at the heart of signalling networks that govern proliferation, differentiation and cell survival. Although the basic regulatory steps have been elucidated, many features of this pathway are only beginning to emerge. This review focuses on the role of protein-protein interactions in the regulation of this pathway, and how they contribute to co-ordinate activation steps, subcellular redistribution, substrate phosphorylation and cross-talk with other signalling pathways. PMID:11023813

  12. R-Ras Signals through Specific Integrin α Cytoplasmic Domains to Promote Migration and Invasion of Breast Epithelial Cells

    PubMed Central

    Keely, Patricia J.; Rusyn, Elena V.; Cox, Adrienne D.; Parise, Leslie V.

    1999-01-01

    Specificity and modulation of integrin function have important consequences for cellular responses to the extracellular matrix, including differentiation and transformation. The Ras-related GTPase, R-Ras, modulates integrin affinity, but little is known of the signaling pathways and biological functions downstream of R-Ras. Here we show that stable expression of activated R-Ras or the closely related TC21 (R-Ras 2) induced integrin-mediated migration and invasion of breast epithelial cells through collagen and disrupted differentiation into tubule structures, whereas dominant negative R-Ras had opposite effects. These results imply novel roles for R-Ras and TC21 in promoting a transformed phenotype and in the basal migration and polarization of these cells. Importantly, R-Ras induced an increase in cellular adhesion and migration on collagen but not fibronectin, suggesting that R-Ras signals to specific integrins. This was further supported by experiments in which R-Ras enhanced the migration of cells expressing integrin chimeras containing the α2, but not the α5, cytoplasmic domain. In addition, a transdominant inhibition previously noted only between integrin β cytoplasmic domains was observed for the α2 cytoplasmic domain; α2β1-mediated migration was inhibited by the expression of excess α2 but not α5 cytoplasmic domain-containing chimeras, suggesting the existence of limiting factors that bind the integrin α subunit. Using pharmacological inhibitors, we found that R-Ras induced migration on collagen through a combination of phosphatidylinositol 3-kinase and protein kinase C, but not MAPK, which is distinct from the other Ras family members, Rac, Cdc42, and N- and K-Ras. Thus, R-Ras communicates with specific integrin α cytoplasmic domains through a unique combination of signaling pathways to promote cell migration and invasion. PMID:10352023

  13. Aurora kinase A interacts with H-Ras and potentiates Ras-MAPK signaling | Office of Cancer Genomics

    Cancer.gov

    In cancer, upregulated Ras promotes cellular transformation and proliferation in part through activation of oncogenic Ras-MAPK signaling. While directly inhibiting Ras has proven challenging, new insights into Ras regulation through protein-protein interactions may offer unique opportunities for therapeutic intervention. Here we report the identification and validation of Aurora kinase A (Aurora A) as a novel Ras binding protein. We demonstrate that the kinase domain of Aurora A mediates the interaction with the N-terminal domain of H-Ras.

  14. Extracellular matrix-modulated Heartless signaling in Drosophila blood progenitors regulates their differentiation via a Ras/ETS/FOG pathway and target of rapamycin function

    PubMed Central

    Dragojlovic-Munther, Michelle; Martinez-Agosto, Julian A

    2014-01-01

    Maintenance of hematopoietic progenitors ensures a continuous supply of blood cells during the lifespan of an organism. Thus, understanding the molecular basis for progenitor maintenance is a continued focus of investigation. A large pool of undifferentiated blood progenitors are maintained in the Drosophila hematopoietic organ, the larval lymph gland, by a complex network of signaling pathways that are mediated by niche-, progenitor-, or differentiated hemocyte-derived signals. In this study we examined the function of the Drosophila fibroblast growth factor receptor (FGFR), Heartless, a critical regulator of early lymph gland progenitor specification in the late embryo, during larval lymph gland hematopoiesis. Activation of Heartless signaling in hemocyte progenitors by its two ligands, Pyramus and Thisbe, is both required and sufficient to induce progenitor differentiation and formation of the plasmatocyte-rich lymph gland cortical zone. We identify two transcriptional regulators that function downstream of Heartless signaling in lymph gland progenitors, the ETS protein, Pointed, and the Friend-of-GATA (FOG) protein, U-shaped, which are required for this Heartless-induced differentiation response. Furthermore, cross-talk of Heartless and target of rapamycin signaling in hemocyte progenitors is required for lamellocyte differentiation downstream of Thisbe-mediated Heartless activation. Finally, we identify the Drosophila heparan sulfate proteoglycan, Trol, as a critical negative regulator of Heartless ligand signaling in the lymph gland, demonstrating that sequestration of differentiation signals by the extracellular matrix is a unique mechanism employed in blood progenitor maintenance that is of potential relevance to many other stem cell niches. PMID:23603494

  15. RAS and downstream RAF-MEK and PI3K-AKT signaling in neuronal development, function and dysfunction

    PubMed Central

    Zhong, Jian

    2016-01-01

    In postmitotic neurons, the activation of RAS family small GTPases regulates survival, growth and differentiation. Dysregulation of RAS or its major effector pathway, the cascade of RAF-, mitogen-activated and extracellular-signal regulated kinase kinases (MEK), and extracellular-signal regulated kinases (ERK) causes the Rasopathies, a group of neurodevelopmental disorders whose pathogenic mechanisms are the subject of intense research. I here summarize the functions of RAS – RAF – MEK – ERK signaling in neurons in vivo, and discuss perspectives for harnessing this pathway to enable novel treatments for nervous system injury, the Rasopathies, and possibly other neurological conditions. PMID:26760308

  16. MicroRNA-126 inhibits colon cancer cell proliferation and invasion by targeting the chemokine (C-X-C motif) receptor 4 and Ras homolog gene family, member A, signaling pathway

    PubMed Central

    Yuan, Wei; Guo, Ye-Qing; Li, Xia-Yu; Deng, Min-Zi; Shen, Zhao-Hua; Bo, Chi-Bin; Dai, Ya-Fei; Huang, Ming-Yu; Yang, Zhen-Yu; Quan, Yong-Sheng; Tian, Li; Wang, Xiaoyan

    2016-01-01

    MicroRNA-126 (miR-126) suppresses the migration, proliferation and invasion of colon cancer cells. However, the underlying mechanisms of miR-126 in colon cancer have not been fully elucidated. In this study, in vivo experiments revealed that miR-126 inhibits colon cancer growth and metastasis. Furthermore, miR-126 was down-regulated in human colon cancer tissue, and its expression was inversely correlated with TNM stage and metastasis of patients. Low level of miR-126 identified patients with poor prognosis. And we found that miR-126 expression was negatively correlated with the expression levels of chemokine (C-X-C motif) receptor 4 (CXCR4) and components of signaling pathway of Ras homolog gene family, member A (RhoA) in vitro and in vivo. Moreover, we verified that miR-126 negatively regulated CXCR4 and RhoA signaling in vitro. In addition, either in miR-126-overexpressing or in miR- 126-silenced colon cancer cells, the restoration of CXCR4 could significantly reverse the proliferation and invasion, as well as abolish the effects of miR-126 on RhoA signaling pathway. Collectively, these results demonstrated that miR-126 acts as a tumor suppressor by inactivating RhoA signaling via CXCR4 in colon cancer. And miR-126 may serve as a prognostic marker for monitoring and treating colon cancer. PMID:27517626

  17. MicroRNA-126 inhibits colon cancer cell proliferation and invasion by targeting the chemokine (C-X-C motif) receptor 4 and Ras homolog gene family, member A, signaling pathway.

    PubMed

    Yuan, Wei; Guo, Ye-Qing; Li, Xia-Yu; Deng, Min-Zi; Shen, Zhao-Hua; Bo, Chi-Bin; Dai, Ya-Fei; Huang, Ming-Yu; Yang, Zhen-Yu; Quan, Yong-Sheng; Tian, Li; Wang, Xiaoyan

    2016-09-13

    MicroRNA-126 (miR-126) suppresses the migration, proliferation and invasion of colon cancer cells. However, the underlying mechanisms of miR-126 in colon cancer have not been fully elucidated. In this study, in vivo experiments revealed that miR-126 inhibits colon cancer growth and metastasis. Furthermore, miR-126 was down-regulated in human colon cancer tissue, and its expression was inversely correlated with TNM stage and metastasis of patients. Low level of miR-126 identified patients with poor prognosis. And we found that miR-126 expression was negatively correlated with the expression levels of chemokine (C-X-C motif) receptor 4 (CXCR4) and components of signaling pathway of Ras homolog gene family, member A (RhoA) in vitro and in vivo. Moreover, we verified that miR-126 negatively regulated CXCR4 and RhoA signaling in vitro. In addition, either in miR-126-overexpressing or in miR- 126-silenced colon cancer cells, the restoration of CXCR4 could significantly reverse the proliferation and invasion, as well as abolish the effects of miR-126 on RhoA signaling pathway. Collectively, these results demonstrated that miR-126 acts as a tumor suppressor by inactivating RhoA signaling via CXCR4 in colon cancer. And miR-126 may serve as a prognostic marker for monitoring and treating colon cancer.

  18. RAS/ERK pathway transcriptional regulation through ETS/AP-1 binding sites.

    PubMed

    Hollenhorst, Peter C

    2012-01-01

    The RAS/RAF/MEK/ERK signaling pathway is activated by mutation in many cancers. Neighboring ETS and AP-1 DNA binding sequences can act as response elements for transcriptional activation by this pathway. ERK phosphorylation of an ETS transcription factor is one mechanism of activating the RAS/ERK gene expression program that can promote cancer cell phenotypes such as proliferation, invasion, and metastasis. Recent genome-wide mapping of ETS proteins over-expressed by chromosomal rearrangement in prostate cancer reveals a second mechanism for activation of this gene expression program. An oncogenic subset of ETS transcription factors can activate RAS/ERK target genes even in the absence of RAS/ERK pathway activation by binding ETS/AP-1 sequences. Thus, regulation of cancer cell invasion and metastasis via ETS/AP-1 sequence elements depends on which ETS protein is bound, and the status of the RAS/ERK pathway. This commentary will focus on what is known about the selectivity of ETS/AP-1 sequences for different ETS transcription factors and the transcriptional consequences of ETS protein selection.

  19. Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

    PubMed Central

    Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

    2001-01-01

    Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype. PMID:11606538

  20. Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

    PubMed

    Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

    2001-10-01

    Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype.

  1. Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes.

    PubMed

    Cailotto, Frederic; Bianchi, Arnaud; Sebillaud, Sylvie; Venkatesan, Narayanan; Moulin, David; Jouzeau, Jean-Yves; Netter, Patrick

    2007-01-01

    ANK is a multipass transmembrane protein transporter thought to play a role in the export of intracellular inorganic pyrophosphate and so to contribute to the pathophysiology of chondrocalcinosis. As transforming growth factor-beta-1 (TGF-beta1) was shown to favor calcium pyrophosphate dihydrate deposition, we investigated the contribution of ANK to the production of extracellular inorganic pyrophosphate (ePPi) by chondrocytes and the signaling pathways involved in the regulation of Ank expression by TGF-beta1. Chondrocytes were exposed to 10 ng/mL of TGF-beta1, and Ank expression was measured by quantitative polymerase chain reaction and Western blot. ePPi was quantified in cell supernatants. RNA silencing was used to define the respective roles of Ank and PC-1 in TGF-beta1-induced ePPi generation. Finally, selective kinase inhibitors and dominant-negative/overexpression plasmid strategies were used to explore the contribution of several signaling pathways to Ank induction by TGF-beta1. TGF-beta1 strongly increased Ank expression at the mRNA and protein levels, as well as ePPi production. Using small interfering RNA technology, we showed that Ank contributed approximately 60% and PC-1 nearly 20% to TGF-beta1-induced ePPi generation. Induction of Ank by TGF-beta1 required activation of the extracellular signal-regulated kinase (ERK) pathway but not of p38-mitogen-activated protein kinase or of protein kinase A. In line with the general protein kinase C (PKC) inhibitor calphostin C, Gö6976 (a Ca2+-dependent PKC inhibitor) diminished TGF-beta1-induced Ank expression by 60%, whereas a 10% inhibition was observed with rottlerin (a PKCdelta inhibitor). These data suggest a regulatory role for calcium in TGF-beta1-induced Ank expression. Finally, we demonstrated that the stimulatory effect of TGF-beta1 on Ank expression was inhibited by the suppression of the Ras/Raf-1 pathway, while being enhanced by their constitutive activation. Transient overexpression of Smad 7, an

  2. Growth hormone signaling pathways.

    PubMed

    Carter-Su, Christin; Schwartz, Jessica; Argetsinger, Lawrence S

    2016-06-01

    Over 20years ago, our laboratory showed that growth hormone (GH) signals through the GH receptor-associated tyrosine kinase JAK2. We showed that GH binding to its membrane-bound receptor enhances binding of JAK2 to the GHR, activates JAK2, and stimulates tyrosyl phosphorylation of both JAK2 and GHR. The activated JAK2/GHR complex recruits a variety of signaling proteins, thereby initiating multiple signaling pathways and cellular responses. These proteins and pathways include: 1) Stat transcription factors implicated in the expression of multiple genes, including the gene encoding insulin-like growth factor 1; 2) Shc adapter proteins that lead to activation of the grb2-SOS-Ras-Raf-MEK-ERK1,2 pathway; 3) insulin receptor substrate proteins implicated in the phosphatidylinositol-3-kinase and Akt pathway; 4) signal regulatory protein α, a transmembrane scaffold protein that recruits proteins including the tyrosine phosphatase SHP2; and 5) SH2B1, a scaffold protein that can activate JAK2 and enhance GH regulation of the actin cytoskeleton. Our recent work has focused on the function of SH2B1. We have shown that SH2B1β is recruited to and phosphorylated by JAK2 in response to GH. SH2B1 localizes to the plasma membrane, cytoplasm and focal adhesions; it also cycles through the nucleus. SH2B1 regulates the actin cytoskeleton and promotes GH-dependent motility of RAW264.7 macrophages. Mutations in SH2B1 have been found in humans exhibiting severe early-onset childhood obesity and insulin resistance. These mutations impair SH2B1 enhancement of GH-induced macrophage motility. As SH2B1 is expressed ubiquitously and is also recruited to a variety of receptor tyrosine kinases, our results raise the possibility that effects of SH2B1 on the actin cytoskeleton in various cell types, including neurons, may play a role in regulating body weight.

  3. RAS signalling through PI3-Kinase controls cell migration via modulation of Reelin expression

    PubMed Central

    Castellano, Esther; Molina-Arcas, Miriam; Krygowska, Agata Adelajda; East, Philip; Warne, Patricia; Nicol, Alastair; Downward, Julian

    2016-01-01

    RAS signalling through phosphoinositide 3-kinase (PI3-Kinase) has been shown to have an essential role in tumour initiation and maintenance. RAS also regulates cell motility and tumour invasiveness, but the role of direct RAS binding to PI3-Kinase in this remains uncertain. Here, we provide evidence that disruption of RAS interaction with PI3-Kinase p110α decreases cell motility and prevents activation of Rac GTPase. Analysis of gene expression in cells lacking RAS interaction with p110α reveals increased levels of the extracellular matrix glycoprotein Reelin and activation of its downstream pathway resulting in upregulation of E-cadherin expression. Induction of the Reelin/E-cadherin axis is also observed in Kras mutant lung tumours that are regressing due to blockade of RAS interaction with PI3-Kinase. Furthermore, loss of Reelin correlates with decreased survival of lung and breast cancer patients. Reelin thus plays a role in restraining RAS and PI3-kinase promotion of cell motility and potentially tumour metastasis. PMID:27071537

  4. Chaperone-mediated specificity in Ras and Rap signaling.

    PubMed

    Azoulay-Alfaguter, Inbar; Strazza, Marianne; Mor, Adam

    2015-01-01

    Ras and Rap proteins are closely related small guanosine triphosphatase (GTPases) that share similar effector-binding domains but operate in a very different signaling networks; Ras has a dominant role in cell proliferation, while Rap mediates cell adhesion. Ras and Rap proteins are regulated by several shared processes such as post-translational modification, phosphorylation, activation by guanine exchange factors and inhibition by GTPase-activating proteins. Sub-cellular localization and trafficking of these proteins to and from the plasma membrane are additional important regulatory features that impact small GTPases function. Despite its importance, the trafficking mechanisms of Ras and Rap proteins are not completely understood. Chaperone proteins play a critical role in trafficking of GTPases and will be the focus of the discussion in this work. We will review several aspects of chaperone biology focusing on specificity toward particular members of the small GTPase family. Understanding this specificity should provide key insights into drug development targeting individual small GTPases.

  5. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    SciTech Connect

    Naumov, Inna; Kazanov, Dina; Lisiansky, Victoria; Starr, Alex; Aroch, Ilan; Shapira, Shiran; Kraus, Sarah; Arber, Nadir

    2012-01-15

    Background: Functional 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. Results: A 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 {approx} 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 {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective 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.

  6. RAS/MAPK Activation Drives Resistance to Smo Inhibition, Metastasis, and Tumor Evolution in Shh Pathway-Dependent Tumors.

    PubMed

    Zhao, Xuesong; Ponomaryov, Tatyana; Ornell, Kimberly J; Zhou, Pengcheng; Dabral, Sukriti K; Pak, Ekaterina; Li, Wei; Atwood, Scott X; Whitson, Ramon J; Chang, Anne Lynn S; Li, Jiang; Oro, Anthony E; Chan, Jennifer A; Kelleher, Joseph F; Segal, Rosalind A

    2015-09-01

    Aberrant Shh signaling promotes tumor growth in diverse cancers. The importance of Shh signaling is particularly evident in medulloblastoma and basal cell carcinoma (BCC), where inhibitors targeting the Shh pathway component Smoothened (Smo) show great therapeutic promise. However, the emergence of drug resistance limits long-term efficacy, and the mechanisms of resistance remain poorly understood. Using new medulloblastoma models, we identify two distinct paradigms of resistance to Smo inhibition. Sufu mutations lead to maintenance of the Shh pathway in the presence of Smo inhibitors. Alternatively activation of the RAS-MAPK pathway circumvents Shh pathway dependency, drives tumor growth, and enhances metastatic behavior. Strikingly, in BCC patients treated with Smo inhibitor, squamous cell cancers with RAS/MAPK activation emerged from the antecedent BCC tumors. Together, these findings reveal a critical role of the RAS-MAPK pathway in drug resistance and tumor evolution of Shh pathway-dependent tumors.

  7. RAS/MAPK activation drives resistance to Smo inhibition, metastasis and tumor evolution in Shh pathway-dependent tumors

    PubMed Central

    Zhao, Xuesong; Ponomaryov, Tatyana; Ornell, Kimberly J.; Zhou, Pengcheng; Dabral, Sukriti K.; Pak, Ekaterina; Li, Wei; Atwood, Scott X.; Whitson, Ramon J.; Chang, Anne Lynn S.; Li, Jiang; Oro, Anthony E.; Chan, Jennifer A.; Kelleher, Joseph F.; Segal, Rosalind A.

    2015-01-01

    Aberrant Shh signaling promotes tumor growth in diverse cancers. The importance of Shh signaling is particularly evident in medulloblastoma and basal cell carcinoma (BCC), where inhibitors targeting the Shh pathway component Smoothened (Smo) show great therapeutic promise. However, the emergence of drug resistance limits long-term efficacy and the mechanisms of resistance remain poorly understood. Using new medulloblastoma models, we identify two distinct paradigms of resistance to Smo inhibition. Sufu mutations lead to maintenance of the Shh pathway in the presence of Smo inhibitors. Alternatively activation of the RAS/MAPK pathway circumvents Shh pathway-dependency, drives tumor growth and enhances metastatic behavior. Strikingly, in BCC patients treated with Smo inhibitor, squamous cell cancers with RAS/MAPK activation emerged from the antecedent BCC tumors. Together these findings reveal a critical role of RAS/MAPK pathway in drug resistance and tumor evolution of Shh pathway-dependent tumors. PMID:26130651

  8. Atorvastatin augments temozolomide's efficacy in glioblastoma via prenylation-dependent inhibition of Ras signaling.

    PubMed

    Peng, Peng; Wei, Wei; Long, Cheng; Li, Jingwen

    2017-07-29

    Ras signaling is often dysregulated and plays essential roles for the maintenance of glioblastoma. The proper function of Ras depends largely on the appropriate post-translational modification termed prenylation. Targeting protein prenylation therefore represents an alternative therapeutic strategy in glioblastoma. In this study, we demonstrate that prenylation inhibition by atorvastatin is active against glioblastoma. Atorvastatin alone dose-dependently inhibits growth and survival of multiple glioblastoma cell lines. Its combination with temozolomide significantly enhances temozolomide's efficacy in in vitro cultured cell system as well as in vivo xenograft glioblastoma tumor model. We further show that this is achieved by the inhibition of Ras prenylation, leading to decreased activation of Ras and its downstream signaling pathways, including Erk, rS6 and eIF4E. Our findings suggest that inhibition of Ras activity by atorvastatin effectively targets the MEK and other signaling pathways. Our study provides a fundamental evidence to repurpose atorvastatin for a potential treatment of glioblastoma. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Small molecule stabilization of the KSR inactive state antagonizes oncogenic Ras signalling

    PubMed Central

    Dhawan, Neil S.; scopton, Alex P.; Dar, Arvin C.

    2016-01-01

    Deregulation of the Ras–mitogen activated protein kinase (MAPK) pathway is an early event in many different cancers and a key driver of resistance to targeted therapies1. Sustained signalling through this pathway is caused most often by mutations in K-Ras, which biochemically favours the stabilization of active RAF signalling complexes2. Kinase suppressor of Ras (KSR) is a MAPK scaffold3–5 that is subject to allosteric regulation through dimerization with RAF6,7. Direct targeting of KSR could have important therapeutic implications for cancer; however, testing this hypothesis has been difficult owing to a lack of small-molecule antagonists of KSR function. Guided by KSR mutations that selectively suppress oncogenic, but not wild-type, Ras signalling, we developed a class of compounds that stabilize a previously unrecognized inactive state of KSR. These compounds, exemplified by APS-2-79, modulate KSR-dependent MAPK signalling by antagonizing RAF heterodimerization as well as the conformational changes required for phosphorylation and activation of KSR-bound MEK (mitogen-activated protein kinase kinase). Furthermore, APS-2-79 increased the potency of several MEK inhibitors specifically within Ras-mutant cell lines by antagonizing release of negative feedback signalling, demonstrating the potential of targeting KSR to improve the efficacy of current MAPK inhibitors. These results reveal conformational switching in KSR as a druggable regulator of oncogenic Ras, and further suggest co-targeting of enzymatic and scaffolding activities within Ras–MAPK signalling complexes as a therapeutic strategy for overcoming Ras-driven cancers. PMID:27556948

  10. Overexpressed Galectin-3 in Pancreatic Cancer Induces Cell Proliferation and Invasion by Binding Ras and Activating Ras Signaling

    PubMed Central

    Song, Shumei; Ji, Baoan; Ramachandran, Vijaya; Wang, Huamin; Hafley, Margarete; Logsdon, Craig; Bresalier, Robert S.

    2012-01-01

    Pancreatic cancer (PDAC) is a lethal disease with a five-year survival of 3–5%. Mutations in K-Ras are found in nearly all cases, but K-Ras mutations alone are not sufficient for the development of PDAC. Additional factors contribute to activation of Ras signaling and lead to tumor formation. Galectin-3 (Gal-3), a multifunctional β-galactoside-binding protein, is highly expressed in PDAC. We therefore investigated the functional role of Gal-3 in pancreatic cancer progression and its relationship to Ras signaling. Expression of Gal-3 was determined by immunohistochemistry, Q-PCR and immunoblot. Functional studies were performed using pancreatic cell lines genetically engineered to express high or low levels of Gal-3. Ras activity was examined by Raf pull-down assays. Co-immunoprecipitation and immunofluorescence were used to assess protein-protein interactions. In this study, we demonstrate that Gal-3 was highly up-regulated in human tumors and in a mutant K-Ras mouse model of PDAC. Down-regulation of Gal-3 by lentivirus shRNA decreased PDAC cell proliferation and invasion in vitro and reduced tumor volume and size in an orthotopic mouse model. Gal-3 bound Ras and maintained Ras activity; down-regulation of Gal-3 decreased Ras activity as well as Ras down-stream signaling including phosphorylation of ERK and AKT and Ral A activity. Transfection of Gal-3 cDNA into PDAC cells with low-level Gal-3 augmented Ras activity and its down-stream signaling. These results suggest that Gal-3 contributes to pancreatic cancer progression, in part, by binding Ras and activating Ras signaling. Gal-3 may therefore be a potential novel target for this deadly disease. PMID:22900040

  11. Investigating RAS Signaling in Cancer | Office of Cancer Clinical Proteomics Research

    Cancer.gov

    CPTAC expertise has been charged to develop RAS specific targeted proteomic assays to study the important pathways of human cancer. The oncogene RAS is linked to 30 percent of human cancers, but the search for a targeted therapy for RAS has remained elusive. To advance our understanding of this oncogene and to develop improved targeted therapies against RAS pathway, the National Cancer Institute (NCI) has launched a RAS Initiative.

  12. Deregulated Ras signaling compromises DNA damage checkpoint recovery in S. cerevisiae

    PubMed Central

    Wood, Matthew D

    2010-01-01

    The DNA damage checkpoint maintains genome stability by arresting the cell cycle and promoting DNA repair under genotoxic stress. Cells must downregulate the checkpoint signaling pathways in order to resume cell division after completing DNA repair. While the mechanisms of checkpoint activation have been well-characterized, the process of checkpoint recovery, and the signals regulating it, has only recently been investigated. We have identified a new role for the Ras signaling pathway as a regulator of DNA damage checkpoint recovery. Here we report that in budding yeast, deletion of the IRA1 and IRA2 genes encoding negative regulators of Ras prevents cellular recovery from a DNA damage induced arrest. the checkpoint kinase Rad53 is dephosphorylated in an IRA-deficient strain, indicating that recovery failure is not caused by constitutive checkpoint pathway activation. the ira1Δ ira2Δ recovery defect requires the checkpoint kinase Chk1 and the cAMP-dependent protein kinase (PKA) catalytic subunit Tpk2. Furthermore, PKA phosphorylation sites on the anaphase promoting complex specificity factor Cdc20 are required for the recovery defect, indicating a link between the recovery defect and PKA regulation of mitosis. This work identifies a new signaling pathway that can regulate DNA damage checkpoint recovery and implicates the Ras signaling pathway as an important regulator of mitotic events. PMID:20716966

  13. Loco signaling pathway in longevity.

    PubMed

    Lin, Yuh-Ru; Parikh, Hardik; Park, Yongkyu

    2011-05-01

    Despite the various roles of regulator of G protein signaling (RGS) protein in the G protein signaling pathway that have been defined, the function of RGS has not been characterized in longevity signaling pathways. We found that reduced expression of Loco, a Drosophila RGS protein, resulted in a longer lifespan of flies with stronger resistance to stress, higher MnSOD activity and increased fat content. In contrast, overexpression of the loco gene shortened the fly lifespan significantly, lowered stress resistance and reduced fat content, also indicating that the RGS domain containing GTPase-activating protein (GAP) activity is related to the regulation of longevity. Interestingly, expressional changes of yeast RGS2 and rat RGS14, homologs to the fly Loco, also affected oxidative stress resistance and longevity in the respective species. It is known that Loco inactivates inhibitory Gαi•GTP protein to reduce activity of adenylate cyclase (AC) and RGS14 interacts with activated H-Ras and Raf-1 kinases, which subsequently inhibits ERK phosphorylation. We propose that Loco/RGS14 protein may regulate stress resistance and longevity as an activator in AC-cAMP-PKA pathway and/or as a molecular scaffold that sequesters active Ras and Raf from Ras•GTP-Raf-MEK-ERK signaling pathway. Consistently, our data showed that downregulation of Loco significantly diminishes cAMP amounts and increases p-ERK levels with higher resistance to the oxidative stress.

  14. Enhanced MET translation and signaling sustains K-Ras driven proliferation under anchorage-independent growth conditions

    PubMed Central

    Fujita-Sato, Saori; Galeas, Jacqueline; Truitt, Morgan; Pitt, Cameron; Urisman, Anatoly; Bandyopadhyay, Sourav; Ruggero, Davide; McCormick, Frank

    2015-01-01

    Oncogenic K-Ras mutation occurs frequently in several types of cancers including pancreatic and lung cancers. Tumors with K-Ras mutation are resistant to chemotherapeutic drugs as well as molecular targeting agents. Although numerous approaches are ongoing to find effective ways to treat these tumors, there are still no effective therapies for K-Ras mutant cancer patients. Here we report that K-Ras mutant cancers are more dependent on K-Ras in anchorage independent culture conditions than in monolayer culture conditions. In seeking to determine mechanisms that contribute to the K-Ras dependency in anchorage independent culture conditions, we discovered the involvement of Met in K-Ras-dependent, anchorage independent cell growth. The Met signaling pathway is enhanced and plays an indispensable role in anchorage independent growth even in cells in which Met is not amplified. Indeed, Met expression is elevated under anchorage-independent growth conditions and is regulated by K-Ras in a MAPK/ERK kinase (MEK)-dependent manner. Remarkably, in spite of a global down-regulation of mRNA translation during anchorage independent growth, we find that Met mRNA translation is specifically enhanced under these conditions. Importantly, ectopic expression of an active Met mutant rescues K-Ras ablation-derived growth suppression, indicating that K-Ras mediated Met expression drives “K-Ras addiction” in anchorage independent conditions. Our results indicate that enhanced Met expression and signaling is essential for anchorage independent growth of K-Ras mutant cancer cells and suggests that pharmacological inhibitors of Met could be effective for K-Ras mutant tumor patients. PMID:25977330

  15. Ras-Mek-Erk signaling regulates Nf1 heterozygous neointima formation.

    PubMed

    Stansfield, Brian K; Bessler, Waylan K; Mali, Raghuveer; Mund, Julie A; Downing, Brandon D; Kapur, Reuben; Ingram, David A

    2014-01-01

    Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor-suppressor gene, which encodes neurofibromin, a negative regulator of diverse Ras signaling cascades. Arterial stenosis is a nonneoplastic manifestation of NF1 that predisposes some patients to debilitating morbidity and sudden death. Recent murine studies demonstrate that Nf1 heterozygosity (Nf1(+/-)) in monocytes/macrophages significantly enhances intimal proliferation after arterial injury. However, the downstream Ras effector pathway responsible for this phenotype is unknown. Based on in vitro assays demonstrating enhanced extracellular signal-related kinase (Erk) signaling in Nf1(+/-) macrophages and vascular smooth muscle cells and in vivo evidence of Erk amplification without alteration of phosphatidylinositol 3-kinase signaling in Nf1(+/-) neointimas, we tested the hypothesis that Ras-Erk signaling regulates intimal proliferation in a murine model of NF1 arterial stenosis. By using a well-established in vivo model of inflammatory cell migration and standard cell culture, neurofibromin-deficient macrophages demonstrate enhanced sensitivity to growth factor stimulation in vivo and in vitro, which is significantly diminished in the presence of PD0325901, a specific inhibitor of Ras-Erk signaling in phase 2 clinical trials for cancer. After carotid artery injury, Nf1(+/-) mice demonstrated increased intimal proliferation compared with wild-type mice. Daily administration of PD0325901 significantly reduced Nf1(+/-) neointima formation to levels of wild-type mice. These studies identify the Ras-Erk pathway in neurofibromin-deficient macrophages as the aberrant pathway responsible for enhanced neointima formation.

  16. The cornerstone K-RAS mutation in pancreatic adenocarcinoma: From cell signaling network, target genes, biological processes to therapeutic targeting.

    PubMed

    Jonckheere, Nicolas; Vasseur, Romain; Van Seuningen, Isabelle

    2017-03-01

    RAS belongs to the super family of small G proteins and plays crucial roles in signal transduction from membrane receptors in the cell. Mutations of K-RAS oncogene lead to an accumulation of GTP-bound proteins that maintains an active conformation. In the pancreatic ductal adenocarcinoma (PDAC), one of the most deadly cancers in occidental countries, mutations of the K-RAS oncogene are nearly systematic (>90%). Moreover, K-RAS mutation is the earliest genetic alteration occurring during pancreatic carcinogenetic sequence. In this review, we discuss the central role of K-RAS mutations and their tremendous diversity of biological properties by the interconnected regulation of signaling pathways (MAPKs, NF-κB, PI3K, Ral…). In pancreatic ductal adenocarcinoma, transcriptome analysis and preclinical animal models showed that K-RAS mutation alters biological behavior of PDAC cells (promoting proliferation, migration and invasion, evading growth suppressors, regulating mucin pattern, and miRNA expression). K-RAS also impacts tumor microenvironment and PDAC metabolism reprogramming. Finally we discuss therapeutic targeting strategies of K-RAS that have been developed without significant clinical success so far. As K-RAS is considered as the undruggable target, targeting its multiple effectors and target genes should be considered as potential alternatives.

  17. A Fungus-Specific Protein Domain Is Essential for RasA-Mediated Morphogenetic Signaling in Aspergillus fumigatus

    PubMed Central

    Al Abdallah, Qusai; Norton, Tiffany S.; Hill, Amy M.; LeClaire, Lawrence L.

    2016-01-01

    ABSTRACT Ras proteins function as conserved regulators of eukaryotic growth and differentiation and are essential signaling proteins orchestrating virulence in pathogenic fungi. Here, we report the identification of a novel N-terminal domain of the RasA protein in the filamentous fungus Aspergillus fumigatus. Whereas this domain is absent in Ras homologs of higher eukaryotes, the N-terminal extension is conserved among fungi and is characterized by a short string of two to eight amino acids terminating in an invariant arginine. For this reason, we have termed the RasA N-terminal domain the invariant arginine domain (IRD). Through mutational analyses, the IRD was found to be essential for polarized morphogenesis and asexual development, with the invariant arginine residue being most essential. Although IRD truncation resulted in a nonfunctional Ras phenotype, IRD mutation was not associated with mislocalization of the RasA protein or significant changes in steady-state RasA activity levels. Mutation of the RasA IRD diminished protein kinase A (PKA) activation and resulted in decreased interaction with the Rho-type GTPase, Cdc42. Taken together, our findings reveal novel, fungus-specific mechanisms for Ras protein function and signal transduction. IMPORTANCE Aspergillus fumigatus is an important fungal pathogen against which limited treatments exist. During invasive disease, A. fumigatus hyphae grow in a highly polarized fashion, forming filaments that invade blood vessels and disseminate to distant sites. Once invasion and dissemination occur, mortality rates are high. We have previously shown that the Ras signaling pathway is an important regulator of the hyphal growth machinery supporting virulence in A. fumigatus. Here, we show that functional Ras signaling in A. fumigatus requires a novel, fungus-specific domain within the Ras protein. This domain is highly conserved among fungi, yet absent in higher eukaryotes, suggesting a potentially crucial difference in

  18. A Fungus-Specific Protein Domain Is Essential for RasA-Mediated Morphogenetic Signaling in Aspergillus fumigatus.

    PubMed

    Al Abdallah, Qusai; Norton, Tiffany S; Hill, Amy M; LeClaire, Lawrence L; Fortwendel, Jarrod R

    2016-01-01

    Ras proteins function as conserved regulators of eukaryotic growth and differentiation and are essential signaling proteins orchestrating virulence in pathogenic fungi. Here, we report the identification of a novel N-terminal domain of the RasA protein in the filamentous fungus Aspergillus fumigatus. Whereas this domain is absent in Ras homologs of higher eukaryotes, the N-terminal extension is conserved among fungi and is characterized by a short string of two to eight amino acids terminating in an invariant arginine. For this reason, we have termed the RasA N-terminal domain the invariant arginine domain (IRD). Through mutational analyses, the IRD was found to be essential for polarized morphogenesis and asexual development, with the invariant arginine residue being most essential. Although IRD truncation resulted in a nonfunctional Ras phenotype, IRD mutation was not associated with mislocalization of the RasA protein or significant changes in steady-state RasA activity levels. Mutation of the RasA IRD diminished protein kinase A (PKA) activation and resulted in decreased interaction with the Rho-type GTPase, Cdc42. Taken together, our findings reveal novel, fungus-specific mechanisms for Ras protein function and signal transduction. IMPORTANCEAspergillus fumigatus is an important fungal pathogen against which limited treatments exist. During invasive disease, A. fumigatus hyphae grow in a highly polarized fashion, forming filaments that invade blood vessels and disseminate to distant sites. Once invasion and dissemination occur, mortality rates are high. We have previously shown that the Ras signaling pathway is an important regulator of the hyphal growth machinery supporting virulence in A. fumigatus. Here, we show that functional Ras signaling in A. fumigatus requires a novel, fungus-specific domain within the Ras protein. This domain is highly conserved among fungi, yet absent in higher eukaryotes, suggesting a potentially crucial difference in the

  19. NF1 Is a Direct G Protein Effector Essential for Opioid Signaling to Ras in the Striatum.

    PubMed

    Xie, Keqiang; Colgan, Lesley A; Dao, Maria T; Muntean, Brian S; Sutton, Laurie P; Orlandi, Cesare; Boye, Sanford L; Boye, Shannon E; Shih, Chien-Cheng; Li, Yuqing; Xu, Baoji; Smith, Roy G; Yasuda, Ryohei; Martemyanov, Kirill A

    2016-11-21

    It is well recognized that G-protein-coupled receptors (GPCRs) can activate Ras-regulated kinase pathways to produce lasting changes in neuronal function. Mechanisms by which GPCRs transduce these signals and their relevance to brain disorders are not well understood. Here, we identify a major Ras regulator, neurofibromin 1 (NF1), as a direct effector of GPCR signaling via Gβγ subunits in the striatum. We find that binding of Gβγ to NF1 inhibits its ability to inactivate Ras. Deletion of NF1 in striatal neurons prevents the opioid-receptor-induced activation of Ras and eliminates its coupling to Akt-mTOR-signaling pathway. By acting in the striatal medium spiny neurons of the direct pathway, NF1 regulates opioid-induced changes in Ras activity, thereby sensitizing mice to psychomotor and rewarding effects of morphine. These results delineate a novel mechanism of GPCR signaling to Ras pathways and establish a critical role of NF1 in opioid addiction.

  20. Targeting RTK Signaling Pathways in Cancer

    PubMed Central

    Regad, Tarik

    2015-01-01

    The RAS/MAP kinase and the RAS/PI3K/AKT pathways play a key role in the regulation of proliferation, differentiation and survival. The induction of these pathways depends on Receptor Tyrosine Kinases (RTKs) that are activated upon ligand binding. In cancer, constitutive and aberrant activations of components of those pathways result in increased proliferation, survival and metastasis. For instance, mutations affecting RTKs, Ras, B-Raf, PI3K and AKT are common in perpetuating the malignancy of several types of cancers and from different tissue origins. Therefore, these signaling pathways became prime targets for cancer therapy. This review aims to provide an overview about the most frequently encountered mutations, the pathogenesis that results from such mutations and the known therapeutic strategies developed to counteract their aberrant functions. PMID:26404379

  1. SIGNAL TRANSDUCTION. Membrane potential modulates plasma membrane phospholipid dynamics and K-Ras signaling.

    PubMed

    Zhou, Yong; Wong, Ching-On; Cho, Kwang-jin; van der Hoeven, Dharini; Liang, Hong; Thakur, Dhananiay P; Luo, Jialie; Babic, Milos; Zinsmaier, Konrad E; Zhu, Michael X; Hu, Hongzhen; Venkatachalam, Kartik; Hancock, John F

    2015-08-21

    Plasma membrane depolarization can trigger cell proliferation, but how membrane potential influences mitogenic signaling is uncertain. Here, we show that plasma membrane depolarization induces nanoscale reorganization of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate but not other anionic phospholipids. K-Ras, which is targeted to the plasma membrane by electrostatic interactions with phosphatidylserine, in turn undergoes enhanced nanoclustering. Depolarization-induced changes in phosphatidylserine and K-Ras plasma membrane organization occur in fibroblasts, excitable neuroblastoma cells, and Drosophila neurons in vivo and robustly amplify K-Ras-dependent mitogen-activated protein kinase (MAPK) signaling. Conversely, plasma membrane repolarization disrupts K-Ras nanoclustering and inhibits MAPK signaling. By responding to voltage-induced changes in phosphatidylserine spatiotemporal dynamics, K-Ras nanoclusters set up the plasma membrane as a biological field-effect transistor, allowing membrane potential to control the gain in mitogenic signaling circuits.

  2. Hypothyroidism and hyperthyroidism modulates Ras-MAPK intracellular pathway in rat thyroids.

    PubMed

    Leal, Anna Lúcia R C; Pantaleão, Thiago U; Moreira, Débora G; Marassi, Michelle P; Pereira, Valmara S; Rosenthal, Doris; Corrêa da Costa, Vânia Maria

    2007-04-01

    Thyrotrophin induces proliferation and function in thyroid cells acting through a seven transmembrane G protein-coupled receptor. The proliferative pathways induced by thyrotropin (TSH) in thyrocytes in vivo are not completely understood yet. The aim of this work is to evaluate if Ras can be induced by TSH in rat thyroids, and whether extracellular regulated kinase (ERK) may be involved in the subsequent intracellular signalling cascade. We induced hypothyroidism in Wistar rats by methimazole (MMI) treatment (0.03% in the drinking water for 21 days). A subset of the hypothyroid rats received T4 (1 microg/100 g bw) during the last 10 days of MMI treatment. Hyperthyroidism was induced by subcutaneous injections of T4 (10 microg/100 g bw) during 10 days in another group of rats. Our data show that in the hypothyroid rats there is a clear positive Ras modulation, but a decrease in pERK. In contrast, thyroidal pERK increases in T4-induced hyperthyroidism, but without any change in RAS, although these changes did not reach statistical significance. Thus, while the rat thyroid proliferation induced by TSH may involve an increase in RAS signalling, the subsequent cascade does not involve ERK phosphorilation, which in fact, increases during T4-induced hyperthyroidism.

  3. Systemic Regulation of RAS/MAPK Signaling by the Serotonin Metabolite 5-HIAA

    PubMed Central

    Schmid, Tobias; Snoek, L. Basten; Fröhli, Erika; van der Bent, M. Leontien; Kammenga, Jan; Hajnal, Alex

    2015-01-01

    Human cancer is caused by the interplay of mutations in oncogenes and tumor suppressor genes and inherited variations in cancer susceptibility genes. While many of the tumor initiating mutations are well characterized, the effect of genetic background variation on disease onset and progression is less understood. We have used C. elegans genetics to identify genetic modifiers of the oncogenic RAS/MAPK signaling pathway. Quantitative trait locus analysis of two highly diverged C. elegans isolates combined with allele swapping experiments identified the polymorphic monoamine oxidase A (MAOA) gene amx-2 as a negative regulator of RAS/MAPK signaling. We further show that the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA), which is a product of MAOA catalysis, systemically inhibits RAS/MAPK signaling in different organs of C. elegans. Thus, MAOA activity sets a global threshold for MAPK activation by controlling 5-HIAA levels. To our knowledge, 5-HIAA is the first endogenous small molecule that acts as a systemic inhibitor of RAS/MAPK signaling. PMID:25978500

  4. Signaling through mitogen-activated protein kinase and Rac/Rho does not duplicate the effects of activated Ras on skeletal myogenesis.

    PubMed Central

    Ramocki, M B; Johnson, S E; White, M A; Ashendel, C L; Konieczny, S F; Taparowsky, E J

    1997-01-01

    The ability of basic helix-loop-helix muscle regulatory factors (MRFs), such as MyoD, to convert nonmuscle cells to a myogenic lineage is regulated by numerous growth factor and oncoprotein signaling pathways. Previous studies have shown that H-Ras 12V inhibits differentiation to a skeletal muscle lineage by disrupting MRF function via a mechanism that is independent of the dimerization, DNA binding, and inherent transcriptional activation properties of the proteins. To investigate the intracellular signaling pathway(s) that mediates the inhibition of MRF-induced myogenesis by oncogenic Ras, we tested two transformation-defective H-Ras 12V effector domain variants for their ability to alter terminal differentiation. H-Ras 12V,35S retains the ability to activate the Raf/MEK/mitogen-activated protein (MAP) kinase cascade, whereas H-Ras 12V,40C is unable to interact directly with Raf-1 yet still influences other signaling intermediates, including Rac and Rho. Expression of each H-Ras 12V variant in C3H10T1/2 cells abrogates MyoD-induced activation of the complete myogenic program, suggesting that MAP kinase-dependent and -independent Ras signaling pathways individually block myogenesis in this model system. However, additional studies with constitutively activated Rac1 and RhoA proteins revealed no negative effects on MyoD-induced myogenesis. Similarly, treatment of Ras-inhibited myoblasts with the MEK1 inhibitor PD98059 revealed that elevated MAP kinase activity is not a significant contributor to the H-Ras 12V effect. These data suggest that an additional Ras pathway, distinct from the well-characterized MAP kinase and Rac/Rho pathways known to be important for the transforming function of activated Ras, is primarily responsible for the inhibition of myogenesis by H-Ras 12V. PMID:9199290

  5. Inhibition of Ras Signaling in Neurofibromitosis

    DTIC Science & Technology

    2010-07-01

    regulates the level of tyrosine phosphorylation on targets within T cells, among them the critical T cell tyrosine kinase Zap -70 (Boris et al., 2011). In...ASSOCIATED PROTEIN ( ZAP )-70 AND T CELL RECEPTOR (TCR) SIGNALING PATHWAYS” in revision J. Biol. Chem. 4. Qiang Lu, Nicolas Nassar and Jin Wang “An

  6. Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo☆

    PubMed Central

    Newell, Pippa; Toffanin, Sara; Villanueva, Augusto; Chiang, Derek Y.; Minguez, Beatriz; Cabellos, Laia; Savic, Radoslav; Hoshida, Yujin; Lim, Kiat Hon; Melgar-Lesmes, Pedro; Yea, Steven; Peix, Judit; Deniz, Kemal; Fiel, M. Isabel; Thung, Swan; Alsinet, Clara; Tovar, Victoria; Mazzaferro, Vincenzo; Bruix, Jordi; Roayaie, Sasan; Schwartz, Myron; Friedman, Scott L.; Llovet, Josep M.

    2010-01-01

    Background/Aims The success of sorafenib in the treatment of advanced hepatocellular carcinoma (HCC) has focused interest on the role of Ras signaling in this malignancy. We investigated the molecular alterations of the Ras pathway in HCC and the antineoplastic effects of sorafenib in combination with rapamycin, an inhibitor of mTOR pathway, in experimental models. Methods Gene expression (qRT-PCR, oligonucleotide microarray), DNA copy number changes (SNP-array), methylation of tumor suppressor genes (methylation-specific PCR) and protein activation (immunohistochemistry) were analysed in 351 samples. Anti-tumoral effects of combined therapy targeting the Ras and mTOR pathways were evaluated in cell lines and HCC xenografts. Results Different mechanisms accounted for Ras pathway activation in HCC. H-ras was up-regulated during different steps of hepatocarcinogenesis. B-raf was overexpressed in advanced tumors and its expression was associated with genomic amplification. Partial methylation of RASSF1A and NORE1A was detected in 89% and 44% of tumors respectively, and complete methylation was found in 11 and 4% of HCCs. Activation of the pathway (pERK immunostaining) was identified in 10.3% of HCC. Blockade of Ras and mTOR pathways with sorafenib and rapamycin reduced cell proliferation and induced apoptosis in cell lines. In vivo, the combination of both compounds enhanced tumor necrosis and ulceration when compared with sorafenib alone. Conclusions Ras activation results from several molecular alterations, such as methylation of tumor suppressors and amplification of oncogenes (B-raf). Sorafenib blocks signaling and synergizes with rapamycin in vivo, preventing tumor progression. These data provide the rationale for testing this combination in clinical studies. PMID:19665249

  7. Therapeutic Levels of the Hydroxmethylglutaryl-Coenzyme A Reductase Inhibitor Lovastatin Activate Ras Signaling via Phospholipase D2▿

    PubMed Central

    Cho, Kwang-jin; Hill, Michelle M.; Chigurupati, Sravanthi; Du, Guangwei; Parton, Robert G.; Hancock, John F.

    2011-01-01

    Hydroxmethylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) lower serum cholesterol but exhibit pleiotropic biological effects that are difficult to ascribe solely to cholesterol depletion. Here, we investigated the effect of lovastatin on protein prenylation and cell signaling. We show that high concentrations (50 μM) of lovastatin inhibit Ras, Rho, and Rap prenylation but that therapeutic levels of lovastatin (50 nM to 500 nM) do not. In contrast, depletion of cellular cholesterol by therapeutic levels of lovastatin increased Ras GTP loading and mitogen-activated protein kinase (MAPK) activation in human umbilical vein endothelial cells and rodent fibroblasts. Elevated Ras signaling was not seen in statin-treated cells if cholesterol levels were maintained by supplementation. Activation of Ras-MAPK signaling was a consequence of, and dependent on, activation of phospholipase D2 (PLD2). Expression of dominant interfering PLD2 or biochemical inhibition of PLD2 abrogated Ras and MAPK activation induced by lovastatin. In contrast, ectopic expression of wild-type PLD2 enhanced Ras and MAPK activation in response to therapeutic levels of lovastatin. Statin-induced cholesterol depletion also modestly activated the epidermal growth factor receptor (EGFR), resulting in downregulation of EGFR expression. These results suggest that statins modulate key cell signaling pathways as a direct consequence of cholesterol depletion and identify the EGFR-PLD2-Ras-MAPK axis as an important statin target. PMID:21245384

  8. PUF-8 negatively regulates RAS/MAPK signalling to promote differentiation of C. elegans germ cells

    PubMed Central

    Vaid, Samir; Ariz, Mohd; Chaturbedi, Amaresh; Kumar, Ganga Anil; Subramaniam, Kuppuswamy

    2013-01-01

    Signals that promote germ cell self-renewal by preventing premature meiotic entry are well understood. However, signals that control mitotic proliferation to promote meiotic differentiation have not been well characterized. In Caenorhabditis elegans, GLP-1 Notch signalling promotes the proliferative fate by preventing premature meiotic entry. The germline niche cell, which is the source of the ligand for GLP-1, spatially restricts GLP-1 signalling and thus enables the germ cells that have moved away from the niche to enter meiosis. Here, we show that the suppression of RAS/MAP kinase signalling in the mitotic and meiotic-entry regions is essential for the regulation of the mitosis-meiosis switch by niche signalling. We provide evidence that the conserved PUF family RNA-binding protein PUF-8 and the RAS GAP protein GAP-3 function redundantly to suppress the LET-60 RAS in the mitotic and meiotic entry regions. Germ cells missing both PUF-8 and GAP-3 proliferate in an uncontrolled fashion and fail to undergo meiotic development. MPK-1, the MAP kinase downstream of the LET-60 RAS, is prematurely activated in these cells; downregulation of MPK-1 activation eliminates tumours and restores differentiation. Our results further reveal that PUF-8 negatively regulates LET-60 expression at a post-transcriptional step. LET-60 is misexpressed in the puf-8(-) mutant germlines and PUF-8 physically interacts with the let-60 3′ UTR. Furthermore, PUF-8 suppresses let-60 3′ UTR-mediated expression in the germ cells that are transitioning from the mitotic to meiotic fate. These results reveal that PUF-8-mediated inhibition of the RAS/MAPK pathway is essential for mitotic-to-meiotic fate transition. PMID:23487310

  9. PUF-8 negatively regulates RAS/MAPK signalling to promote differentiation of C. elegans germ cells.

    PubMed

    Vaid, Samir; Ariz, Mohd; Chaturbedi, Amaresh; Kumar, Ganga Anil; Subramaniam, Kuppuswamy

    2013-04-01

    Signals that promote germ cell self-renewal by preventing premature meiotic entry are well understood. However, signals that control mitotic proliferation to promote meiotic differentiation have not been well characterized. In Caenorhabditis elegans, GLP-1 Notch signalling promotes the proliferative fate by preventing premature meiotic entry. The germline niche cell, which is the source of the ligand for GLP-1, spatially restricts GLP-1 signalling and thus enables the germ cells that have moved away from the niche to enter meiosis. Here, we show that the suppression of RAS/MAP kinase signalling in the mitotic and meiotic-entry regions is essential for the regulation of the mitosis-meiosis switch by niche signalling. We provide evidence that the conserved PUF family RNA-binding protein PUF-8 and the RAS GAP protein GAP-3 function redundantly to suppress the LET-60 RAS in the mitotic and meiotic entry regions. Germ cells missing both PUF-8 and GAP-3 proliferate in an uncontrolled fashion and fail to undergo meiotic development. MPK-1, the MAP kinase downstream of the LET-60 RAS, is prematurely activated in these cells; downregulation of MPK-1 activation eliminates tumours and restores differentiation. Our results further reveal that PUF-8 negatively regulates LET-60 expression at a post-transcriptional step. LET-60 is misexpressed in the puf-8(-) mutant germlines and PUF-8 physically interacts with the let-60 3' UTR. Furthermore, PUF-8 suppresses let-60 3' UTR-mediated expression in the germ cells that are transitioning from the mitotic to meiotic fate. These results reveal that PUF-8-mediated inhibition of the RAS/MAPK pathway is essential for mitotic-to-meiotic fate transition.

  10. Signal Integration by Lipid-Mediated Spatial Cross Talk between Ras Nanoclusters

    PubMed Central

    Zhou, Yong; Liang, Hong; Rodkey, Travis; Ariotti, Nicholas; Parton, Robert G.

    2014-01-01

    Lipid-anchored Ras GTPases form transient, spatially segregated nanoclusters on the plasma membrane that are essential for high-fidelity signal transmission. The lipid composition of Ras nanoclusters, however, has not previously been investigated. High-resolution spatial mapping shows that different Ras nanoclusters have distinct lipid compositions, indicating that Ras proteins engage in isoform-selective lipid sorting and accounting for different signal outputs from different Ras isoforms. Phosphatidylserine is a common constituent of all Ras nanoclusters but is only an obligate structural component of K-Ras nanoclusters. Segregation of K-Ras and H-Ras into spatially and compositionally distinct lipid assemblies is exquisitely sensitive to plasma membrane phosphatidylserine levels. Phosphatidylserine spatial organization is also modified by Ras nanocluster formation. In consequence, Ras nanoclusters engage in remote lipid-mediated communication, whereby activated H-Ras disrupts the assembly and operation of spatially segregated K-Ras nanoclusters. Computational modeling and experimentation reveal that complex effects of caveolin and cortical actin on Ras nanoclustering are similarly mediated through regulation of phosphatidylserine spatiotemporal dynamics. We conclude that phosphatidylserine maintains the lateral segregation of diverse lipid-based assemblies on the plasma membrane and that lateral connectivity between spatially remote lipid assemblies offers important previously unexplored opportunities for signal integration and signal processing. PMID:24366544

  11. RAS signaling promotes resistance to JAK inhibitors by suppressing BAD-mediated apoptosis.

    PubMed

    Winter, Peter S; Sarosiek, Kristopher A; Lin, Kevin H; Meggendorfer, Manja; Schnittger, Susanne; Letai, Anthony; Wood, Kris C

    2014-12-23

    Myeloproliferative neoplasms (MPNs) frequently have an activating mutation in the gene encoding Janus kinase 2 (JAK2). Thus, targeting the pathway mediated by JAK and its downstream substrate, signal transducer and activator of transcription (STAT), may yield clinical benefit for patients with MPNs containing the JAK2(V617F) mutation. Although JAK inhibitor therapy reduces splenomegaly and improves systemic symptoms in patients, this treatment does not appreciably reduce the number of neoplastic cells. To identify potential mechanisms underlying this inherent resistance phenomenon, we performed pathway-centric, gain-of-function screens in JAK2(V617F) hematopoietic cells and found that the activation of the guanosine triphosphatase (GTPase) RAS or its effector pathways [mediated by the kinases AKT and ERK (extracellular signal-regulated kinase)] renders cells insensitive to JAK inhibition. Resistant MPN cells became sensitized to JAK inhibitors when also exposed to inhibitors of the AKT or ERK pathways. Mechanistically, in JAK2(V617F) cells, a JAK2-mediated inactivating phosphorylation of the proapoptotic protein BAD [B cell lymphoma 2 (BCL-2)-associated death promoter] promoted cell survival. In sensitive cells, exposure to a JAK inhibitor resulted in dephosphorylation of BAD, enabling BAD to bind and sequester the prosurvival protein BCL-XL (BCL-2-like 1), thereby triggering apoptosis. In resistant cells, RAS effector pathways maintained BAD phosphorylation in the presence of JAK inhibitors, yielding a specific dependence on BCL-XL for survival. In patients with MPNs, activating mutations in RAS co-occur with the JAK2(V617F) mutation in the malignant cells, suggesting that RAS effector pathways likely play an important role in clinically observed resistance.

  12. RAS signaling and anti-RAS therapy: lessons learned from genetically engineered mouse models, human cancer cells, and patient-related studies.

    PubMed

    Fang, Bingliang

    2016-01-01

    Activating mutations of oncogenic RAS genes are frequently detected in human cancers. The studies in genetically engineered mouse models (GEMMs) reveal that Kras-activating mutations predispose mice to early onset tumors in the lung, pancreas, and gastrointestinal tract. Nevertheless, most of these tumors do not have metastatic phenotypes. Metastasis occurs when tumors acquire additional genetic changes in other cancer driver genes. Studies on clinical specimens also demonstrated that KRAS mutations are present in premalignant tissues and that most of KRAS mutant human cancers have co-mutations in other cancer driver genes, including TP53, STK11, CDKN2A, and KMT2C in lung cancer; APC, TP53, and PIK3CA in colon cancer; and TP53, CDKN2A, SMAD4, and MED12 in pancreatic cancer. Extensive efforts have been devoted to develop therapeutic agents that target enzymes involved in RAS posttranslational modifications, that inhibit downstream effectors of RAS signaling pathways, and that kill RAS mutant cancer cells through synthetic lethality. Recent clinical studies have revealed that sorafenib, a pan-RAF and VEGFR inhibitor, has impressive benefits for KRAS mutant lung cancer patients. Combination therapy of MEK inhibitors with either docetaxel, AKT inhibitors, or PI3K inhibitors also led to improved clinical responses in some KRAS mutant cancer patients. This review discusses knowledge gained from GEMMs, human cancer cells, and patient-related studies on RAS-mediated tumorigenesis and anti-RAS therapy. Emerging evidence demonstrates that RAS mutant cancers are heterogeneous because of the presence of different mutant alleles and/or co-mutations in other cancer driver genes. Effective subclassifications of RAS mutant cancers may be necessary to improve patients' outcomes through personalized precision medicine. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology

  13. EGFR/ARF6 regulation of Hh signalling stimulates oncogenic Ras tumour overgrowth.

    PubMed

    Chabu, Chiswili; Li, Da-Ming; Xu, Tian

    2017-03-10

    Multiple signalling events interact in cancer cells. Oncogenic Ras cooperates with Egfr, which cannot be explained by the canonical signalling paradigm. In turn, Egfr cooperates with Hedgehog signalling. How oncogenic Ras elicits and integrates Egfr and Hedgehog signals to drive overgrowth remains unclear. Using a Drosophila tumour model, we show that Egfr cooperates with oncogenic Ras via Arf6, which functions as a novel regulator of Hh signalling. Oncogenic Ras induces the expression of Egfr ligands. Egfr then signals through Arf6, which regulates Hh transport to promote Hh signalling. Blocking any step of this signalling cascade inhibits Hh signalling and correspondingly suppresses the growth of both, fly and human cancer cells harbouring oncogenic Ras mutations. These findings highlight a non-canonical Egfr signalling mechanism, centered on Arf6 as a novel regulator of Hh signalling. This explains both, the puzzling requirement of Egfr in oncogenic Ras-mediated overgrowth and the cooperation between Egfr and Hedgehog.

  14. EGFR/ARF6 regulation of Hh signalling stimulates oncogenic Ras tumour overgrowth

    PubMed Central

    Chabu, Chiswili; Li, Da-Ming; Xu, Tian

    2017-01-01

    Multiple signalling events interact in cancer cells. Oncogenic Ras cooperates with Egfr, which cannot be explained by the canonical signalling paradigm. In turn, Egfr cooperates with Hedgehog signalling. How oncogenic Ras elicits and integrates Egfr and Hedgehog signals to drive overgrowth remains unclear. Using a Drosophila tumour model, we show that Egfr cooperates with oncogenic Ras via Arf6, which functions as a novel regulator of Hh signalling. Oncogenic Ras induces the expression of Egfr ligands. Egfr then signals through Arf6, which regulates Hh transport to promote Hh signalling. Blocking any step of this signalling cascade inhibits Hh signalling and correspondingly suppresses the growth of both, fly and human cancer cells harbouring oncogenic Ras mutations. These findings highlight a non-canonical Egfr signalling mechanism, centered on Arf6 as a novel regulator of Hh signalling. This explains both, the puzzling requirement of Egfr in oncogenic Ras-mediated overgrowth and the cooperation between Egfr and Hedgehog. PMID:28281543

  15. AMPK and Endothelial Nitric Oxide Synthase Signaling Regulates K-Ras Plasma Membrane Interactions via Cyclic GMP-Dependent Protein Kinase 2.

    PubMed

    Cho, Kwang-Jin; Casteel, Darren E; Prakash, Priyanka; Tan, Lingxiao; van der Hoeven, Dharini; Salim, Angela A; Kim, Choel; Capon, Robert J; Lacey, Ernest; Cunha, Shane R; Gorfe, Alemayehu A; Hancock, John F

    2016-12-15

    K-Ras must localize to the plasma membrane and be arrayed in nanoclusters for biological activity. We show here that K-Ras is a substrate for cyclic GMP-dependent protein kinases (PKGs). In intact cells, activated PKG2 selectively colocalizes with K-Ras on the plasma membrane and phosphorylates K-Ras at Ser181 in the C-terminal polybasic domain. K-Ras phosphorylation by PKG2 is triggered by activation of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase. Phosphorylated K-Ras reorganizes into distinct nanoclusters that retune the signal output. Phosphorylation acutely enhances K-Ras plasma membrane affinity, but phosphorylated K-Ras is progressively lost from the plasma membrane via endocytic recycling. Concordantly, chronic pharmacological activation of AMPK → PKG2 signaling with mitochondrial inhibitors, nitric oxide, or sildenafil inhibits proliferation of K-Ras-positive non-small cell lung cancer cells. The study shows that K-Ras is a target of a metabolic stress-signaling pathway that can be leveraged to inhibit oncogenic K-Ras function.

  16. Nox4-Derived H2O2 Mediates Endoplasmic Reticulum Signaling through Local Ras Activation▿

    PubMed Central

    Wu, Ru-Feng; Ma, Zhenyi; Liu, Zhe; Terada, Lance S.

    2010-01-01

    The unfolded-protein response (UPR) of the endoplasmic reticulum (ER) has been linked to oxidant production, although the molecular details and functional significance of this linkage are poorly understood. Using a ratiometric H2O2 sensor targeted to different subcellular compartments, we demonstrate specific production of H2O2 by the ER in response to the stressors tunicamycin and HIV-1 Tat, but not to thapsigargin or dithiothreitol. Knockdown of the oxidase Nox4, expressed on ER endomembranes, or expression of ER-targeted catalase blocked ER H2O2 production by tunicamycin and Tat and prevented the UPR following exposure to these two agonists, but not to thapsigargin or dithiothreitol. Tat also triggered Nox4-dependent, sustained activation of Ras leading to ERK, but not phosphatidylinositol 3-kinase (PI3K)/mTOR, pathway activation. Cell fractionation studies and green fluorescent protein (GFP) fusions of GTPase effector binding domains confirmed selective activation of endogenous RhoA and Ras on the ER surface, with ER-associated K-Ras acting upstream of the UPR and downstream of Nox4. Notably, the Nox4/Ras/ERK pathway induced autophagy, and suppression of autophagy unmasked cell death and prevented differentiation of endothelial cells in 3-dimensional matrix. We conclude that the ER surface provides a platform to spatially organize agonist-specific Nox4-dependent oxidative signaling events, leading to homeostatic protective mechanisms rather than oxidative stress. PMID:20457808

  17. Bypassing both surface attachment and surface recognition requirements for appressorium formation by overactive ras signaling in Magnaporthe oryzae.

    PubMed

    Zhou, Xiaoying; Zhao, Xinhua; Xue, Chaoyang; Dai, Yafeng; Xu, Jin-Rong

    2014-09-01

    Magnaporthe oryzae forms a highly specialized infection structure called an appressorium for plant penetration. In M. oryzae and many other plant-pathogenic fungi, surface attachment and surface recognition are two essential requirements for appressorium formation. Development of appressoria in the air has not been reported. In this study, we found that expression of a dominant active MoRAS2(G18V) allele in M. oryzae resulted in the formation of morphologically abnormal appressoria on nonconducive surfaces, in liquid suspensions, and on aerial hyphae without attachment to hard surfaces. Both the Pmk1 mitogen-activated protein kinase cascade and cAMP signaling pathways that regulate surface recognition and appressorium morphogenesis in M. oryzae were overactivated in the MoRAS2(G18V) transformant. In mutants deleted of PMK1 or CPKA, expression of MoRAS2(G18V) had no significant effects on appressorium morphogenesis. Furthermore, expression of dominant MoRAS2 in Colletotrichum graminicola and C. gloeosporioides also caused the formation of appressorium-like structures in aerial hyphae. Overall, our data indicate that MoRas2 functions upstream from both the cAMP-PKA and Pmk1 pathways and overactive Ras signaling leads to improper activation of these two pathways and appressorium formation without surface attachment in appressorium-forming pathogens.

  18. Defined spatiotemporal features of RAS-ERK signals dictate cell fate in MCF-7 mammary epithelial cells.

    PubMed

    Herrero, Ana; Casar, Berta; Colón-Bolea, Paula; Agudo-Ibáñez, Lorena; Crespo, Piero

    2016-06-15

    Signals conveyed through the RAS-ERK pathway are essential for the determination of cell fate. It is well established that signal variability is achieved in the different microenvironments in which signals unfold. It is also known that signal duration is critical for decisions concerning cell commitment. However, it is unclear how RAS-ERK signals integrate time and space in order to elicit a given biological response. To investigate this, we used MCF-7 cells, in which EGF-induced transient ERK activation triggers proliferation, whereas sustained ERK activation in response to heregulin leads to adipocytic differentiation. We found that both proliferative and differentiating signals emanate exclusively from plasma membrane-disordered microdomains. Of interest, the EGF signal can be transformed into a differentiating stimulus by HRAS overexpression, which prolongs ERK activation, but only if HRAS localizes at disordered membrane. On the other hand, HRAS signals emanating from the Golgi complex induce apoptosis and can prevent heregulin-induced differentiation. Our results indicate that within the same cellular context, RAS can exert different, even antagonistic, effects, depending on its sublocalization. Thus cell destiny is defined by the ability of a stimulus to activate RAS at the appropriate sublocalization for an adequate period while avoiding switching on opposing RAS signals.

  19. Defined spatiotemporal features of RAS-ERK signals dictate cell fate in MCF-7 mammary epithelial cells

    PubMed Central

    Herrero, Ana; Casar, Berta; Colón-Bolea, Paula; Agudo-Ibáñez, Lorena; Crespo, Piero

    2016-01-01

    Signals conveyed through the RAS-ERK pathway are essential for the determination of cell fate. It is well established that signal variability is achieved in the different microenvironments in which signals unfold. It is also known that signal duration is critical for decisions concerning cell commitment. However, it is unclear how RAS-ERK signals integrate time and space in order to elicit a given biological response. To investigate this, we used MCF-7 cells, in which EGF-induced transient ERK activation triggers proliferation, whereas sustained ERK activation in response to heregulin leads to adipocytic differentiation. We found that both proliferative and differentiating signals emanate exclusively from plasma membrane–disordered microdomains. Of interest, the EGF signal can be transformed into a differentiating stimulus by HRAS overexpression, which prolongs ERK activation, but only if HRAS localizes at disordered membrane. On the other hand, HRAS signals emanating from the Golgi complex induce apoptosis and can prevent heregulin-induced differentiation. Our results indicate that within the same cellular context, RAS can exert different, even antagonistic, effects, depending on its sublocalization. Thus cell destiny is defined by the ability of a stimulus to activate RAS at the appropriate sublocalization for an adequate period while avoiding switching on opposing RAS signals. PMID:27099370

  20. DNA segment in chromosome 9q34 which is duplicated in a sporadic case of Tuberous sclerosis encodes a new gene with homology to oncogenes in the Ras signaling pathway

    SciTech Connect

    Smith, M.; Handa, K.; Bengstsson, U.

    1994-09-01

    The TSC1 gene has been mapped to a 1 megabase region on chromosome 9q34. This region is flanked by markers D9S125 and D9S114. D9S10 and D9S66 loci map within this region. In a sporadic case of Tuberous sclerosis we demonstrated the presence of a DNA duplication using probes for the D9S10, and D9S66 loci and cosmids which overlap the telomeric end of the D9S66 cosmid. The duplication was demonstrated by analysis of the D9S66 polymorphism which revealed the presence of two maternal alleles in the patient. It was also demonstrated using FISH to analyze metaphase and interphase chromosomes, and by pulsed field gel electrophoresis which revealed novel M1u1 fragments in patient cellular DNA examined using as probes unique sequence regions of D9S10, or unique sequence regions of cosmids flanking the telomeric end of D9S66. There was no evidence for duplication of DBH or ABL. We carried out DNA sequence analysis of regions of D9S10 and cosmids which overlap D9S10 at its telomeric end, and determined that these cosmids contain exons with regions of high homology to portions of the Vav oncogene. We used a unique sequence region of D9S10 to examine mRNA and demonstrated a transcript of 4 kb. These studies are important since they have refined the map position of the TSC1 locus and secondly because they have led to the identification of a new oncogene with high homology to oncogenes in the RAS signalling pathway.

  1. Solution phase parallel synthesis and evaluation of MAPK inhibitory activities of close structural analogues of a Ras pathway modulator.

    PubMed

    Lu, Yingchun; Sakamuri, Sukumar; Chen, Quin-Zene; Keng, Yen-Fang; Khazak, Vladimir; Illgen, Katrin; Schabbert, Silke; Weber, Lutz; Menon, Sanjay R

    2004-08-02

    A solution phase parallel synthesis approach was undertaken to rapidly explore the structure-activity relationship of an inhibitor of the Ras/Raf protein interaction identified from a small molecule compound library. Evaluation of the MAPK pathway signaling inhibitory activity of the synthesized analogues as well as their antiproliferative activity and ability to inhibit soft agar growth were performed.

  2. RAS signaling promotes resistance to JAK inhibitors by suppressing BAD-mediated apoptosis

    PubMed Central

    Winter, Peter S.; Sarosiek, Kristopher A.; Lin, Kevin H.; Meggendorfer, Manja; Schnittger, Susanne; Letai, Anthony

    2015-01-01

    Myeloproliferative neoplasms (MPNs) frequently have an activating mutation in the gene encoding Janus kinase 2 (JAK2). Thus, targeting the pathway mediated by JAK and its downstream substrate, signal transducer and activator of transcription (STAT), may yield clinical benefit for patients with MPNs containing the JAK2V617F mutation. Although JAK inhibitor therapy reduces splenomegaly and improves systemic symptoms in patients, this treatment does not appreciably reduce the number of neoplastic cells. To identify potential mechanisms underlying this inherent resistance phenomenon, we performed pathway-centric, gain-of-function screens in JAK2V617F hematopoietic cells and found that the activation of the guanosine triphosphatase (GTPase) RAS or its effector pathways (mediated by the kinases AKT and ERK) renders cells insensitive to JAK inhibition. Resistant MPN cells became sensitized to JAK inhibitors when also exposed to inhibitors of the AKT or ERK pathways. Mechanistically, in JAK2V617F cells a JAK2-mediated inactivating phosphorylation of the pro-apoptotic protein BAD [B-cell lymphoma 2 (BCL-2)-associated death promoter] promoted cell survival. In sensitive cells, exposure to a JAK inhibitor resulted in dephosphorylation of BAD, enabling BAD to bind and sequester the pro-survival protein BCL-XL (also known as BCL2-like 1), thereby triggering apoptosis. In resistant cells, RAS effector pathways maintained BAD phosphorylation in the presence of JAK inhibitors, yielding a specific dependence on BCL-XL for survival. BCL-XL inhibitors potently induced apoptosis in JAK inhibitor-resistant cells. In patients with MPNs, activating mutations in RAS co-occur with the JAK2V617F mutation in the malignant cells, suggesting that RAS effector pathways likely play an important role in clinically observed resistance. PMID:25538080

  3. Plant farnesyltransferase can restore yeast Ras signaling and mating

    SciTech Connect

    Yalovsky, S.; Callan, K.L.; Narita, J.O.

    1997-04-01

    Farnesyltransferase (FTase) is a heterodimeric enzyme that modifies a group of proteins, including Ras, in mammals and yeasts. Plant FTase {alpha} and {beta} subunits were cloned from tomato and expressed in the yeast Saccharomyces cerevisiae to assess their functional conservation in farnesylating Ras and a-factor proteins, which are important for cell growth and mating. The tomato FTase {beta} subunit (LeFTB) alone was unable to complement the growth defect of ram1{del} mutant yeast strains in which the chromosomal FTase {beta} subunit gene was deleted, but coexpression of LeFTB with the plant {alpha} subunit gene (LeFTA) restored normal growth, Ras membrane association, and mating. LeFTB contains a novel 66-amino-acid sequence domain whose deletion reduces the efficiency of tomato FTase to restore normal growth to yeast ram1{del} strains. Coexpression of LeFTA and LeFTB in either yeast or insect cells yielded a functional enzyme that correctly farnesylated CaaX-motif-containing peptides. Despite their low degree of sequence homology, yeast and plant FTases shared similar in vivo and in vitro substrate specificities, demonstrating that this enzymatic modification of proteins with intermediates from the isoprenoid biosynthesis pathway is conserved in evolutionarily divergent eukaryotes. 56 refs., 7 figs., 1 tab.

  4. Plant farnesyltransferase can restore yeast Ras signaling and mating.

    PubMed Central

    Yalovsky, S; Trueblood, C E; Callan, K L; Narita, J O; Jenkins, S M; Rine, J; Gruissem, W

    1997-01-01

    Farnesyltransferase (FTase) is a heterodimeric enzyme that modifies a group of proteins, including Ras, in mammals and yeasts. Plant FTase alpha and beta subunits were cloned from tomato and expressed in the yeast Saccharomyces cerevisiae to assess their functional conservation in farnesylating Ras and a-factor proteins, which are important for cell growth and mating. The tomato FTase beta subunit (LeFTB) alone was unable to complement the growth defect of ram1 delta mutant yeast strains in which the chromosomal FTase beta subunit gene was deleted, but coexpression of LeFTB with the plant alpha subunit gene (LeFTA) restored normal growth, Ras membrane association, and mating. LeFTB contains a novel 66-amino-acid sequence domain whose deletion reduces the efficiency of tomato FTase to restore normal growth to yeast ram1 delta strains. Coexpression of LeFTA and LeFTB in either yeast or insect cells yielded a functional enzyme that correctly farnesylated CaaX-motif-containing peptides. Despite their low degree of sequence homology, yeast and plant FTases shared similar in vivo and in vitro substrate specificities, demonstrating that this enzymatic modification of proteins with intermediates from the isoprenoid biosynthesis pathway is conserved in evolutionarily divergent eukaryotes. PMID:9121446

  5. Mitochondrial Activity and Cyr1 Are Key Regulators of Ras1 Activation of C. albicans Virulence Pathways

    PubMed Central

    Grahl, Nora; Demers, Elora G.; Lindsay, Allia K.; Harty, Colleen E.; Willger, Sven D.; Piispanen, Amy E.; Hogan, Deborah A.

    2015-01-01

    Candida albicans is both a major fungal pathogen and a member of the commensal human microflora. The morphological switch from yeast to hyphal growth is associated with disease and many environmental factors are known to influence the yeast-to-hyphae switch. The Ras1-Cyr1-PKA pathway is a major regulator of C. albicans morphogenesis as well as biofilm formation and white-opaque switching. Previous studies have shown that hyphal growth is strongly repressed by mitochondrial inhibitors. Here, we show that mitochondrial inhibitors strongly decreased Ras1 GTP-binding and activity in C. albicans and similar effects were observed in other Candida species. Consistent with there being a connection between respiratory activity and GTP-Ras1 binding, mutants lacking complex I or complex IV grew as yeast in hypha-inducing conditions, had lower levels of GTP-Ras1, and Ras1 GTP-binding was unaffected by respiratory inhibitors. Mitochondria-perturbing agents decreased intracellular ATP concentrations and metabolomics analyses of cells grown with different respiratory inhibitors found consistent perturbation of pyruvate metabolism and the TCA cycle, changes in redox state, increased catabolism of lipids, and decreased sterol content which suggested increased AMP kinase activity. Biochemical and genetic experiments provide strong evidence for a model in which the activation of Ras1 is controlled by ATP levels in an AMP kinase independent manner. The Ras1 GTPase activating protein, Ira2, but not the Ras1 guanine nucleotide exchange factor, Cdc25, was required for the reduction of Ras1-GTP in response to inhibitor-mediated reduction of ATP levels. Furthermore, Cyr1, a well-characterized Ras1 effector, participated in the control of Ras1-GTP binding in response to decreased mitochondrial activity suggesting a revised model for Ras1 and Cyr1 signaling in which Cyr1 and Ras1 influence each other and, together with Ira2, seem to form a master-regulatory complex necessary to integrate

  6. Crosstalk between the Ras2p-controlled Mitogen-activated Protein Kinase and cAMP Pathways during Invasive Growth of Saccharomyces cerevisiae

    PubMed Central

    Mösch, Hans-Ulrich; Kübler, Eric; Krappmann, Sven; Fink, Gerald R.; Braus, Gerhard H.

    1999-01-01

    The two highly conserved RAS genes of the budding yeast Saccharomyces cerevisiae are redundant for viability. Here we show that haploid invasive growth development depends on RAS2 but not RAS1. Ras1p is not sufficiently expressed to induce invasive growth. Ras2p activates invasive growth using either of two downstream signaling pathways, the filamentation MAPK (Cdc42p/Ste20p/MAPK) cascade or the cAMP-dependent protein kinase (Cyr1p/cAMP/PKA) pathway. This signal branch point can be uncoupled in cells expressing Ras2p mutant proteins that carry amino acid substitutions in the adenylyl cyclase interaction domain and therefore activate invasive growth solely dependent on the MAPK cascade. Both Ras2p-controlled signaling pathways stimulate expression of the filamentation response element-driven reporter gene depending on the transcription factors Ste12p and Tec1p, indicating a crosstalk between the MAPK and the cAMP signaling pathways in haploid cells during invasive growth. PMID:10233147

  7. The C-terminus of H-Ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways.

    PubMed

    Oeste, Clara L; Díez-Dacal, Beatriz; Bray, Francesca; García de Lacoba, Mario; de la Torre, Beatriz G; Andreu, David; Ruiz-Sánchez, Antonio J; Pérez-Inestrosa, Ezequiel; García-Domínguez, Carlota A; Rojas, José M; Pérez-Sala, Dolores

    2011-01-06

    Ras proteins are crucial players in differentiation and oncogenesis and constitute important drug targets. The localization and activity of Ras proteins are highly dependent on posttranslational modifications at their C-termini. In addition to an isoprenylated cysteine, H-Ras, but not other Ras proteins, possesses two cysteine residues (C181 and C184) in the C-terminal hypervariable domain that act as palmitoylation sites in cells. Cyclopentenone prostaglandins (cyPG) are reactive lipidic mediators that covalently bind to H-Ras and activate H-Ras dependent pathways. Dienone cyPG, such as 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) and Δ(12)-PGJ(2) selectively bind to the H-Ras hypervariable domain. Here we show that these cyPG bind simultaneously C181 and C184 of H-Ras, thus potentially altering the conformational tendencies of the hypervariable domain. Based on these results, we have explored the capacity of several bifunctional cysteine reactive small molecules to bind to the hypervariable domain of H-Ras proteins. Interestingly, phenylarsine oxide (PAO), a widely used tyrosine phosphatase inhibitor, and dibromobimane, a cross-linking agent used for cysteine mapping, effectively bind H-Ras hypervariable domain. The interaction of PAO with H-Ras takes place in vitro and in cells and blocks modification of H-Ras by 15d-PGJ(2). Moreover, PAO treatment selectively alters H-Ras membrane partition and the pattern of H-Ras activation in cells, from the plasma membrane to endomembranes. These results identify H-Ras as a novel target for PAO. More importantly, these observations reveal that small molecules or reactive intermediates interacting with spatially vicinal cysteines induce intramolecular cross-linking of H-Ras C-terminus potentially contributing to the modulation of Ras-dependent pathways.

  8. Calcium activation of Ras mediated by neuronal exchange factor Ras-GRF.

    PubMed

    Farnsworth, C L; Freshney, N W; Rosen, L B; Ghosh, A; Greenberg, M E; Feig, L A

    1995-08-10

    Tyrosine kinase receptors stimulate the Ras signalling pathway by enhancing the activity of the SOS nucleotide-exchange factor. This occurs, at least in part, by the recruitment of an SOS-GRB2 complex to Ras in the plasma membrane. Here we describe a different signalling pathway to Ras that involves activation of the Ras-GRF exchange factor in response to Ca2+ influx. In particular, we show that the ability of Ras-GRF to activate Ras in vivo is markedly enhanced by raised Ca2+ concentrations. Activation is mediated by calmodulin binding to an IQ motif in Ras-GRF, because substitutions in conserved amino acids in this motif prevent both calmodulin binding to Ras-GRF and Ras-GRF activation in vivo. So far, full-length Ras-GRF has been detected only in brain neurons. Our findings implicate Ras-GRF in the regulation of neuronal functions that are influenced by Ca2+ signals.

  9. TOR and RAS pathways regulate desiccation tolerance in Saccharomyces cerevisiae

    PubMed Central

    Welch, Aaron Z.; Gibney, Patrick A.; Botstein, David; Koshland, Douglas E.

    2013-01-01

    Tolerance to desiccation in cultures of Saccharomyces cerevisiae is inducible; only one in a million cells from an exponential culture survive desiccation compared with one in five cells in stationary phase. Here we exploit the desiccation sensitivity of exponentially dividing cells to understand the stresses imposed by desiccation and their stress response pathways. We found that induction of desiccation tolerance is cell autonomous and that there is an inverse correlation between desiccation tolerance and growth rate in glucose-, ammonia-, or phosphate-limited continuous cultures. A transient heat shock induces a 5000–fold increase in desiccation tolerance, whereas hyper-ionic, -reductive, -oxidative, or -osmotic stress induced much less. Furthermore, we provide evidence that the Sch9p-regulated branch of the TOR and Ras-cAMP pathway inhibits desiccation tolerance by inhibiting the stress response transcription factors Gis1p, Msn2p, and Msn4p and by activating Sfp1p, a ribosome biogenesis transcription factor. Among 41 mutants defective in ribosome biogenesis, a subset defective in 60S showed a dramatic increase in desiccation tolerance independent of growth rate. We suggest that reduction of a specific intermediate in 60S biogenesis, resulting from conditions such as heat shock and nutrient deprivation, increases desiccation tolerance. PMID:23171550

  10. Mutated K-ras(Asp12) promotes tumourigenesis in Apc(Min) mice more in the large than the small intestines, with synergistic effects between K-ras and Wnt pathways.

    PubMed

    Luo, Feijun; Brooks, David G; Ye, Hongtao; Hamoudi, Rifat; Poulogiannis, George; Patek, Charles E; Winton, Douglas J; Arends, Mark J

    2009-10-01

    Summary K-ras mutations are found in 40-50% of human colorectal adenomas and carcinomas, but their functional contribution remains incompletely understood. Here, we show that a conditional mutant K-ras mouse model (K-ras(Asp12)/Cre), with transient intestinal Cre activation by beta-Naphthoflavone (beta-NF) treatment, displayed transgene recombination and K-ras(Asp12) expression in the murine intestines, but developed few intestinal adenomas over 2 years. However, when crossed with Apc(Min/+) mice, the K-ras(Asp12)/Cre/Apc(Min/+) offspring showed acceleration of intestinal tumourigenesis with significantly changed average lifespan (P < 0.05) decreased to 18.4 +/- 5.4 weeks from 20.9 +/- 4.7 weeks (control Apc(Min/+) mice). The numbers of adenomas in the small intestine and large intestine were significantly (P < 0.01) increased by 1.5-fold and 5.7-fold, respectively, in K-ras(Asp12)/Cre/Apc(Min/+) mice compared with Apc(Min/+) mice, with the more marked increase in adenoma prevalence in the large intestine. To explore possible mechanisms for K-ras(Asp12) and Apc(Min) co-operation, the Mitogen-activated protein kinase (Mapk), Akt and Wnt signalling pathways, including selected target gene expression levels, were evaluated in normal large intestine and large intestinal tumours. K-ras(Asp12) increased activation of Mapk and Akt signalling pathway targets phospho-extracellular signal-regulated kinase (pErk) and pAkt, and increased relative expression levels of Wnt pathway targets vascular endothelial growth factor (VEGF), gastrin, cyclo-oxygenase 2 (Cox2) and T-cell lymphoma invasion and metastasis 1 (Tiam1) in K-ras(Asp12)/Cre/Apc(Min/+) adenomas compared with that of Apc(Min/+) adenomas, although other Wnt signalling pathway target genes such as Peroxisome proliferator-activated receptor delta (PPARd), matrix metalloproteinase 7 (MMP7), protein phosphatase 1 alpha (PP1A) and c-myc remained unchanged. In conclusion, intestinal expression of K-ras(Asp12) promotes mutant

  11. Ras activation of Erk restores impaired tonic BCR signaling and rescues immature B cell differentiation

    PubMed Central

    Rowland, Sarah L.; DePersis, Corinne L.; Torres, Raul M.

    2010-01-01

    B cell receptors (BCRs) generate tonic signals critical for B cell survival and early B cell development. To determine whether these signals also mediate the development of transitional and mature B cells, we examined B cell development using a mouse strain in which nonautoreactive immunoglobulin heavy and light chain–targeted B cells express low surface BCR levels. We found that reduced BCR expression translated into diminished tonic BCR signals that strongly impaired the development of transitional and mature B cells. Constitutive expression of Bcl-2 did not rescue the differentiation of BCR-low B cells, suggesting that this defect was not related to decreased cell survival. In contrast, activation of the Ras pathway rescued the differentiation of BCR-low immature B cells both in vitro and in vivo, whereas extracellular signal-regulated kinase (Erk) inhibition impaired the differentiation of normal immature B cells. These results strongly suggest that tonic BCR signaling mediates the differentiation of immature into transitional and mature B cells via activation of Erk, likely through a pathway requiring Ras. PMID:20176802

  12. MiR-21/RASA1 axis affects malignancy of colon cancer cells via RAS pathways

    PubMed Central

    Gong, Bo; Liu, Wan-Wei; Nie, Wen-Jing; Li, Dong-Feng; Xie, Zi-Jun; Liu, Chao; Liu, Yan-Hui; Mei, Ping; Li, Zi-Jun

    2015-01-01

    AIM: To determine how the oncogene miR-21 regulates the RAS signaling pathways and affects colon cancer cell behaviors. METHODS: RAS p21 GTPase activating protein 1 (RASA1) protein expression in six colon cancer cell lines was assessed by Western blot. Colon cancer RKO cells were chosen for transfection because they are KRAS wild type colon cancer cells whose RASA1 expression is significantly decreased. RKO cells were transfected with vectors overexpressing or down-regulating either miR-21 or RASA1. Furthermore, a luciferase reporter assay was used to determine whether RASA1 is a gene target of miR-21. Then, changes in mRNA and protein levels of RASA1, RAS-GTP, and other components of the RAS signaling pathways were assessed in transfected RKO cells by real-time quantitative reverse transcription-polymerase chain reaction, Western blot and immunoprecipitation. Finally, cell proliferation, apoptosis, invasion, and tumor formation ability were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye assay, flow cytometry, transwell assay, and animal experiment, respectively. RESULTS: RASA1 protein levels were significantly decreased in RKO cells compared with the other 5 colon cancer cell lines, and RASA1 was confirmed as a target gene of miR-21. Interestingly, RASA1 mRNA and protein levels in pre-miR-21-LV (up-regulation of miR-21) cells were lower than those in anti-miR-21-LV (down-regulation of miR-21) cells (P < 0.05). In addition, pre-miR-21-LV or siRASA1 (down-regulation of RASA1) cells showed higher cell proliferation, reduced apoptosis, increased expression of RAS-GTP, p-AKT, Raf-1, KRAS, and p-ERK1/2, and higher invasion and tumor formation ability, compared with control, anti-miR-21-LV or pcDNA3.1-RASA1 (up-regulation of RASA1) cells (P < 0.05). CONCLUSION: RASA1 is a target gene of miR-21, which promotes malignant behaviors of RKO cells through regulation of RASA1 expression. PMID:25663768

  13. Dissecting the senescence-like program in tumor cells activated by Ras signaling.

    PubMed

    Bihani, Teeru; Chicas, Agustin; Lo, Crystal Pui-Kwan; Lin, Athena W

    2007-01-26

    Activated Ras signaling can induce a permanent growth arrest in osteosarcoma cells. Here, we report that a senescence-like growth inhibition is also achieved in human carcinoma cells upon the transduction of H-Ras(V12). Ras-induced tumor senescence can be recapitulated by the transduction of activated, but not wild-type, MEK. The ability for H-Ras(V12) to suppress tumor cell growth is drastically compromised in cells that harbor endogenous activating ras mutations. Notably, growth inhibition of tumor cells containing ras mutations can be achieved through the introduction of activated MEK. Tumor senescence induced by Ras signaling can occur in the absence of p16 or Rb and is not interrupted by the inactivation of Rb, p107, or p130 via short hairpin RNA or the transduction with HPV16 E7. In contrast, inactivation of p21 via short hairpin RNA disrupts Ras-induced tumor senescence. In summary, this study uncovers a senescence-like program activated by Ras signaling to inhibit cancer cell growth. This program appears to be intact in cancer cells that do not harbor ras mutations. Moreover, cancer cells that carry ras mutations remain susceptible to tumor senescence induced by activated MEK. These novel findings can potentially lead to the development of innovative cancer intervention.

  14. K-Ras promotes growth transformation and invasion of immortalized human pancreatic cells by Raf and phosphatidylinositol 3-kinase signaling.

    PubMed

    Campbell, Paul M; Groehler, Angela L; Lee, Kwang M; Ouellette, Michel M; Khazak, Vladimir; Der, Channing J

    2007-03-01

    Mutational activation of the K-Ras oncogene is well established as a key genetic step in the development and growth of pancreatic adenocarcinomas. However, the mechanism by which aberrant Ras signaling promotes uncontrolled pancreatic tumor cell growth remains to be fully elucidated. The recent use of primary human cells to study Ras-mediated oncogenesis provides important model cell systems to dissect this mechanism. We have used a model of telomerase-immortalized human pancreatic duct-derived cells (E6/E7/st) to study mechanisms of Ras growth transformation. First, we found that human papillomavirus E6 and E7 oncogenes, which block the function of the p53 and Rb tumor suppressors, respectively, and SV40 small t antigen were required to allow mutant K-Ras(12D) growth transformation. Second, K-Ras(12D) caused growth transformation in vitro, including enhanced growth rate and loss of density dependency for growth, anchorage independence, and invasion through reconstituted basement membrane proteins, and tumorigenic transformation in vivo. Third, we determined that the Raf, phosphatidylinositol 3-kinase (PI3K), and Ral guanine nucleotide exchange factor effector pathways were activated, although extracellular signal-regulated kinase (ERK) activity was not up-regulated persistently. Finally, pharmacologic inhibition of Raf/mitogen-activated protein kinase/ERK and PI3K signaling impaired K-Ras-induced anchorage-independent growth and invasion. In summary, our studies established, characterized, and validated E6/E7/st cells for the study of Ras-induced oncogenesis.

  15. Oncogenic K-Ras signals through epidermal growth factor receptor and wild-type H-Ras to promote radiation survival in pancreatic and colorectal carcinoma cells.

    PubMed

    Cengel, Keith A; Voong, K Rahn; Chandrasekaran, Sanjay; Maggiorella, Laurence; Brunner, Thomas B; Stanbridge, Eric; Kao, Gary D; McKenna, W Gillies; Bernhard, Eric J

    2007-04-01

    Pancreatic and colorectal carcinomas frequently express oncogenic/mutant K-Ras that contributes to both tumorigenesis and clinically observed resistance to radiation treatment. We have previously shown that farnesyltransferase inhibitors (FTI) radiosensitize many pancreatic and colorectal cancer cell lines that express oncogenic K-ras at doses that inhibit the prenylation and activation of H-Ras but not K-Ras. In the present study, we have examined the mechanism of FTI-mediated radiosensitization in cell lines that express oncogenic K-Ras and found that wild-type H-Ras is a contributor to radiation survival in tumor cells that express oncogenic K-Ras. In these experiments, inhibiting the expression of oncogenic K-Ras, wild-type H-Ras, or epidermal growth factor receptor (EGFR) led to similar levels of radiosensitization as treatment with the FTI tipifarnib. Treatment with the EGFR inhibitor gefitinib led to similar levels of radiosensitization, and the combinations of tipifarnib or gefitinib plus inhibition of K-Ras, H-Ras, or EGFR expression did not provide additional radiosensitization compared with tipifarnib or gefitinib alone. Finally, supplementing culture medium with the EGFR ligand transforming growth factor alpha was able to reverse the radiosensitizing effect of inhibiting K-ras expression. Taken together, these findings suggest that EGFR-activated H-Ras signaling is initiated by oncogenic K-Ras to promote radiation survival in pancreatic and colorectal cancers.

  16. Oncogenic K-Ras Signals through Epidermal Growth Factor Receptor and Wild-Type H-Ras to Promote Radiation Survival in Pancreatic and Colorectal Carcinoma Cells1

    PubMed Central

    Cengel, Keith A.; Voong, K. Rahn; Chandrasekaran, Sanjay; Maggiorella, Laurence; Brunner, Thomas B.; Stanbridge, Eric; Kao, Gary D.; McKenna, W. Gillies; Bernhard, Eric J.

    2007-01-01

    Pancreatic and colorectal carcinomas frequently express oncogenic/mutant K-Ras that contributes to both tumorigenesis and clinically observed resistance to radiation treatment. We have previously shown that farnesyltransferase inhibitors (FTI) radiosensitize many pancreatic and colorectal cancer cell lines that express oncogenic K-ras at doses that inhibit the prenylation and activation of H-Ras but not K-Ras. In the present study, we have examined the mechanism of FTI-mediated radiosensitization in cell lines that express oncogenic K-Ras and found that wild-type H-Ras is a contributor to radiation survival in tumor cells that express oncogenic K-Ras. In these experiments, inhibiting the expression of oncogenic K-Ras, wild-type H-Ras, or epidermal growth factor receptor (EGFR) led to similar levels of radiosensitization as treatment with the FTI tipifarnib. Treatment with the EGFR inhibitor gefitinib led to similar levels of radiosensitization, and the combinations of tipifarnib or gefitinib plus inhibition of K-Ras, H-Ras, or EGFR expression did not provide additional radiosensitization compared with tipifarnib or gefitinib alone. Finally, supplementing culture medium with the EGFR ligand transforming growth factor α was able to reverse the radiosensitizing effect of inhibiting K-ras expression. Taken together, these findings suggest that EGFR-activated H-Ras signaling is initiated by oncogenic K-Ras to promote radiation survival in pancreatic and colorectal cancers. PMID:17460778

  17. Mapping the functional versatility and fragility of Ras GTPase signaling circuits through in vitro network reconstitution.

    PubMed

    Coyle, Scott M; Lim, Wendell A

    2016-01-14

    The Ras-superfamily GTPases are central controllers of cell proliferation and morphology. Ras signaling is mediated by a system of interacting molecules: upstream enzymes (GEF/GAP) regulate Ras's ability to recruit multiple competing downstream effectors. We developed a multiplexed, multi-turnover assay for measuring the dynamic signaling behavior of in vitro reconstituted H-Ras signaling systems. By including both upstream regulators and downstream effectors, we can systematically map how different network configurations shape the dynamic system response. The concentration and identity of both upstream and downstream signaling components strongly impacted the timing, duration, shape, and amplitude of effector outputs. The distorted output of oncogenic alleles of Ras was highly dependent on the balance of positive (GAP) and negative (GEF) regulators in the system. We found that different effectors interpreted the same inputs with distinct output dynamics, enabling a Ras system to encode multiple unique temporal outputs in response to a single input. We also found that different Ras-to-GEF positive feedback mechanisms could reshape output dynamics in distinct ways, such as signal amplification or overshoot minimization. Mapping of the space of output behaviors accessible to Ras provides a design manual for programming Ras circuits, and reveals how these systems are readily adapted to produce an array of dynamic signaling behaviors. Nonetheless, this versatility comes with a trade-off of fragility, as there exist numerous paths to altered signaling behaviors that could cause disease.

  18. Orchestration of Morphogenesis in Filamentous Fungi: Conserved Roles for Ras Signaling Networks

    PubMed Central

    Fortwendel, Jarrod R.

    2015-01-01

    Filamentous fungi undergo complex developmental programs including conidial germination, polarized morphogenesis, and differentiation of sexual and asexual structures. For many fungi, the coordinated completion of development is required for pathogenicity, as specialized morphological structures must be produced by the invading fungus. Ras proteins are highly conserved GTPase signal transducers and function as major regulators of growth and development in eukaryotes. Filamentous fungi typically express two Ras homologues, comprising distinct groups of Ras1-like and Ras2-like proteins based on sequence homology. Recent evidence suggests shared roles for both Ras1 and Ras2 homologues, but also supports the existence of unique functions in the areas of stress response and virulence. This review focuses on the roles played by both Ras protein groups during growth, development, and pathogenicity of a diverse array of filamentous fungi. PMID:26257821

  19. [Growth hormone signaling pathways].

    PubMed

    Zych, Sławomir; Szatkowska, Iwona; Czerniawska-Piatkowska, Ewa

    2006-01-01

    The substantial improvement in the studies on a very complicated mechanism-- growth hormone signaling in a cell, has been noted in last decade. GH-induced signaling is characterized by activation of several pathways, including extracellular signal-regulated kinase (ERK), the signal transducer and activator of transcription and phosphatidylinositol-3 kinase (PI3) pathways. This review shows a current model of the growth hormone receptor dimerization, rotation of subunits and JAK2 kinase activation as the initial steps in the cascade of events. In the next stages of the signaling process, the GH-(GHR)2-(JAK2)2 complex may activate signaling molecules such as Stat, IRS-1 and IRS-2, and particularly all cascade proteins that activate MAP kinase. These pathways regulate basal cellular functions including target gene transcription, enzymatic activity and metabolite transport. Therefore growth hormone is considered as a major regulator of postnatal growth and metabolism, probably for mammary gland growth and development too.

  20. Eve-3: a liver enriched suppressor of Ras/MAPK signaling.

    PubMed

    King, James A J; Corcoran, Niall M; D'Abaco, Giovanna M; Straffon, Andrew F; Smith, Craig T; Poon, Carole L C; Buchert, Michael; I, Stacey; Hall, Nathan E; Lock, Peter; Hovens, Christopher M

    2006-04-01

    The developed liver is able to tightly control cellular proliferation, rapidly switching from quiescence to growth in response to specific stimuli. This suggests that growth inhibitors may be involved in the control of liver growth. We analyzed the role of the Spred-family of growth inhibitors in the liver. We screened human EST databases for Spred-related sequences. Clones were isolated, sequenced, epitope-tagged and expressed. Subcellular localization of clones were determined and their effects on cellular signaling pathways analysed using specific antibodies. Cell cycle progression assays and protein interaction studies were initiated. Organ distribution of transcripts and their expression throughout liver development and in primary hepatocytes were recorded. We have identified a new, liver-restricted protein, Eve-3, containing a single Ena Vasp homology (EVH1) domain that can potently block activation of the Ras/MAPK pathway. Eve-3 is specific in inhibiting the Ras/MAPK pathway. Eve-3 can block serum-mediated cell cycle progression and its expression is highly regulated during liver development. The liver is the only organ that can regulate its growth and mass. Eve-3 may act as an inhibitor of proliferation pathways in the mature liver and be involved in modulating the unique regenerative capacity of this organ.

  1. Oleylethanolamide activates Ras-Erk pathway and improves myocardial function in doxorubicin-induced heart failure.

    PubMed

    Su, Hou-Fen; Samsamshariat, Ahmad; Fu, Jin; Shan, Yue-Xin; Chen, Yung-Hsiang; Piomelli, Daniele; Wang, Ping H

    2006-02-01

    Oleylethanolamide (OEA) is a natural fatty acid ethanolamide produced in the heart, but its biological actions in myocardium have not yet been defined. This study was carried out to determine whether OEA could be used to prevent the development of heart failure or improve evolving heart failure. We studied in vivo and in vitro actions of OEA in cardiac muscle. In an animal model of doxorubicin cardiomyopathy, OEA showed robust effects and attenuated the progression of systolic/diastolic dysfunction and ventricular remodeling. During evolving doxorubicin cardiomyopathy, a therapeutic course of OEA treatment partially restored myocardial function. The preventive and therapeutic effects of OEA were associated with significant improvement of survival. To investigate the mechanism of OEA action in cardiac muscle, we have carried out in vitro experiments in cultured cardiomyocytes. The results showed that OEA, through activation of Ras-Raf-1-Mek-Erk signaling, inhibited doxorubicin-induced apoptosis. Additional experiments showed that OEA activation of the Erk pathway involved activation of Neu/ErbB2 receptor, which suggests OEA actions in cardiac muscle might require activation of Neu/ErbB2. In summary, OEA improved ventricular remodeling and augmented cardiac function in doxorubicin cardiomyopathy, possibly involving activation of Neu/ErbB2 and Ras-Erk signaling. These findings suggest OEA is a novel cardioprotective compound that may be used to develop new strategies for the management of cardiomyopathy.

  2. A Unique Platform for H-Ras Signaling Involving Clathrin-independent Endocytosis

    PubMed Central

    Porat-Shliom, Natalie; Kloog, Yoel

    2008-01-01

    Trafficking of H-Ras was examined to determine whether it can enter cells through clathrin-independent endocytosis (CIE). H-Ras colocalized with the CIE cargo protein, class I major histocompatibility complex, and it was sequestered in vacuoles that formed upon expression of an active mutant of Arf6, Q67L. Activation of Ras, either through epidermal growth factor stimulation or the expression of an active mutant of Ras, G12V, induced plasma membrane ruffling and macropinocytosis, a stimulated form of CIE. Live imaging of cells expressing H-RasG12V and fluorescent protein chimeras with pleckstrin homology domains that recognize specific phosphoinositides showed that incoming macropinosomes contained phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatiylinositol 3,4,5-trisphosphate (PIP3). PIP2 loss from the macropinosome was followed by the recruitment of Rab5, a downstream target of Ras, and then PIP3 loss. Our studies support a model whereby Ras can signal on macropinosomes that pass through three distinct stages: PIP2/PIP3, PIP3/Rab5, and Rab5. Vacuoles that form in cells expressing Arf6Q67L trap Ras signaling in the first stage, recruiting the active form of the Ras effectors extracellular signal-regulated kinase and protein kinase B (Akt) but not Rab5. Arf6 stimulation of macropinocytosis also involves passage through the distinct lipid phases, but recruitment of Akt is not observed. PMID:18094044

  3. Spatio-temporal segregation of Ras signals: one ship, three anchors, many harbors.

    PubMed

    Rocks, Oliver; Peyker, Anna; Bastiaens, Philippe I H

    2006-08-01

    Dynamic assembly of spatially separated signaling platforms enables a cell to tune cellular outputs in response to different input stimuli. Understanding how a vast diversity in signaling responses can be generated from a limited protein repertoire requires knowledge of how cells maintain the segregation of proteins and thereby orchestrate their local activities. Ras proteins are subject to this type of precise regulation of localization, and thus activity, in space and time. A model emerges where different lipid anchors dynamically shuttle Ras between specific membrane compartments, where differences in the accessibility of signaling environments and in the residence time of Ras therein account for isoform-specific signaling responses.

  4. v-K-ras leads to preferential farnesylation of p21ras in FRTL-5 cells: Multiple interference with the isoprenoid pathway

    PubMed Central

    Laezza, Chiara; Di Marzo, Vincenzo; Bifulco, Maurizio

    1998-01-01

    The isoprenoid pathway in FRTL-5 thyroid cells was found to be deeply altered on transformation with v-K-ras. A dramatic overall reduction of protein prenylation was found in v-K-ras-transformed cells in comparison with the parent FRTL-5 cells, as shown by labeling cells with [3H]mevalonic acid. This phenomenon was accompanied by a relative increase of p21ras farnesylation and by a decrease of the ratio between the amounts of geranylgeraniol and farnesol bound to prenylated proteins. Analysis of protein prenylation in FRTL-5 cells transformed by a temperature-sensitive mutant of the v-K-ras oncogene indicated that these variations represent an early and specific marker of active K-ras. Conversely, FRTL-5 cells transformed with Harvey-ras showed a pattern of [3H]-mevalonate (MVA)-labeled proteins similar to that of nontransformed cells. The K-ras oncogene activation also resulted in an overall decrease of [3H]-MVA incorporation into isopentenyl-tRNA together with an increase of unprocessed [3H]-MVA and no alteration in [3H]-MVA uptake. The effects of v-K-ras on protein prenylation could be mimicked in FRTL-5 cells by lowering the concentration of exogenous [3H]-MVA whereas increasing the [3H]-MVA concentration did not revert the alterations observed in transformed cells. Accordingly, v-K-ras expression was found to: (i) down-regulate mevalonate kinase; (ii) induce farnesyl-pyrophosphate synthase expression; and (iii) augment protein farnesyltransferase but not protein geranylgeranyl-transferase-I activity. Among these events, mevalonate kinase down-regulation appeared to be related strictly to differential protein prenylation. This study represents an example of how expression of the v-K-ras oncogene, through multiple interferences with the isoprenoid metabolic pathway, may result in the preferential farnesylation of the ras oncogene product p21ras. PMID:9811854

  5. Signaling through RAS-RAF-MEK-ERK: from basics to bedside.

    PubMed

    Zebisch, Armin; Czernilofsky, Armin P; Keri, György; Smigelskaite, Julja; Sill, Heinz; Troppmair, Jakob

    2007-01-01

    Aberrant signaling caused by mutations in the RAS-RAF-MEK-ERK pathway and its upstream activators critically contributes to human tumor development. Strategies, which aim at inhibiting hyperactive signaling molecules, appear conceptually straight forward, but their translation into clinical practice has been hampered by many setbacks. Understanding structure, function and regulation of this intracellular pathway as well as its crosstalk with other signaling activities in the cell will be essential to ensure reasonable usage of new therapeutic possibilities. This review provides an understanding of this signaling cascade as revealed by genetic and biochemical approaches and discusses the existing or arising possibilities to interfere with unphysiological activation in cancer. Signaling aberrations and signal transduction therapies will be discussed exemplary for two types of hematological neoplasia, acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS). In the future understanding the role of tumor stem cells, both as a source of tumor recurrence and tumor heterogeneity, the signals controlling their fate as well as epigenetic changes in cancer will be the next critical steps to further advance the applicability of these novel therapeutic strategies.

  6. Transcriptomic analysis of the role of RasGEF1B circular RNA in the TLR4/LPS pathway.

    PubMed

    Ng, Wei Lun; Marinov, Georgi K; Chin, Yoon-Ming; Lim, Yat-Yuen; Ea, Chee-Kwee

    2017-09-25

    Circular RNAs (circRNAs) have recently emerged as a large class of novel non-coding RNA species. However, the detailed functional significance of the vast majority of them remains to be elucidated. Most functional characterization studies targeting circRNAs have been limited to resting cells, leaving their role in dynamic cellular responses to stimuli largely unexplored. In this study, we focus on the LPS-induced cytoplasmic circRNA, mcircRasGEF1B, and combine targeted mcircRasGEF1B depletion with high-throughput transcriptomic analysis to gain insight into its function during the cellular response to LPS stimulation. We show that knockdown of mcircRasGEF1B results in altered expression of a wide array of genes. Pathway analysis revealed an overall enrichment of genes involved in cell cycle progression, mitotic division, active metabolism, and of particular interest, NF-κB, LPS signaling pathways, and macrophage activation. These findings expand the set of functionally characterized circRNAs and support the regulatory role of mcircRasGEF1B in immune response during macrophage activation and protection against microbial infections.

  7. Honokiol suppresses survival signals mediated by Ras-dependent phospholipase D activity in human cancer cells.

    PubMed

    Garcia, Avalon; Zheng, Yang; Zhao, Chen; Toschi, Alfredo; Fan, Judy; Shraibman, Natalie; Brown, H Alex; Bar-Sagi, Dafna; Foster, David A; Arbiser, Jack L

    2008-07-01

    Elevated phospholipase D (PLD) activity provides a survival signal in several human cancer cell lines and suppresses apoptosis when cells are subjected to the stress of serum withdrawal. Thus, targeting PLD survival signals has potential to suppress survival in cancer cells that depend on PLD for survival. Honokiol is a compound that suppresses tumor growth in mouse models. The purpose of this study was to investigate the effect of honokiol on PLD survival signals and the Ras dependence of these signals. The effect of honokiol upon PLD activity was examined in human cancer cell lines where PLD activity provides a survival signal. The dependence of PLD survival signals on Ras was investigated, as was the effect of honokiol on Ras activation. We report here that honokiol suppresses PLD activity in human cancer cells where PLD has been shown to suppress apoptosis. PLD activity is commonly elevated in response to the stress of serum withdrawal, and, importantly, the stress-induced increase in PLD activity is selectively suppressed by honokiol. The stress-induced increase in PLD activity was accompanied by increased Ras activation, and the stress-induced increase in PLD activity in MDA-MB-231 breast cancer cells was dependent on a Ras. The PLD activity was also dependent on the GTPases RalA and ADP ribosylation factor. Importantly, honokiol suppressed Ras activation. The data provided here indicate that honokiol may be a valuable therapeutic reagent for targeting a large number of human cancers that depend on Ras and PLD for their survival.

  8. Circadian and photic regulation of MAP kinase by Ras- and protein phosphatase-dependent pathways in the chick pineal gland.

    PubMed

    Hayashi, Y; Sanada, K; Fukada, Y

    2001-02-23

    Chick pineal mitogen-activated protein kinase (MAPK) exhibits circadian activation and light-dependent deactivation at nighttime. Here we report that, in the chick pineal gland, levels of active forms of MAPK, MEK, Raf-1 and Ras exhibited synchronous circadian rhythms with peaks during the subjective night, suggesting a sequential activation of components in the classical Ras-MAPK pathway in a circadian manner. In contrast, the light-dependent deactivation of MAPK was not accompanied by any change of MEK activity, but it was attributed to the light-dependent activation of protein phosphatase dephosphorylating MAPK. These results indicate that the photic and clock signals regulate MAPK activity via independent pathways, and suggest a pivotal role of MAPK in photic entrainment and maintenance of the circadian oscillation.

  9. Prospero and Pax2 combinatorially control neural cell fate decisions by modulating Ras- and Notch-dependent signaling

    PubMed Central

    2011-01-01

    Background The concept of an equivalence group, a cluster of cells with equal potential to adopt the same specific fate, has served as a useful paradigm to understand neural cell type specification. In the Drosophila eye, a set of five cells, called the 'R7 equivalence group', generates a single photoreceptor neuron and four lens-secreting epithelial cells. This choice between neuronal versus non-neuronal cell fates rests on differential requirements for, and cross-talk between, Notch/Delta- and Ras/mitogen-activated protein kinase (MAPK)-dependent signaling pathways. However, many questions remain unanswered related to how downstream events of these two signaling pathways mediate distinct cell fate decisions. Results Here, we demonstrate that two direct downstream targets of Ras and Notch signaling, the transcription factors Prospero and dPax2, are essential regulators of neuronal versus non-neuronal cell fate decisions in the R7 equivalence group. Prospero controls high activated MAPK levels required for neuronal fate, whereas dPax2 represses Delta expression to prevent neuronal fate. Importantly, activity from both factors is required for proper cell fate decisions to occur. Conclusions These data demonstrate that Ras and Notch signaling are integrated during cell fate decisions within the R7 equivalence group through the combinatorial and opposing activities of Pros and dPax2. Our study provides one of the first examples of how the differential expression and synergistic roles of two independent transcription factors determine cell fate within an equivalence group. Since the integration of Ras and Notch signaling is associated with many developmental and cancer models, these findings should provide new insights into how cell specificity is achieved by ubiquitously used signaling pathways in diverse biological contexts. PMID:21539742

  10. Oncogenic Ras and B-Raf proteins positively regulate death receptor 5 expression through co-activation of ERK and JNK signaling.

    PubMed

    Oh, You-Take; Yue, Ping; Zhou, Wei; Balko, Justin M; Black, Esther P; Owonikoko, Taofeek K; Khuri, Fadlo R; Sun, Shi-Yong

    2012-01-02

    Oncogenic mutations of ras and B-raf frequently occur in many cancer types and are critical for cell transformation and tumorigenesis. Death receptor 5 (DR5) is a cell surface pro-apoptotic death receptor for tumor necrosis factor-related apoptosis-inducing ligand and has been targeted in cancer therapy. The current study has demonstrated induction of DR5 expression by the oncogenic proteins Ras and B-Raf and revealed the underlying mechanisms. We demonstrated that both Ras and B-Raf induce DR5 expression by enforced expression of oncogenic Ras (e.g. H-Ras12V or K-Ras12V) or B-Raf (i.e. V600E) in cells and by analyzing gene expression array data generated from cancer cell lines and from human cancer tissues. This finding is further supported by our results that knockdown of endogenous K-Ras or B-Raf (V600E) reduced the expression of DR5. Importantly, we have elucidated that Ras induces DR5 expression through co-activation of ERK/RSK and JNK signaling pathways and subsequent cooperative effects among the transcriptional factors CHOP, Elk1, and c-Jun to enhance DR5 gene transcription. Moreover, we found that the majority of cancer cell lines highly sensitive to the DR5 agonistic antibody AMG655 have either Ras or B-Raf mutations. Our findings warrant further study on the biology of DR5 regulation by Ras and B-Raf, which may provide new insight into the biology of Ras and B-Raf, and on the potential impact of Ras or B-Raf mutations on the outcome of DR5-targeted cancer therapy.

  11. The Akt signaling pathway

    PubMed Central

    Madhunapantula, SubbaRao V; Mosca, Paul J

    2011-01-01

    Studies using cultured melanoma cells and patient tumor biopsies have demonstrated deregulated PI3 kinase-Akt3 pathway activity in ∼70% of melanomas. Furthermore, targeting Akt3 and downstream PRAS40 has been shown to inhibit melanoma tumor development in mice. Although these preclinical studies and several other reports using small interfering RNAs and pharmacological agents targeting key members of this pathway have been shown to retard melanoma development, analysis of early Phase I and Phase II clinical trials using pharmacological agents to target this pathway demonstrate the need for (1) selection of patients whose tumors have PI3 kinase-Akt pathway deregulation, (2) further optimization of therapeutic agents for increased potency and reduced toxicity, (3) the identification of additional targets in the same pathway or in other signaling cascades that synergistically inhibit the growth and progression of melanoma, and (4) better methods for targeted delivery of pharmaceutical agents inhibiting this pathway. In this review we discuss key potential targets in PI3K-Akt3 signaling, the status of pharmacological agents targeting these proteins, drugs under clinical development, and strategies to improve the efficacy of therapeutic agents targeting this pathway. PMID:22157148

  12. The role of the RAS pathway in iAMP21-ALL

    PubMed Central

    Ryan, S L; Matheson, E; Grossmann, V; Sinclair, P; Bashton, M; Schwab, C; Towers, W; Partington, M; Elliott, A; Minto, L; Richardson, S; Rahman, T; Keavney, B; Skinner, R; Bown, N; Haferlach, T; Vandenberghe, P; Haferlach, C; Santibanez-Koref, M; Moorman, A V; Kohlmann, A; Irving, J A E; Harrison, C J

    2016-01-01

    Intrachromosomal amplification of chromosome 21 (iAMP21) identifies a high-risk subtype of acute lymphoblastic leukaemia (ALL), requiring intensive treatment to reduce their relapse risk. Improved understanding of the genomic landscape of iAMP21-ALL will ascertain whether these patients may benefit from targeted therapy. We performed whole-exome sequencing of eight iAMP21-ALL samples. The mutation rate was dramatically disparate between cases (average 24.9, range 5–51) and a large number of novel variants were identified, including frequent mutation of the RAS/MEK/ERK pathway. Targeted sequencing of a larger cohort revealed that 60% (25/42) of diagnostic iAMP21-ALL samples harboured 42 distinct RAS pathway mutations. High sequencing coverage demonstrated heterogeneity in the form of multiple RAS pathway mutations within the same sample and diverse variant allele frequencies (VAFs) (2–52%), similar to other subtypes of ALL. Constitutive RAS pathway activation was observed in iAMP21 samples that harboured mutations in the predominant clone (⩾35% VAF). Viable iAMP21 cells from primary xenografts showed reduced viability in response to the MEK1/2 inhibitor, selumetinib, in vitro. As clonal (⩾35% VAF) mutations were detected in 26% (11/42) of iAMP21-ALL, this evidence of response to RAS pathway inhibitors may offer the possibility to introduce targeted therapy to improve therapeutic efficacy in these high-risk patients. PMID:27168466

  13. Requirement for Plk2 in orchestrated Ras and Rap signaling, homeostatic structural plasticity, and memory

    PubMed Central

    Lee, Kea Joo; Lee, Yeunkum; Rozeboom, Aaron; Lee, Ji-Yun; Udagawa, Noriko; Hoe, Hyang-Sook; Pak, Daniel T.S.

    2011-01-01

    Summary Ras and Rap small GTPases are important for synaptic plasticity and memory. However, their roles in homeostatic plasticity are unknown. Here, we report that polo-like kinase 2 (Plk2), a homeostatic suppressor of overexcitation, governs the activity of Ras and Rap via coordination of their regulatory proteins. Plk2 directs elimination of Ras activator RasGRF1 and Rap inhibitor SPAR via phosphorylation-dependent ubiquitin-proteasome degradation. Conversely, Plk2 phosphorylation stimulates Ras inhibitor SynGAP and Rap activator PDZGEF1. These Ras/Rap regulators perform complementary functions to downregulate dendritic spines and AMPA receptors following elevated activity, and their collective regulation by Plk2 profoundly stimulates Rap and suppresses Ras. Furthermore, perturbation of Plk2 disrupts Ras and Rap signaling, prevents homeostatic shrinkage and loss of dendritic spines, and impairs proper memory formation. Our study demonstrates a critical role of Plk2 in the synchronized tuning of Ras and Rap, and underscores the functional importance of this regulation in homeostatic synaptic plasticity. PMID:21382555

  14. Requirement for Plk2 in orchestrated ras and rap signaling, homeostatic structural plasticity, and memory.

    PubMed

    Lee, Kea Joo; Lee, Yeunkum; Rozeboom, Aaron; Lee, Ji-Yun; Udagawa, Noriko; Hoe, Hyang-Sook; Pak, Daniel T S

    2011-03-10

    Ras and Rap small GTPases are important for synaptic plasticity and memory. However, their roles in homeostatic plasticity are unknown. Here, we report that polo-like kinase 2 (Plk2), a homeostatic suppressor of overexcitation, governs the activity of Ras and Rap via coordination of their regulatory proteins. Plk2 directs elimination of Ras activator RasGRF1 and Rap inhibitor SPAR via phosphorylation-dependent ubiquitin-proteasome degradation. Conversely, Plk2 phosphorylation stimulates Ras inhibitor SynGAP and Rap activator PDZGEF1. These Ras/Rap regulators perform complementary functions to downregulate dendritic spines and AMPA receptors following elevated activity, and their collective regulation by Plk2 profoundly stimulates Rap and suppresses Ras. Furthermore, perturbation of Plk2 disrupts Ras and Rap signaling, prevents homeostatic shrinkage and loss of dendritic spines, and impairs proper memory formation. Our study demonstrates a critical role of Plk2 in the synchronized tuning of Ras and Rap and underscores the functional importance of this regulation in homeostatic synaptic plasticity. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. A p53-inducible microRNA-34a downregulates Ras signaling by targeting IMPDH

    SciTech Connect

    Kim, Hwa-Ryeon; Roe, Jae-Seok; Lee, Ji-Eun; Hwang, In-Young; Cho, Eun-Jung; Youn, Hong-Duk

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer p53 downregulates IMPDH. Black-Right-Pointing-Pointer p53-dependent miR-34a transactivation inhibits IMPDH transcription. Black-Right-Pointing-Pointer miR-34a-mediated inhibition of IMPDH downregulates GTP-dependent Ras signal. -- Abstract: p53 is a well-known transcription factor that controls cell cycle arrest and cell death in response to a wide range of stresses. Moreover, p53 regulates glucose metabolism and its mutation results in the metabolic switch to the Warburg effect found in cancer cells. Nucleotide biosynthesis is also critical for cell proliferation and the cell division cycle. Nonetheless, little is known about whether p53 regulates nucleotide biosynthesis. Here we demonstrated that p53-inducible microRNA-34a (miR-34a) repressed inosine 5 Prime -monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme of de novo GTP biosynthesis. Treatment with anti-miR-34a inhibitor relieved the expression of IMPDH upon DNA damage. Ultimately, miR-34a-mediated inhibition of IMPDH resulted in repressed activation of the GTP-dependent Ras signaling pathway. In summary, we suggest that p53 has a novel function in regulating purine biosynthesis, aided by miR-34a-dependent IMPDH repression.

  16. Functional Cross-talk between Ras and Rho Pathways

    PubMed Central

    Jaiswal, Mamta; Dvorsky, Radovan; Amin, Ehsan; Risse, Sarah L.; Fansa, Eyad K.; Zhang, Si-Cai; Taha, Mohamed S.; Gauhar, Aziz R.; Nakhaei-Rad, Saeideh; Kordes, Claus; Koessmeier, Katja T.; Cirstea, Ion C.; Olayioye, Monilola A.; Häussinger, Dieter; Ahmadian, Mohammad R.

    2014-01-01

    The three deleted in liver cancer genes (DLC1–3) encode Rho-specific GTPase-activating proteins (RhoGAPs). Their expression is frequently silenced in a variety of cancers. The RhoGAP activity, which is required for full DLC-dependent tumor suppressor activity, can be inhibited by the Src homology 3 (SH3) domain of a Ras-specific GAP (p120RasGAP). Here, we comprehensively investigated the molecular mechanism underlying cross-talk between two distinct regulators of small GTP-binding proteins using structural and biochemical methods. We demonstrate that only the SH3 domain of p120 selectively inhibits the RhoGAP activity of all three DLC isoforms as compared with a large set of other representative SH3 or RhoGAP proteins. Structural and mutational analyses provide new insights into a putative interaction mode of the p120 SH3 domain with the DLC1 RhoGAP domain that is atypical and does not follow the classical PXXP-directed interaction. Hence, p120 associates with the DLC1 RhoGAP domain by targeting the catalytic arginine finger and thus by competitively and very potently inhibiting RhoGAP activity. The novel findings of this study shed light on the molecular mechanisms underlying the DLC inhibitory effects of p120 and suggest a functional cross-talk between Ras and Rho proteins at the level of regulatory proteins. PMID:24443565

  17. PAQR10 and PAQR11 mediate Ras signaling in the Golgi apparatus.

    PubMed

    Jin, Ting; Ding, Qiurong; Huang, Heng; Xu, Daqian; Jiang, Yuhui; Zhou, Ben; Li, Zhenghu; Jiang, Xiaomeng; He, Jing; Liu, Weizhong; Zhang, Yixuan; Pan, Yi; Wang, Zhenzhen; Thomas, Walter G; Chen, Yan

    2012-04-01

    Ras plays a pivotal role in many cellular activities, and its subcellular compartmentalization provides spatial and temporal selectivity. Here we report a mode of spatial regulation of Ras signaling in the Golgi apparatus by two highly homologous proteins PAQR10 and PAQR11 of the progestin and AdipoQ receptors family. PAQR10 and PAQR11 are exclusively localized in the Golgi apparatus. Overexpression of PAQR10/PAQR11 stimulates basal and EGF-induced ERK phosphorylation and increases the expression of ERK target genes in a dose-dependent manner. Overexpression of PAQR10/PAQR11 markedly elevates Golgi localization of HRas, NRas and KRas4A, but not KRas4B. PAQR10 and PAQR11 can also interact with HRas, NRas and KRas4A, but not KRas4B. The increased Ras protein at the Golgi apparatus by overexpression of PAQR10/PAQR11 is in an active state. Consistently, knockdown of PAQR10 and PAQR11 reduces EGF-stimulated ERK phosphorylation and Ras activation at the Golgi apparatus. Intriguingly, PAQR10 and PAQR11 are able to interact with RasGRP1, a guanine nucleotide exchange protein of Ras, and increase Golgi localization of RasGRP1. The C1 domain of RasGRP1 is both necessary and sufficient for the interaction of RasGRP1 with PAQR10/PAQR11. The simulation of ERK phosphorylation by overexpressed PAQR10/PAQR11 is abrogated by downregulation of RasGRP1. Furthermore, differentiation of PC12 cells is significantly enhanced by overexpression of PAQR10/PAQR11. Collectively, this study uncovers a new paradigm of spatial regulation of Ras signaling in the Golgi apparatus by PAQR10 and PAQR11.

  18. PAQR10 and PAQR11 mediate Ras signaling in the Golgi apparatus

    PubMed Central

    Jin, Ting; Ding, Qiurong; Huang, Heng; Xu, Daqian; Jiang, Yuhui; Zhou, Ben; Li, Zhenghu; Jiang, Xiaomeng; He, Jing; Liu, Weizhong; Zhang, Yixuan; Pan, Yi; Wang, Zhenzhen; Thomas, Walter G; Chen, Yan

    2012-01-01

    Ras plays a pivotal role in many cellular activities, and its subcellular compartmentalization provides spatial and temporal selectivity. Here we report a mode of spatial regulation of Ras signaling in the Golgi apparatus by two highly homologous proteins PAQR10 and PAQR11 of the progestin and AdipoQ receptors family. PAQR10 and PAQR11 are exclusively localized in the Golgi apparatus. Overexpression of PAQR10/PAQR11 stimulates basal and EGF-induced ERK phosphorylation and increases the expression of ERK target genes in a dose-dependent manner. Overexpression of PAQR10/PAQR11 markedly elevates Golgi localization of HRas, NRas and KRas4A, but not KRas4B. PAQR10 and PAQR11 can also interact with HRas, NRas and KRas4A, but not KRas4B. The increased Ras protein at the Golgi apparatus by overexpression of PAQR10/PAQR11 is in an active state. Consistently, knockdown of PAQR10 and PAQR11 reduces EGF-stimulated ERK phosphorylation and Ras activation at the Golgi apparatus. Intriguingly, PAQR10 and PAQR11 are able to interact with RasGRP1, a guanine nucleotide exchange protein of Ras, and increase Golgi localization of RasGRP1. The C1 domain of RasGRP1 is both necessary and sufficient for the interaction of RasGRP1 with PAQR10/PAQR11. The simulation of ERK phosphorylation by overexpressed PAQR10/PAQR11 is abrogated by downregulation of RasGRP1. Furthermore, differentiation of PC12 cells is significantly enhanced by overexpression of PAQR10/PAQR11. Collectively, this study uncovers a new paradigm of spatial regulation of Ras signaling in the Golgi apparatus by PAQR10 and PAQR11. PMID:21968647

  19. Centaurin-α1-Ras-Elk-1 Signaling at Mitochondria Mediates β-Amyloid-Induced Synaptic Dysfunction

    PubMed Central

    Szatmari, Erzsebet M.; Oliveira, Ana F.; Sumner, Elizabeth J.

    2013-01-01

    Alzheimer's disease is thought to be caused by β-amyloid peptide (Aβ)-dependent synaptic dysfunction. However, the signaling pathways connecting Aβ and synaptic dysfunction remain elusive. Here we report that Aβ transiently increases the expression level of centaurin-α1 (CentA1) in neurons, which induces a Ras-dependent association of Elk-1 with mitochondria, leading to mitochondrial and synaptic dysfunction in organotypic hippocampal slices of rats. Downregulation of the CentA1–Ras-Elk-1 pathway restored normal mitochondrial activity, spine structural plasticity, spine density, and the amplitude and frequency of miniature EPSCs in Aβ-treated neurons, whereas upregulation of the pathway was sufficient to decrease spine density. Elevations of CentA1 and association of Elk-1 with mitochondria were also observed in transgenic mice overexpressing a human mutant form of amyloid precursor protein. Therefore, the CentA1–Ras-Elk-1 signaling pathway acts on mitochondria to regulate dendritic spine density and synaptic plasticity in response to Aβ in hippocampal neurons, providing new pharmacological targets for Alzheimer's disease. PMID:23516302

  20. Centaurin-α1-Ras-Elk-1 signaling at mitochondria mediates β-amyloid-induced synaptic dysfunction.

    PubMed

    Szatmari, Erzsebet M; Oliveira, Ana F; Sumner, Elizabeth J; Yasuda, Ryohei

    2013-03-20

    Alzheimer's disease is thought to be caused by β-amyloid peptide (Aβ)-dependent synaptic dysfunction. However, the signaling pathways connecting Aβ and synaptic dysfunction remain elusive. Here we report that Aβ transiently increases the expression level of centaurin-α1 (CentA1) in neurons, which induces a Ras-dependent association of Elk-1 with mitochondria, leading to mitochondrial and synaptic dysfunction in organotypic hippocampal slices of rats. Downregulation of the CentA1-Ras-Elk-1 pathway restored normal mitochondrial activity, spine structural plasticity, spine density, and the amplitude and frequency of miniature EPSCs in Aβ-treated neurons, whereas upregulation of the pathway was sufficient to decrease spine density. Elevations of CentA1 and association of Elk-1 with mitochondria were also observed in transgenic mice overexpressing a human mutant form of amyloid precursor protein. Therefore, the CentA1-Ras-Elk-1 signaling pathway acts on mitochondria to regulate dendritic spine density and synaptic plasticity in response to Aβ in hippocampal neurons, providing new pharmacological targets for Alzheimer's disease.

  1. Novel aspects of Ras proteins biology: regulation and implications.

    PubMed

    Pérez-Sala, D; Rebollo, A

    1999-08-01

    The importance of Ras proteins as crucial crossroads in cellular signaling pathways has been well established. In spite of the elucidation of the mechanism of RAS activation by growth factors and the delineation of MAP kinase cascades, the overall framework of Ras interactions is far from being complete. Novel regulators of Ras GDP/GTP exchange have been identified that may mediate the activation of Ras in response to changes in intracellular calcium and diacylglycerol. The direct activation of Ras by free radicals such as nitric oxide also suggests potential regulation of Ras function by the cellular redox state. In addition, the array of Ras effectors continues to expand, uncovering links between Ras and other cellular signaling pathways. Ras is emerging as a dual regulator of cellular functions, playing either positive or negative roles in the regulation of proliferation and apoptosis. The signals transmitted by Ras may be modulated by other pathways triggered in parallel, resulting in the final order for proliferation or apoptosis. The diversity of ras-mediated effects may be related in part to differential involvement of Ras homologues in distinct cellular processes. The study of Ras posttranslational modifications has yielded a broad battery of inhibitors that have been envisaged as anti-cancer agents. Although an irreversible modification, Ras isoprenylation appears to be modulated by growth factors and by the activity of the isoprenoid biosynthetic pathway, which may lead to changes in Ras activity.

  2. Ras-ERK Signaling in Behavior: Old Questions and New Perspectives.

    PubMed

    Fasano, Stefania; Brambilla, Riccardo

    2011-01-01

    The role of Ras-ERK signaling in behavioral plasticity is well established. Inhibition studies using the blood-brain barrier permeable drug SL327 have conclusively demonstrated that this neuronal cell signaling cascade is a crucial component of the synaptic machinery implicated in the formation of various forms of long-term memory, from spatial learning to fear and operant conditioning. However, abnormal Ras-ERK signaling has also been linked to a number of neuropsychiatric conditions, including mental retardation syndromes ("RASopathies"), drug addiction, and l-DOPA induced dyskinesia (LID). The work recently done on these brain disorders has pointed to previously underappreciated roles of Ras-ERK in specific subsets of neurons, like GABAergic interneurons of the hippocampus or the cortex, as well as in the medium spiny neurons of the striatum. Here we will highlight the open questions related to Ras-ERK signaling in these behavioral manifestations and propose crucial experiments for the future.

  3. Membrane potential modulates plasma membrane phospholipid dynamics and K-Ras signaling

    PubMed Central

    Zhou, Yong; Wong, Ching-On; Cho, Kwang-jin; van der Hoeven, Dharini; Liang, Hong; Thakur, Dhananiay P.; Luo, Jialie; Babic, Milos; Zinsmaier, Konrad E.; Zhu, Michael X.; Hu, Hongzhen; Venkatachalam, Kartik; Hancock, John F.

    2015-01-01

    Plasma membrane depolarization can trigger cell proliferation, but how membrane potential influences mitogenic signaling is uncertain. Here, we show that plasma membrane depolarization induces nanoscale reorganization of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate but not other anionic phospholipids. K-Ras, which is targeted to the plasma membrane by electrostatic interactions with phosphatidylserine, in turn undergoes enhanced nanoclustering. Depolarization-induced changes in phosphatidylserine and K-Ras plasma membrane organization occur in fibroblasts, excitable neuroblastoma cells, and Drosophila neurons in vivo and robustly amplify K-Ras–dependent mitogen-activated protein kinase (MAPK) signaling. Conversely, plasma membrane repolarization disrupts K-Ras nanoclustering and inhibits MAPK signaling. By responding to voltage-induced changes in phosphatidylserine spatiotemporal dynamics, K-Ras nanoclusters set up the plasma membrane as a biological field-effect transistor, allowing membrane potential to control the gain in mitogenic signaling circuits. PMID:26293964

  4. Argos induces programmed cell death in the developing Drosophila eye by inhibition of the Ras pathway.

    PubMed

    Sawamoto, K; Taguchi, A; Hirota, Y; Yamada, C; Jin, M H; Okano, H

    1998-04-01

    We studied the role of Ras signaling in the regulation of cell death during Drosophila eye development. Overexpression of Argos, a diffusible inhibitor of the EGF receptor and Ras signaling, caused excessive cell death in developing eyes at pupal stages. The Argos-induced cell death was suppressed by coexpression of the anti-apoptotic genes p35, diap1, or diap2 in the eye as well as by the Df(3L)H99 chromosomal deletion that lacks three apoptosis-inducing genes, reaper, head involution defective (hid) and grim. Transient misexpression of the activated Ras1 protein (Ras1V12) later in pupal development suppressed the Argos-induced cell death. Thus, Argos-induced cell death seemed to have resulted from the suppression of the anti-apoptotic function of Ras. Conversely, cell death induced by overexpression of Hid was suppressed by gain-of-function mutations of the genes coding for MEK and ERK. These results support the idea that Ras signaling functions in two distinct processes during eye development, first triggering the recruitment of cells and later negatively regulating cell death.

  5. Inhibition of Ras signalling reduces neutrophil infiltration and tissue damage in severe acute pancreatitis.

    PubMed

    Yu, Changhui; Merza, Mohammed; Luo, Lingtao; Thorlacius, Henrik

    2015-01-05

    Neutrophil recruitment is known to be a rate-limiting step in mediating tissue injury in severe acute pancreatitis (AP). However, the signalling mechanisms controlling inflammation and organ damage in AP remain elusive. Herein, we examined the role of Ras signalling in AP. Male C57BL/6 mice were treated with a Ras inhibitor (farnesylthiosalicylic acid, FTS) before infusion of taurocholate into the pancreatic duct. Pancreatic and lung tissues as well as blood were collected 24 h after pancreatitis induction. Pretreatment with FTS decreased serum amylase levels by 82% and significantly attenuated acinar cell necrosis, tissue haemorrhage and oedema formation in taurocholate-induced pancreatitis. Inhibition of Ras signalling reduced myeloperoxidase (MPO) levels in the inflamed pancreas by 42%. In addition, administration of FTS decreased pancreatic levels of CXC chemokines as well as circulating levels of interleukin-6 and high-mobility group box 1 in animals exposed to taurocholate. Moreover, treatment with FTS reduced taurocholate-induced MPO levels in the lung. Inhibition of Ras signalling had no effect on neutrophil expression of Mac-1 in mice with pancreatitis. Moreover, FTS had no direct impact on trypsin activation in isolated pancreatic acinar cells. These results indicate that Ras signalling controls CXC chemokine formation, neutrophil recruitment and tissue injury in severe AP. Thus, our findings highlight a new signalling mechanism regulating neutrophil recruitment in the pancreas and suggest that inhibition of Ras signalling might be a useful strategy to attenuate local and systemic inflammation in severe AP.

  6. RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3*

    PubMed Central

    Cailliau, Katia; Lescuyer, Arlette; Burnol, Anne-Françoise; Cuesta-Marbán, Álvaro; Widmann, Christian; Browaeys-Poly, Edith

    2015-01-01

    Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G2/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G2/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G2/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling. PMID:26109071

  7. Differential Role of gp130-Dependent STAT and Ras Signalling for Haematopoiesis Following Bone-Marrow Transplantation

    PubMed Central

    Kroy, Daniela C.; Hebing, Lisa; Sander, Leif E.; Gassler, Nikolaus; Erschfeld, Stephanie; Sackett, Sara; Galm, Oliver; Trautwein, Christian; Streetz, Konrad L.

    2012-01-01

    Introduction Bone marrow transplantation (BMT) is a complex process regulated by different cytokines and growth factors. The pleiotropic cytokine IL-6 (Interleukin-6) and related cytokines of the same family acting on the common signal transducer gp130 are known to play a key role in bone marrow (BM) engraftment. In contrast, the exact signalling events that control IL-6/gp130-driven haematopoietic stem cell development during BMT remain unresolved. Methods Conditional gp130 knockout and knockin mice were used to delete gp130 expression (gp130ΔMx), or to selectively disrupt gp130-dependent Ras (gp130ΔMxRas) or STAT signalling (gp130ΔMxSTAT) in BM cells. BM derived from the respective strains was transplanted into irradiated wildtype hosts and repopulation of various haematopoietic lineages was monitored by flow cytometry. Results BM derived from gp130 deficient donor mice (gp130ΔMx) displayed a delayed engraftment, as evidenced by reduced total white blood cells (WBC), marked thrombocytopenia and anaemia in the early phase after BMT. Lineage analysis unravelled a restricted development of CD4(+) and CD8(+) T-cells, CD19(+) B-cells and CD11b(+) myeloid cells after transplantation of gp130-deficient BM grafts. To further delineate the two major gp130-induced signalling cascades, Ras-MAPK and STAT1/3-signalling respectively, we used gp130ΔMxRas and gp130ΔMxSTAT donor BM. BMT of gp130ΔMxSTAT cells significantly impaired engraftment of CD4(+), CD8(+), CD19(+) and CD11b(+) cells, whereas gp130ΔMxRas BM displayed a selective impairment in early thrombopoiesis. Importantly, gp130-STAT1/3 signalling deficiency in BM grafts severely impaired survival of transplanted mice, thus demonstrating a pivotal role for this pathway in BM graft survival and function. Conclusion Our data unravel a vital function of IL-6/gp130-STAT1/3 signals for BM engraftment and haematopoiesis, as well as for host survival after transplantation. STAT1/3 and ras-dependent pathways thereby exert

  8. Interplay Between HGF/SF-Met-Ras Signaling, Tumor Metabolism and Blood Flow as a Potential Target for Breast Cancer Therapy.

    PubMed

    Natan, Sari; Tsarfaty, Galia; Horev, Judith; Haklai, Roni; Kloog, Yoel; Tsarfaty, Ilan

    2014-01-01

    High glucose uptake and increase blood flow is a characteristic of most metastatic tumors. Activation of Ras signaling increases glycolytic flux into lactate, de novo nucleic acid synthesis and uncoupling of ATP synthase from the proton gradient. Met tyrosine kinase receptor signaling upon activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF), increases glycolysis, oxidative phosporylation, oxygen consumption, and tumor blood volume. Ras is a key factor in Met signaling. Using the Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS), we investigated interplay between HGF/SF-Met-Ras signaling, metabolism, and tumor blood-flow regulation. In vitro, HGF/SF-activated Met increased Ras activity, Erk phosphorylation, cell motility and glucose uptake, but did not affect ATP. FTS inhibited basal and HGF/SF-induced signaling and cell motility, while further increasing glucose uptake and inhibiting ATP production. In vivo, HGF/SF rapidly increased tumor blood volume. FTS did not affect basal blood-flow but abolished the HGF/SF effect. Our results further demonstrate the complex interplay between growth-factor-receptor signaling and cellular and tumor metabolism, as reflected in blood flow. Inhibition of Ras signaling does not affect glucose consumption or basal tumor blood flow but dramatically decreases ATP synthesis and the HGF/SF induced increase in tumor blood volume. These findings demonstrate that the HGF/SF-Met-Ras pathway critically influences tumor-cell metabolism and tumor blood-flow regulation. This pathway could potentially be used to individualize tumor therapy based on functional molecular imaging, and for combined signaling/anti-metabolic targeted therapy.

  9. Hydrolysis of phosphatidylcholine couples Ras to activation of Raf protein kinase during mitogenic signal transduction.

    PubMed Central

    Cai, H; Erhardt, P; Troppmair, J; Diaz-Meco, M T; Sithanandam, G; Rapp, U R; Moscat, J; Cooper, G M

    1993-01-01

    We have investigated the relationship between hydrolysis of phosphatidylcholine (PC) and activation of the Raf-1 protein kinase in Ras-mediated transduction of mitogenic signals. As previously reported, cotransfection of a PC-specific phospholipase C (PC-PLC) expression plasmid bypassed the block to cell proliferation resulting from expression of the dominant inhibitory mutant Ras N-17. In contrast, PC-PLC failed to bypass the inhibitory effect of dominant negative Raf mutants, suggesting that PC-PLC functions downstream of Ras but upstream of Raf. Consistent with this hypothesis, treatment of quiescent cells with exogenous PC-PLC induced Raf activation, even when normal Ras function was blocked by Ras N-17 expression. Further, activation of Raf in response to mitogenic growth factors was blocked by inhibition of endogenous PC-PLC. Taken together, these results indicate that hydrolysis of PC mediates Raf activation in response to mitogenic growth factors. Images PMID:8246981

  10. The Fourth International Symposium on Genetic Disorders of the Ras/MAPK Pathway

    PubMed Central

    Stevenson, David A.; Schill, Lisa; Schoyer, Lisa; Andresen, Brage S.; Bakker, Annette; Bayrak-Toydemir, Pinar; Burkitt-Wright, Emma; Chatfield, Kathryn; Elefteriou, Florent; Elgersma, Ype; Fisher, Michael J.; Franz, David; Gelb, Bruce D.; Goriely, Anne; Gripp, Karen W.; Hardan, Antonio Y.; Keppler-Noreuil, Kim M.; Kerr, Bronwyn; Korf, Bruce; Leoni, Chiara; McCormick, Frank; Plotkin, Scott R.; Rauen, Katherine A.; Reilly, Karlyne; Roberts, Amy; Sandler, Abby; Siegel, Dawn; Walsh, Karin; Widemann, Brigitte C.

    2016-01-01

    The RASopathies are a group of disorders due to variations of genes associated with the Ras/MAPK pathway. Some of the RASopathies include neurofibromatosis type 1 (NF1), Noonan syndrome, Noonan syndrome with multiple lentigines, cardiofaciocutaneous (CFC) syndrome, Costello syndrome, Legius syndrome, and capillary malformation–arteriovenous malformation (CM-AVM) syndrome. In combination, the RASopathies are a frequent group of genetic disorders. This report summarizes the proceedings of the 4th International Symposium on Genetic Disorders of the Ras/MAPK pathway and highlights gaps in the field. PMID:27155140

  11. Mapping the functional versatility and fragility of Ras GTPase signaling circuits through in vitro network reconstitution

    PubMed Central

    Coyle, Scott M; Lim, Wendell A

    2016-01-01

    The Ras-superfamily GTPases are central controllers of cell proliferation and morphology. Ras signaling is mediated by a system of interacting molecules: upstream enzymes (GEF/GAP) regulate Ras’s ability to recruit multiple competing downstream effectors. We developed a multiplexed, multi-turnover assay for measuring the dynamic signaling behavior of in vitro reconstituted H-Ras signaling systems. By including both upstream regulators and downstream effectors, we can systematically map how different network configurations shape the dynamic system response. The concentration and identity of both upstream and downstream signaling components strongly impacted the timing, duration, shape, and amplitude of effector outputs. The distorted output of oncogenic alleles of Ras was highly dependent on the balance of positive (GAP) and negative (GEF) regulators in the system. We found that different effectors interpreted the same inputs with distinct output dynamics, enabling a Ras system to encode multiple unique temporal outputs in response to a single input. We also found that different Ras-to-GEF positive feedback mechanisms could reshape output dynamics in distinct ways, such as signal amplification or overshoot minimization. Mapping of the space of output behaviors accessible to Ras provides a design manual for programming Ras circuits, and reveals how these systems are readily adapted to produce an array of dynamic signaling behaviors. Nonetheless, this versatility comes with a trade-off of fragility, as there exist numerous paths to altered signaling behaviors that could cause disease. DOI: http://dx.doi.org/10.7554/eLife.12435.001 PMID:26765565

  12. Shengmai Formula suppressed over-activated Ras/MAPK pathway in C. elegans by opening mitochondrial permeability transition pore via regulating cyclophilin D

    PubMed Central

    Liu, Yan; Zhi, Dejuan; Li, Menghui; Liu, Dongling; Wang, Xin; Wu, Zhengrong; Zhang, Zhanxin; Fei, Dongqing; Li, Yang; Zhu, Hongmei; Xie, Qingjian; Yang, Hui; Li, Hongyu

    2016-01-01

    Since about 30% of all human cancers contain mutationally activated Ras, down regulating the over-activation of Ras/MAPK pathway represents a viable approach for treating cancers. Over-activation of Ras/MAPK pathway is accompanied by accumulation of reactive oxygen species (ROS). One approach for developing anti-cancer drugs is to target ROS production and their accumulation. To test this idea, we have employed C. elegans of let-60 (gf) mutant, which contain over-activated let-60 (the homolog of mammalian ras) and exhibit tumor-like symptom of multivulva phenotype, to determine whether anti-oxidants can affect their tumor-like phenotype. Specifically we studied the effect of Shengmai formula (SM), a traditional Chinese medicine that has strong anti-oxidant activity, on the physiology of let-60 (gf) mutants. Unexpectedly, we found that SM treatment led to the opening of mitochondrial permeability transition pore by regulating cyclophilin D and then triggered oxidative stress and related signaling pathway activation, including p53, JNK, and p38/MAPK pathways. Finally, SM induced mitochondrial pathway of apoptosis and inhibited the tumor-like symptom of the multivulva phenotype of let-60(gf) mutants. Our results provide evidences to support that SM act as a pro-oxidant agent and could serve as a potential drug candidate for combating over-activated Ras-related cancer. PMID:27982058

  13. Shengmai Formula suppressed over-activated Ras/MAPK pathway in C. elegans by opening mitochondrial permeability transition pore via regulating cyclophilin D.

    PubMed

    Liu, Yan; Zhi, Dejuan; Li, Menghui; Liu, Dongling; Wang, Xin; Wu, Zhengrong; Zhang, Zhanxin; Fei, Dongqing; Li, Yang; Zhu, Hongmei; Xie, Qingjian; Yang, Hui; Li, Hongyu

    2016-12-16

    Since about 30% of all human cancers contain mutationally activated Ras, down regulating the over-activation of Ras/MAPK pathway represents a viable approach for treating cancers. Over-activation of Ras/MAPK pathway is accompanied by accumulation of reactive oxygen species (ROS). One approach for developing anti-cancer drugs is to target ROS production and their accumulation. To test this idea, we have employed C. elegans of let-60 (gf) mutant, which contain over-activated let-60 (the homolog of mammalian ras) and exhibit tumor-like symptom of multivulva phenotype, to determine whether anti-oxidants can affect their tumor-like phenotype. Specifically we studied the effect of Shengmai formula (SM), a traditional Chinese medicine that has strong anti-oxidant activity, on the physiology of let-60 (gf) mutants. Unexpectedly, we found that SM treatment led to the opening of mitochondrial permeability transition pore by regulating cyclophilin D and then triggered oxidative stress and related signaling pathway activation, including p53, JNK, and p38/MAPK pathways. Finally, SM induced mitochondrial pathway of apoptosis and inhibited the tumor-like symptom of the multivulva phenotype of let-60(gf) mutants. Our results provide evidences to support that SM act as a pro-oxidant agent and could serve as a potential drug candidate for combating over-activated Ras-related cancer.

  14. A lin-45 raf enhancer screen identifies eor-1, eor-2 and unusual alleles of Ras pathway genes in Caenorhabditis elegans.

    PubMed

    Rocheleau, Christian E; Howard, Robyn M; Goldman, Alissa P; Volk, Mandy L; Girard, Laura J; Sundaram, Meera V

    2002-05-01

    In Caenorhabditis elegans, the Ras/Raf/MEK/ERK signal transduction pathway controls multiple processes including excretory system development, P12 fate specification, and vulval cell fate specification. To identify positive regulators of Ras signaling, we conducted a genetic screen for mutations that enhance the excretory system and egg-laying defects of hypomorphic lin-45 raf mutants. This screen identified unusual alleles of several known Ras pathway genes, including a mutation removing the second SH3 domain of the sem-5/Grb2 adaptor, a temperature-sensitive mutation in the helical hairpin of let-341/Sos, a gain-of-function mutation affecting a potential phosphorylation site of the lin-1 Ets domain transcription factor, a dominant-negative allele of ksr-1, and hypomorphic alleles of sur-6/PP2A-B, sur-2/Mediator, and lin-25. In addition, this screen identified multiple alleles of two newly identified genes, eor-1 and eor-2, that play a relatively weak role in vulval fate specification but positively regulate Ras signaling during excretory system development and P12 fate specification. The spectrum of identified mutations argues strongly for the specificity of the enhancer screen and for a close involvement of eor-1 and eor-2 in Ras signaling.

  15. RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma.

    PubMed

    Chen, Xu; Wu, Qiuxia; Depeille, Philippe; Chen, Peirong; Thornton, Sophie; Kalirai, Helen; Coupland, Sarah E; Roose, Jeroen P; Bastian, Boris C

    2017-05-08

    Constitutive activation of Gαq signaling by mutations in GNAQ or GNA11 occurs in over 80% of uveal melanomas (UMs) and activates MAPK. Protein kinase C (PKC) has been implicated as a link, but the mechanistic details remained unclear. We identified PKC δ and ɛ as required and sufficient to activate MAPK in GNAQ mutant melanomas. MAPK activation depends on Ras and is caused by RasGRP3, which is significantly and selectively overexpressed in response to GNAQ/11 mutation in UM. RasGRP3 activation occurs via PKC δ- and ɛ-dependent phosphorylation and PKC-independent, DAG-mediated membrane recruitment, possibly explaining the limited effect of PKC inhibitors to durably suppress MAPK in UM. The findings nominate RasGRP3 as a therapeutic target for cancers driven by oncogenic GNAQ/11. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Kinase suppressor of Ras1 compartmentalizes hippocampal signal transduction and subserves synaptic plasticity and memory formation.

    PubMed

    Shalin, Sara C; Hernandez, Caterina M; Dougherty, Michele K; Morrison, Deborah K; Sweatt, J David

    2006-06-01

    The ERK/MAP kinase cascade is important for long-term memory formation and synaptic plasticity, with a myriad of upstream signals converging upon ERK activation. Despite this convergence of signaling, neurons routinely activate appropriate biological responses to different stimuli. Scaffolding proteins represent a mechanism to achieve compartmentalization of signaling and the appropriate targeting of ERK-dependent processes. We report that kinase suppressor of Ras (KSR1) functions biochemically in the hippocampus to scaffold the components of the ERK cascade, specifically regulating the cascade when a membrane fraction of ERK is activated via a PKC-dependent pathway but not via a cAMP/PKA-dependent pathway. Specificity of KSR1-dependent signaling also extends to specific downstream targets of ERK. Behaviorally and physiologically, we found that the absence of KSR1 leads to deficits in associative learning and theta burst stimulation-induced LTP. Our report provides novel insight into the endogenous scaffolding role of KSR1 in controlling kinase activation within the nervous system.

  17. Photochemical Modulation of Ras-Mediated Signal Transduction using Caged Farnesyltransferase Inhibitors: Activation via One- and Two-Photon Excitation

    PubMed Central

    Abate-Pella, Daniel; Zeliadt, Nicholette A.; Ochocki, Joshua D.; Warmka, Janel K.; Dore, Timothy M.; Blank, David A.; Wattenberg, Elizabeth V.; Distefano, Mark D.

    2012-01-01

    The creation of caged molecules involves the attachment of protecting groups to biologically active compounds such as ligands, substrates, and drugs that can be removed under specific conditions. Photoremovable caging groups are the most common due to their ability to be removed with high spatial and temporal resolution. Here, the synthesis and photochemistry of a caged inhibitor of protein farnesyltransferase, Bhc-FTI, is described. The inhibitor was caged by alkylation of a critical thiol functional group with a Bhc moiety; while Bhc is well established as a protecting group for carboxylates and phosphates, it has not been extensively used to cage sulfhydryls. The resulting caged molecule, Bhc-FTI, can be photolyzed with UV light to release the inhibitor (FTI) that prevents Ras farnesylation, Ras membrane localization and downstream signaling. Finally, it is shown that Bhc-FTI can be uncaged by two-photon excitation to produce FTI at levels sufficient to inhibit Ras localization and alter cell morphology. Given the widespread involvement of Ras proteins in signal transduction pathways, this caged inhibitor should be useful in a plethora of studies. PMID:22492666

  18. Photochemical modulation of Ras-mediated signal transduction using caged farnesyltransferase inhibitors: activation by one- and two-photon excitation.

    PubMed

    Abate-Pella, Daniel; Zeliadt, Nicholette A; Ochocki, Joshua D; Warmka, Janel K; Dore, Timothy M; Blank, David A; Wattenberg, Elizabeth V; Distefano, Mark D

    2012-05-07

    The creation of caged molecules involves the attachment of protecting groups to biologically active compounds such as ligands, substrates and drugs that can be removed under specific conditions. Photoremovable caging groups are the most common due to their ability to be removed with high spatial and temporal resolution. Here, the synthesis and photochemistry of a caged inhibitor of protein farnesyltransferase is described. The inhibitor, FTI, was caged by alkylation of a critical thiol group with a bromohydroxycoumarin (Bhc) moiety. While Bhc is well established as a protecting group for carboxylates and phosphates, it has not been extensively used to cage sulfhydryl groups. The resulting caged molecule, Bhc-FTI, can be photolyzed with UV light to release the inhibitor that prevents Ras farnesylation, Ras membrane localization and downstream signaling. Finally, it is shown that Bhc-FTI can be uncaged by two-photon excitation to produce FTI at levels sufficient to inhibit Ras localization and alter cell morphology. Given the widespread involvement of Ras proteins in signal transduction pathways, this caged inhibitor should be useful in a plethora of studies.

  19. Constitutively active RAS signaling reduces 1,25 dihydroxyvitamin D-mediated gene transcription in intestinal epithelial cells by reducing vitamin D receptor expression.

    PubMed

    DeSmet, Marsha L; Fleet, James C

    2017-01-16

    High vitamin D status is associated with reduced colon cancer risk but these studies ignore the diversity in the molecular etiology of colon cancer. RAS activating mutations are common in colon cancer and they activate pro-proliferative signaling pathways. We examined the impact of RAS activating mutations on 1,25 dihydroxyvitamin D (1,25(OH)2D)-mediated gene expression in cultured colon and intestinal cell lines. Transient transfection of Caco-2 cells with a constitutively active mutant K-RAS (G12 V) significantly reduced 1,25(OH)2D-induced activity of both a human 25-hydroxyvitamin D, 24 hydroxyase (CYP24A1) promoter-luciferase and an artificial 3X vitamin D response element (VDRE) promoter-luciferase reporter gene. Young Adult Mouse Colon (YAMC) and Rat Intestinal Epithelial (RIE) cell lines with stable expression of mutant H-RAS had suppressed 1,25(OH)2D-mediated induction of CYP24A1 mRNA. The RAS effects were associated with lower Vitamin D receptor (VDR) mRNA and protein levels in YAMC and RIE cells and they could be partially reversed by VDR overexpression. RAS-mediated suppression of VDR levels was not due to either reduced VDR mRNA stability or increased VDR gene methylation. However, chromatin accessibility to the VDR gene at the proximal promoter (-300bp), an enhancer region at -6kb, and an enhancer region located in exon 3 was significantly reduced in RAS transformed YAMC cells (YAMC-RAS). These data show that constitutively active RAS signaling suppresses 1,25(OH)2D-mediated gene transcription in colon epithelial cells by reducing VDR gene transcription but the mechanism for this suppression is not yet known. These data suggest that cancers with RAS-activating mutations may be less responsive to vitamin D mediated treatment or chemoprevention.

  20. Ras-GRF1 signaling is required for normal β-cell development and glucose homeostasis

    PubMed Central

    Font de Mora, Jaime; Esteban, Luis Miguel; Burks, Deborah J.; Núñez, Alejandro; Garcés, Carmen; García-Barrado, María José; Iglesias-Osma, María Carmen; Moratinos, Julio; Ward, Jerrold M.; Santos, Eugenio

    2003-01-01

    Development of diabetes generally reflects an inadequate mass of insulin-producing β-cells. β-cell proliferation and differentiation are regulated by a variety of growth factors and hormones, including insulin-like growth factor I (IGF-I). GRF1 is a Ras-guanine nucleotide exchange factor known previously for its restricted expression in brain and its role in learning and memory. Here we demonstrate that GRF1 is also expressed in pancreatic islets. Interest ingly, our GRF1-deficient mice exhibit reduced body weight, hypoinsulinemia and glucose intolerance owing to a reduction of β-cells. Whereas insulin resistance is not detected in peripheral tissues, GRF1 knockout mice are leaner due to increased lipid catabolism. The reduction in circulating insulin does not reflect defective glucose sensing or insulin production but results from impaired β-cell proliferation and reduced neogenesis. IGF-I treatment of isolated islets from GRF1 knockouts fails to activate critical downstream signals such as Akt and Erk. The observed phenotype is similar to manifestations of preclinical type 2 diabetes. Thus, our observations demonstrate a novel and specific role for Ras-GRF1 pathways in the development and maintenance of normal β-cell number and function. PMID:12805218

  1. Effects of mutant human Ki-ras{sup G12C} gene dosage on murine lung tumorigenesis and signaling to its downstream effectors

    SciTech Connect

    Dance-Barnes, Stephanie T.; Kock, Nancy D.; Floyd, Heather S.; Moore, Joseph E.; Mosley, Libyadda J.; D'Agostino, Ralph B.; Pettenati, Mark J.; Miller, Mark Steven

    2008-08-15

    Studies in cell culture have suggested that the level of RAS expression can influence the transformation of cells and the signaling pathways stimulated by mutant RAS expression. However, the levels of RAS expression in vivo appear to be subject to feedback regulation, limiting the total amount of RAS protein that can be expressed. We utilized a bitransgenic mouse lung tumor model that expressed the human Ki-ras{sup G12C} allele in a tetracycline-inducible, lung-specific manner. Treatment for 12 months with 500 {mu}g/ml of doxycycline (DOX) allowed for maximal expression of the human Ki-ras{sup G12C} allele in the lung, and resulted in the development of focal hyperplasia and adenomas. We determined if different levels of mutant RAS expression would influence the phenotype of the lung lesions. Treatment with 25, 100 and 500 {mu}g/ml of DOX resulted in dose-dependent increases in transgene expression and tumor multiplicity. Microscopic analysis of the lungs of mice treated with the 25 {mu}g/ml dose of DOX revealed infrequent foci of hyperplasia, whereas mice treated with the 100 and 500 {mu}g/ml doses exhibited numerous hyperplastic foci and also adenomas. Immunohistochemical and RNA analysis of the downstream effector pathways demonstrated that different levels of mutant RAS transgene expression resulted in differences in the expression and/or phosphorylation of specific signaling molecules. Our results suggest that the molecular alterations driving tumorigenesis may differ at different levels of mutant Ki-ras{sup G12C} expression, and this should be taken into consideration when inducible transgene systems are utilized to promote tumorigenesis in mouse models.

  2. Nitric oxide induces thioredoxin-1 nuclear translocation: Possible association with the p21Ras survival pathway

    SciTech Connect

    Arai, Roberto J.; Yodoi, J.; Debbas, V.; Laurindo, Francisco R.; Stern, A.; Monteiro, Hugo P. . E-mail: hpmonte@uol.com.br

    2006-10-06

    One of the major redox-regulating molecules with thiol reducing activity is thioredoxin-1 (TRX-1). TRX-1 is a multifunctional protein that exists in the extracellular millieu, cytoplasm, and nucleus, and has a distinct role in each environment. It is well known that TRX-1 promptly migrates to the nuclear compartment in cells exposed to oxidants. However, the intracellular location of TRX-1 in cells exposed to nitrosothiols has not been investigated. Here, we demonstrated that the exposure of HeLa cells to increasing concentrations of the nitrosothiol S-nitroso-N-acetylpenicillamine (SNAP) promoted TRX-1 nuclear accumulation. The SNAP-induced TRX-1 translocation to the nucleus was inhibited by FPTIII, a selective inhibitor of p21Ras. Furthermore, TRX-1 migration was attenuated in cells stably transfected with NO insensitive p21Ras (p21{sup RasC118S}). Downstream to p21Ras, the MAP Kinases ERK1/2 were activated by SNAP under conditions that promote TRX-1 nuclear translocation. Inhibition of MEK prevented SNAP-stimulated ERK1/2 activation and TRX-1 nuclear migration. In addition, cells treated with p21Ras or MEK inhibitor showed increased susceptibility to cell death induced by SNAP. In conclusion, our observations suggest that the nuclear translocation of TRX-1 is induced by SNAP involving p21Ras survival pathway.

  3. Ras in Cancer and Developmental Diseases

    PubMed Central

    Fernández-Medarde, Alberto; Santos, Eugenio

    2011-01-01

    Somatic, gain-of-function mutations in ras genes were the first specific genetic alterations identified in human cancer about 3 decades ago. Studies during the last quarter century have characterized the Ras proteins as essential components of signaling networks controlling cellular proliferation, differentiation, or survival. The oncogenic mutations of the H-ras, N-ras, or K-ras genes frequently found in human tumors are known to throw off balance the normal outcome of those signaling pathways, thus leading to tumor development. Oncogenic mutations in a number of other upstream or downstream components of Ras signaling pathways (including membrane RTKs or cytosolic kinases) have been detected more recently in association with a variety of cancers. Interestingly, the oncogenic Ras mutations and the mutations in other components of Ras/MAPK signaling pathways appear to be mutually exclusive events in most tumors, indicating that deregulation of Ras-dependent signaling is the essential requirement for tumorigenesis. In contrast to sporadic tumors, separate studies have identified germline mutations in Ras and various other components of Ras signaling pathways that occur in specific association with a number of different familial, developmental syndromes frequently sharing common phenotypic cardiofaciocutaneous features. Finally, even without being a causative force, defective Ras signaling has been cited as a contributing factor to many other human illnesses, including diabetes and immunological and inflammatory disorders. We aim this review at summarizing and updating current knowledge on the contribution of Ras mutations and altered Ras signaling to development of various tumoral and nontumoral pathologies. PMID:21779504

  4. CPI-17 drives oncogenic Ras signaling in human melanomas via Ezrin-Radixin-Moesin family proteins

    PubMed Central

    Riecken, Lars Björn; Zoch, Ansgar; Wiehl, Ulrike; Reichert, Sabine; Scholl, Ingmar; Cui, Yan; Ziemer, Mirjana; Anderegg, Ulf; Hagel, Christian; Morrison, Helen

    2016-01-01

    Hyperactive Ras signaling has strong oncogenic effects causing several different forms of cancer. Hyperactivity is frequently induced by mutations within Ras itself, which account for up to 30% of all human cancers. In addition, hyperactive Ras signaling can also be triggered independent of Ras by either mutation or by misexpression of various upstream regulators and immediate downstream effectors. We have previously reported that C-kinase potentiated protein phosphatase-1 inhibitor of 17 kDa (CPI-17) can drive Ras activity and promote tumorigenic transformation by inhibition of the tumor suppressor Merlin. We now describe an additional element of this oncogenic mechanism in the form of the ezrin-radixin-moesin (ERM) protein family, which exhibits opposing roles in Ras activity control. Thus, CPI-17 drives Ras activity and tumorigenesis in a two-fold way; inactivation of the tumor suppressor merlin and activation of the growth promoting ERM family. The in vivo significance of this oncogenic switch is highlighted by demonstrating CPI-17's involvement in human melanoma pathogenesis. PMID:27793041

  5. Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells.

    PubMed Central

    Migliaccio, A; Di Domenico, M; Castoria, G; de Falco, A; Bontempo, P; Nola, E; Auricchio, F

    1996-01-01

    The mechanism by which estradiol acts on cell multiplication is still unclear. Under conditions of estradiol-dependent growth, estradiol treatment of human mammary cancer MCF-7 cells triggers rapid and transient activation of the mitogen-activated (MAP) kinases, erk-1 and erk-2, increases the active form of p21ras, tyrosine phosphorylation of Shc and p190 protein and induces association of p190 to p21ras-GAP. Both Shc and p190 are substrates of activated src and once phosphorylated, they interact with other proteins and upregulate p21ras. Estradiol activates the tyrosine kinase/p21ras/MAP-kinase pathway in MCF-7 cells with kinetics which are similar to those of peptide mitogens. It is only after introduction of the human wild-type 67 kDa estradiol receptor cDNA that Cos cells become estradiol-responsive in terms of erk-2 activity. This finding, together with the inhibition by the pure anti-estrogen ICI 182 780 of the stimulatory effect of estradiol on each step of the pathway in MCF-7 cells proves that the classic estradiol receptor is responsible for the transduction pathway activation. Transfection experiments of Cos cells with the estradiol receptor cDNA and in vitro experiments with c-src show that the estradiol receptor activates c-src and this activation requires occupancy of the receptor by hormone. Our experiments suggest that c-src is an initial and integral part of the signaling events mediated by the estradiol receptor. Images PMID:8635462

  6. Targeting the RAS/MAPK pathway with miR-181a in acute myeloid leukemia

    PubMed Central

    Santhanam, Ramasamy; Eisfeld, Ann-Kathrin; Chiang, Chi-ling; Lankenau, Malori; Yu, Bo; Hoellerbauer, Pia; Jin, Yan; Tarighat, Somayeh S.; Khalife, Jihane; Walker, Alison; Perrotti, Danilo; Bloomfield, Clara D.; Wang, Hongyan; Lee, Robert J.; Lee, Ly James; Marcucci, Guido

    2016-01-01

    Deregulation of microRNAs' expression frequently occurs in acute myeloid leukemia (AML). Lower miR-181a expression is associated with worse outcomes, but the exact mechanisms by which miR-181a mediates this effect remain elusive. Aberrant activation of the RAS pathway contributes to myeloid leukemogenesis. Here, we report that miR-181a directly binds to 3′-untranslated regions (UTRs); downregulates KRAS, NRAS and MAPK1; and decreases AML growth. The delivery of miR-181a mimics to target AML cells using transferrin-targeting lipopolyplex nanoparticles (NP) increased mature miR-181a; downregulated KRAS, NRAS and MAPK1; and resulted in decreased phosphorylation of the downstream RAS effectors. NP-mediated upregulation of miR-181a led to reduced proliferation, impaired colony formation and increased sensitivity to chemotherapy. Ectopic expression of KRAS, NRAS and MAPK1 attenuated the anti-leukemic activity of miR-181a mimics, thereby validating the relevance of the deregulated miR-181a-RAS network in AML. Finally, treatment with miR-181a-NP in a murine AML model resulted in longer survival compared to mice treated with scramble-NP control. These data support that targeting the RAS-MAPK-pathway by miR-181a mimics represents a novel promising therapeutic approach for AML and possibly for other RAS-driven cancers. PMID:27517749

  7. MSI1, a negative regulator of the RAS-cAMP pathway in Saccharomyces cerevisiae.

    PubMed Central

    Ruggieri, R; Tanaka, K; Nakafuku, M; Kaziro, Y; Toh-e, A; Matsumoto, K

    1989-01-01

    We have previously demonstrated that the IRA1-encoded protein inhibits the function of the RAS protein in a fashion antagonistic to the function of the CDC25 protein in the RAS-cAMP pathway in Saccharomyces cerevisiae. In an attempt to identify genes involved in the regulation of this pathway, high-copy-number plasmid suppressors of the heat shock sensitivity of the ira1 mutation were isolated. One such suppressor, MSI1, was found to encode a putative protein of 422 amino acids that shows homology to the beta subunit of the mammalian guanine nucleotide-binding regulatory proteins. Overexpression of the MSI1 gene could suppress the heat shock sensitivity and the defect in sporulation caused by the ira1 and RAS2Val19 mutations but not those of the bcy1 mutation. Furthermore, the high level of intracellular cAMP in ira1 and RAS2Val19 cells was reduced by the MSI1 gene carried on a YEp-based plasmid. These results suggest that the MSI1 protein is a negative regulator of the RAS-mediated induction of cAMP in S. cerevisiae. Images PMID:2554329

  8. A Cardiac-enriched MicroRNA, miR-378, Blocks Cardiac Hypertrophy by Targeting Ras Signaling*

    PubMed Central

    Nagalingam, Raghu S.; Sundaresan, Nagalingam R.; Gupta, Mahesh P.; Geenen, David L.; Solaro, R. John; Gupta, Madhu

    2013-01-01

    Understanding the regulation of cardiomyocyte growth is crucial for the management of adverse ventricular remodeling and heart failure. MicroRNA-378 (miR-378) is a newly described member of the cardiac-enriched miRNAs, which is expressed only in cardiac myocytes and not in cardiac fibroblasts. We have previously shown that miR-378 regulates cardiac growth during the postnatal period by direct targeting of IGF1R (Knezevic, I., Patel, A., Sundaresan, N. R., Gupta, M. P., Solaro, R. J., Nagalingam, R. S., and Gupta, M. (2012) J. Biol. Chem. 287, 12913–12926). Here, we report that miR-378 is an endogenous negative regulator of cardiac hypertrophy, and its levels are down-regulated during hypertrophic growth of the heart and during heart failure. In primary cultures of cardiomyocytes, overexpression of miR-378 blocked phenylephrine (PE)-stimulated Ras activity and also prevented activation of two major growth-promoting signaling pathways, PI3K-AKT and Raf1-MEK1-ERK1/2, acting downstream of Ras signaling. Overexpression of miR-378 suppressed PE-induced phosphorylation of S6 ribosomal kinase, pERK1/2, pAKT, pGSK-3β, and nuclear accumulation of NFAT. There was also suppression of the fetal gene program that was induced by PE. Experiments carried out to delineate the mechanism behind the suppression of Ras, led us to identify Grb2, an upstream component of Ras signaling, as a bona fide direct target of miR-378-mediated regulation. Deficiency of miR-378 alone was sufficient to induce fetal gene expression, which was prevented by knocking down Grb2 expression and blocking Ras activation, thus suggesting that miR-378 interferes with Ras activation by targeting Grb2. Our study demonstrates that miR-378 is an endogenous negative regulator of Ras signaling and cardiac hypertrophy and its deficiency contributes to the development of cardiac hypertrophy. PMID:23447532

  9. Arabidopsis brassinosteroid signaling pathway.

    PubMed

    Belkhadir, Youssef; Wang, Xuelu; Chory, Joanne

    2006-12-05

    Plants control their size through the action of several phytohormones. One class of growth-promoting hormones is the brassinosteroids (BRs), the polyhydroxylated steroid hormones of plants. Here, we present the Arabidopsis-specific proteins that are the founding members of key BR signaling pathway components found in all plants. The genetic studies that identified these components are unique to Arabidopsis owing to its rapid generation time, sophisticated genetics, and facile transformation protocols, thereby highlighting the importance of a reference plant for understanding fundamental processes in all land plants.

  10. Low proliferation and high apoptosis of osteoblastic cells on hydrophobic surface are associated with defective Ras signaling

    SciTech Connect

    Chang, Eun-Ju; Kim, Hong-Hee; Huh, Jung-Eun; Kim, In-Ae; Seung Ko, Jea; Chung, Chong-Pyoung; Kim, Hyun-Man . E-mail: hyunmkim@plaza.snu.ac.kr

    2005-02-01

    The hydrophobic (HPB) nature of most polymeric biomaterials has been a major obstacle in using those materials in vivo due to low compatibility with cells. However, there is little knowledge of the molecular detail to explain how surface hydrophobicity affects cell responses. In this study, we compared the proliferation and apoptosis of human osteoblastic MG63 cells adhered to hydrophilic (HPL) and hydrophobic surfaces. On the hydrophobic surface, less formation of focal contacts and actin stress fibers, a delay in cell cycle progression, and an increase in apoptosis were observed. By using fibroblast growth factor 1 (FGF1) as a model growth factor, we also investigated intracellular signaling pathways on hydrophilic and hydrophobic surfaces. The activation of Ras, Akt, and ERK by FGF1 was impaired in MG63 cells on the hydrophobic surface. The overexpression of constitutively active form of Ras and Akt rescued those cells from apoptosis and recovered cell cycle progression. Furthermore, their overexpression also restored the actin cytoskeletal organization on the hydrophobic surface. Finally, the proliferative, antiapoptotic, and cytoskeletal effects of constitutively active Ras in MG63 cells on the hydrophobic surface were blocked by wortmannin and PD98059 that inhibit Akt and ERK activation, respectively. Therefore, our results suggest that the activation of Ras and its downstream molecules Akt and ERK to an appropriate level is one of crucial elements in the determination of osteoblast cell responses. The Ras pathway may represent a cell biological target that should be considered for successful surface modification of biomaterials to induce adequate cell responses in the bone tissue.

  11. Rapid induction of apoptosis by PI3K inhibitors is dependent upon their transient inhibition of RAS-ERK signaling

    PubMed Central

    Will, Marie; Qin, Alice Can Ran; Toy, Weiyi; Yao, Zhan; Rodrik-Outmezguine, Vanessa; Schneider, Claudia; Huang, Xiaodong; Monian, Prashant; Jiang, Xuejun; de Stanchina, Elisa; Baselga, Jose; Liu, Ningshu; Chandarlapaty, Sarat; Rosen, Neal

    2014-01-01

    The effects of selective PI3K and AKT inhibitors were compared in human tumor cell lines in which the pathway is dysregulated. Both caused inhibition of AKT, relief of feedback inhibition of RTKs, and growth arrest. However, only the PI3K inhibitors caused rapid induction of cell death. In seeking a mechanism for this phenomenon, we found that PI3K inhibition, but not AKT inhibition, causes rapid inhibition of wild type RAS and of RAF/MEK/ERK signaling. Inhibition of RAS-ERK signaling is transient, rebounding a few hours after drug addition, and is required for rapid induction of apoptosis. Combined MEK and AKT inhibition also promotes cell death and in murine models of HER2+ cancer, either pulsatile PI3K inhibition or combined MEK and AKT inhibition causes tumor regressions. We conclude that PI3K is upstream of RAS and AKT and that pulsatile inhibition of both pathways is sufficient for effective antitumor activity. PMID:24436048

  12. Rapid induction of apoptosis by PI3K inhibitors is dependent upon their transient inhibition of RAS-ERK signaling.

    PubMed

    Will, Marie; Qin, Alice Can Ran; Toy, Weiyi; Yao, Zhan; Rodrik-Outmezguine, Vanessa; Schneider, Claudia; Huang, Xiaodong; Monian, Prashant; Jiang, Xuejun; de Stanchina, Elisa; Baselga, José; Liu, Ningshu; Chandarlapaty, Sarat; Rosen, Neal

    2014-03-01

    The effects of selective phosphoinositide 3-kinase (PI3K) and AKT inhibitors were compared in human tumor cell lines in which the pathway is dysregulated. Both caused inhibition of AKT, relief of feedback inhibition of receptor tyrosine kinases, and growth arrest. However, only the PI3K inhibitors caused rapid induction of cell death. In seeking a mechanism for this phenomenon, we found that PI3K inhibition, but not AKT inhibition, causes rapid inhibition of wild-type RAS and of RAF-MEK-ERK signaling. Inhibition of RAS-ERK signaling is transient, rebounding a few hours after drug addition, and is required for rapid induction of apoptosis. Combined MEK and AKT inhibition also promotes cell death, and in murine models of HER2(+) cancer, either pulsatile PI3K inhibition or combined MEK and AKT inhibition causes tumor regression. We conclude that PI3K is upstream of RAS and AKT and that pulsatile inhibition of both pathways is sufficient for effective antitumor activity.

  13. Honokiol Suppresses Survival Signals Mediated by Ras-Dependent Phospholipase D Activity in Human Cancer Cells

    PubMed Central

    Garcia, Avalon; Zheng, Yang; Zhao, Chen; Toschi, Alfredo; Fan, Judy; Shraibman, Natalie; Brown, H. Alex; Bar-Sagi, Dafna; Foster, David A.; Arbiser, Jack L.

    2009-01-01

    Purpose Elevated phospholipase D (PLD) activity provides a survival signal in several human cancer cell lines and suppresses apoptosis when cells are subjected to the stress of serum withdrawal. Thus, targeting PLD survival signals has potential to suppress survival in cancer cells that depend on PLD for survival. Honokiol is a compound that suppresses tumor growth in mouse models. The purpose of this study was to investigate the effect of honokiol on PLD survival signals and the Ras dependence of these signals. Experimental Design The effect of honokiol upon PLD activity was examined in human cancer cell lines where PLD activity provides a survival signal. The dependence of PLD survival signals on Ras was investigated, as was the effect of honokiol on Ras activation. Results We report here that honokiol suppresses PLD activity in human cancer cells where PLD has been shown to suppress apoptosis. PLD activity is commonly elevated in response to the stress of serum withdrawal, and, importantly, the stress-induced increase in PLD activity is selectively suppressed by honokiol. The stress-induced increase in PLD activity was accompanied by increased Ras activation, and the stress-induced increase in PLD activity in MDA-MB-231 breast cancer cells was dependent on a Ras. The PLD activity was also dependent on the GTPases RalA and ADP ribosylation factor. Importantly, honokiol suppressed Ras activation. Conclusion The data provided here indicate that honokiol may be a valuable therapeutic reagent for targeting a large number of human cancers that depend on Ras and PLD for their survival. PMID:18594009

  14. Differential Regulation of N-Myc and c-Myc Synthesis, Degradation, and Transcriptional Activity by the Ras/Mitogen-activated Protein Kinase Pathway*

    PubMed Central

    Kapeli, Katannya; Hurlin, Peter J.

    2011-01-01

    Myc transcription factors are important regulators of proliferation and can promote oncogenesis when deregulated. Deregulated Myc expression in cancers can result from MYC gene amplification and translocation but also from alterations in mitogenic signaling pathways that affect Myc levels through both transcriptional and post-transcription mechanisms. For example, mutations in Ras family GTPase proteins that cause their constitutive activation can increase cellular levels of c-Myc by interfering with its rapid proteasomal degradation. Although enhanced protein stability is generally thought to be applicable to other Myc family members, here we show that c-Myc and its paralog N-Myc respond to oncogenic H-Ras (H-RasG12V) in very different ways. H-RasG12V promotes accumulation of both c-Myc and N-Myc, but although c-Myc accumulation is achieved by enhanced protein stability, N-Myc accumulation is associated with an accelerated rate of translation that overcomes a surprising H-RasG12V-mediated destabilization of N-Myc. We show that H-RasG12V-mediated degradation of N-Myc functions independently of key phosphorylation sites in the highly conserved Myc homology box I region that controls c-Myc protein stability by oncogenic Ras. Finally, we found that N-Myc and c-Myc transcriptional activity is associated with their proteasomal degradation but that N-Myc may be uniquely dependent on Ras-stimulated proteolysis for target gene expression. Taken together, these studies provide mechanistic insight into how oncogenic Ras augments N-Myc levels in cells and suggest that enhanced N-Myc translation and degradation-coupled transactivation may contribute to oncogenesis. PMID:21908617

  15. The Fibroblast Growth Factor signaling pathway

    PubMed Central

    Ornitz, David M; Itoh, Nobuyuki

    2015-01-01

    The signaling component of the mammalian Fibroblast Growth Factor (FGF) family is comprised of eighteen secreted proteins that interact with four signaling tyrosine kinase FGF receptors (FGFRs). Interaction of FGF ligands with their signaling receptors is regulated by protein or proteoglycan cofactors and by extracellular binding proteins. Activated FGFRs phosphorylate specific tyrosine residues that mediate interaction with cytosolic adaptor proteins and the RAS-MAPK, PI3K-AKT, PLCγ, and STAT intracellular signaling pathways. Four structurally related intracellular non-signaling FGFs interact with and regulate the family of voltage gated sodium channels. Members of the FGF family function in the earliest stages of embryonic development and during organogenesis to maintain progenitor cells and mediate their growth, differentiation, survival, and patterning. FGFs also have roles in adult tissues where they mediate metabolic functions, tissue repair, and regeneration, often by reactivating developmental signaling pathways. Consistent with the presence of FGFs in almost all tissues and organs, aberrant activity of the pathway is associated with developmental defects that disrupt organogenesis, impair the response to injury, and result in metabolic disorders, and cancer. © 2015 Wiley Periodicals, Inc. PMID:25772309

  16. Phosphoproteomics reveals ALK promote cell progress via RAS/JNK pathway in neuroblastoma

    PubMed Central

    Xu, Guofeng; Zhang, Min; Wu, Yeming; Wu, Zhixiang

    2016-01-01

    Emerging evidence suggests receptor tyrosine kinase ALK as a promising therapeutic target in neuroblastoma. However, clinical trials reveal that a limited proportion of ALK-positive neuroblastoma patients experience clinical benefits from Crizotinib, a clinically approved specific inhibitor of ALK. The precise molecular mechanisms of aberrant ALK activity in neuroblastoma remain elusive, limiting the clinical application of ALK as a therapeutic target in neuroblastoma. Here, we describe a deep quantitative phosphoproteomic approach in which Crizotinib-treated neuroblastoma cell lines bearing aberrant ALK are used to investigate downstream regulated phosphoproteins. We identified more than 19,500—and quantitatively analyzed approximately 10,000—phosphorylation sites from each cell line, ultimately detecting 450–790 significantly-regulated phosphorylation sites. Multiple layers of bioinformatic analysis of the significantly-regulated phosphoproteins identified RAS/JNK as a downstream signaling pathway of ALK, independent of the ALK variant present. Further experiments demonstrated that ALK/JNK signaling could be inactivated by either ALK- or JNK-specific inhibitors, resulting in cell growth inhibition by induction of cell cycle arrest and cell apoptosis. Our study broadly defines the phosphoproteome in response to ALK inhibition and provides a resource for further clinical investigation of ALK as therapeutic target for the treatment of neuroblastoma. PMID:27732954

  17. Assembly and function of the regulator of G protein signaling 14 (RGS14)·H-Ras signaling complex in live cells are regulated by Gαi1 and Gαi-linked G protein-coupled receptors.

    PubMed

    Vellano, Christopher P; Brown, Nicole E; Blumer, Joe B; Hepler, John R

    2013-02-01

    Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates heterotrimeric G protein and H-Ras signaling pathways. RGS14 possesses an RGS domain that binds active Gα(i/o)-GTP subunits to promote GTP hydrolysis and a G protein regulatory (GPR) motif that selectively binds inactive Gα(i1/3)-GDP subunits to form a stable heterodimer at cellular membranes. RGS14 also contains two tandem Ras/Rap binding domains (RBDs) that bind H-Ras. Here we show that RGS14 preferentially binds activated H-Ras-GTP in live cells to enhance H-Ras cellular actions and that this interaction is regulated by inactive Gα(i1)-GDP and G protein-coupled receptors (GPCRs). Using bioluminescence resonance energy transfer (BRET) in live cells, we show that RGS14-Luciferase and active H-Ras(G/V)-Venus exhibit a robust BRET signal at the plasma membrane that is markedly enhanced in the presence of inactive Gα(i1)-GDP but not active Gα(i1)-GTP. Active H-Ras(G/V) interacts with a native RGS14·Gα(i1) complex in brain lysates, and co-expression of RGS14 and Gα(i1) in PC12 cells greatly enhances H-Ras(G/V) stimulatory effects on neurite outgrowth. Stimulation of the Gα(i)-linked α(2A)-adrenergic receptor induces a conformational change in the Gα(i1)·RGS14·H-Ras(G/V) complex that may allow subsequent regulation of the complex by other binding partners. Together, these findings indicate that inactive Gα(i1)-GDP enhances the affinity of RGS14 for H-Ras-GTP in live cells, resulting in a ternary signaling complex that is further regulated by GPCRs.

  18. CAVEOLIN-1 REGULATES HIV-1 TAT-INDUCED ALTERATIONS OF TIGHT JUNCTION PROTEIN EXPRESSION VIA MODULATION OF THE RAS SIGNALING

    PubMed Central

    Zhong, Yu; Smart, Eric J.; Weksler, Babette; Couraud, Pierre-Olivier; Hennig, Bernhard; Toborek, Michal

    2009-01-01

    The blood-brain barrier (BBB) is the critical structure for preventing HIV trafficking into the brain. Specific HIV proteins, such as Tat protein, can contribute to the dysfunction of tight junctions at the BBB and HIV entry into the brain. Tat is released by HIV-1 infected cells and can interact with a variety of cell surface receptors activating several signal transduction pathways, including those localized in caveolae. The present study focused on the mechanisms of Tat-induced caveolae-associated Ras signaling at the level of the BBB. Treatment with Tat activated the Ras pathway in human brain microvascular endothelial cells (HBMEC). However, caveolin-1 silencing markedly attenuated these effects. Because the integrity of the brain endothelium is regulated by intercellular tight junctions, these structural elements of the BBB were also evaluated in the present study. Exposure to Tat diminished the expression of several tight junction proteins, namely, occludin, zonula occludens (ZO)-1, and ZO-2 in the caveolar fraction of HBMEC. These effects were effectively protected by pharmacological inhibition of the Ras signaling and by silencing of caveolin-1. The present data indicate the importance of caveolae-associated signaling in the disruption of tight junctions upon Tat exposure. They also demonstrate that caveolin-1 may constitute an early and critical modulator that controls signaling pathways leading to the disruption of tight junction proteins. Thus, caveolin-1 may provide an effective target to protect against Tat-induced HBMEC dysfunction and the disruption of the BBB in HIV-1-infected patients. PMID:18667611

  19. Modeling and stochastic simulation of the Ras/cAMP/PKA pathway in the yeast Saccharomyces cerevisiae evidences a key regulatory function for intracellular guanine nucleotides pools.

    PubMed

    Cazzaniga, Paolo; Pescini, Dario; Besozzi, Daniela; Mauri, Giancarlo; Colombo, Sonia; Martegani, Enzo

    2008-02-01

    In the yeast Saccharomyces cerevisiae, the Ras/cAMP/PKA pathway is involved in the regulation of metabolism and cell cycle progression. The pathway is tightly regulated by several control mechanisms, as the feedback cycle ruled by the activity of phosphodiesterase. Here, we present a discrete mathematical model for the Ras/cAMP/PKA pathway that considers its principal cytoplasmic components and their mutual interactions. The tau-leaping algorithm is then used to perform stochastic simulations of the model. We investigate this system under various conditions, and we test how different values of several stochastic reaction constants affect the pathway behaviour. Finally, we show that the level of guanine nucleotides, GTP and GDP, could be relevant metabolic signals for the regulation of the whole pathway.

  20. Activated N-Ras signaling regulates arterial-venous specification in zebrafish.

    PubMed

    Ren, Chun-Guang; Wang, Lei; Jia, Xiao-E; Liu, Yi-Jie; Dong, Zhi-Wei; Jin, Yi; Chen, Yi; Deng, Min; Zhou, Yong; Zhou, Yi; Ren, Rui-Bao; Pan, Wei-Jun; Liu, Ting-Xi

    2013-05-12

    The aberrant activation of Ras signaling is associated with human diseases including hematological malignancies and vascular disorders. So far the pathological roles of activated Ras signaling in hematopoiesis and vasculogenesis are largely unknown. A conditional Cre/loxP transgenic strategy was used to mediate the specific expression of a constitutively active form of human N-Ras in zebrafish endothelial and hematopoietic cells driven by the zebrafish lmo2 promoter. The expression of hematopoietic and endothelial marker genes was analyzed both via whole mount in situ hybridization (WISH) assay and real-time quantitative PCR (qPCR). The embryonic vascular morphogenesis was characterized both by living imaging and immunofluorescence on the sections with a confocal microscopy, and the number of endothelial cells in the embryos was quantified by flow cytometry. The functional analyses of the blood circulation were carried out by fluorescence microangiography assay and morpholino injection. In the activated N-Ras transgenic embryos, the primitive hematopoiesis appeared normal, however, the definitive hematopoiesis of these embryos was completely absent. Further analysis of endothelial cell markers confirmed that transcription of arterial marker ephrinB2 was significantly decreased and expression of venous marker flt4 excessively increased, indicating the activated N-Ras signaling promotes the venous development at the expense of arteriogenesis during zebrafish embryogenesis. The activated N-Ras-expressing embryos showed atrophic axial arteries and expansive axial veins, leading to no definitive hematopoietic stem cell formation, the blood circulation failure and subsequently embryonic lethality. Our studies revealed for the first time that activated N-Ras signaling during the endothelial differentiation in vertebrates can disrupt the balance of arterial-venous specification, thus providing new insights into the pathogenesis of the congenital human vascular disease and

  1. RAS/ERK signaling controls proneural genetic programs in cortical development and gliomagenesis.

    PubMed

    Li, Saiqun; Mattar, Pierre; Dixit, Rajiv; Lawn, Samuel O; Wilkinson, Grey; Kinch, Cassandra; Eisenstat, David; Kurrasch, Deborah M; Chan, Jennifer A; Schuurmans, Carol

    2014-02-05

    Neural cell fate specification is well understood in the embryonic cerebral cortex, where the proneural genes Neurog2 and Ascl1 are key cell fate determinants. What is less well understood is how cellular diversity is generated in brain tumors. Gliomas and glioneuronal tumors, which are often localized in the cerebrum, are both characterized by a neoplastic glial component, but glioneuronal tumors also have an intermixed neuronal component. A core abnormality in both tumor groups is overactive RAS/ERK signaling, a pro-proliferative signal whose contributions to cell differentiation in oncogenesis are largely unexplored. We found that RAS/ERK activation levels differ in two distinct human tumors associated with constitutively active BRAF. Pilocytic astrocytomas, which contain abnormal glial cells, have higher ERK activation levels than gangliogliomas, which contain abnormal neuronal and glial cells. Using in vivo gain of function and loss of function in the mouse embryonic neocortex, we found that RAS/ERK signals control a proneural genetic switch, inhibiting Neurog2 expression while inducing Ascl1, a competing lineage determinant. Furthermore, we found that RAS/ERK levels control Ascl1's fate specification properties in murine cortical progenitors--at higher RAS/ERK levels, Ascl1(+) progenitors are biased toward proliferative glial programs, initiating astrocytomas, while at moderate RAS/ERK levels, Ascl1 promotes GABAergic neuronal and less glial differentiation, generating glioneuronal tumors. Mechanistically, Ascl1 is phosphorylated by ERK, and ERK phosphoacceptor sites are necessary for Ascl1's GABAergic neuronal and gliogenic potential. RAS/ERK signaling thus acts as a rheostat to influence neural cell fate selection in both normal cortical development and gliomagenesis, controlling Neurog2-Ascl1 expression and Ascl1 function.

  2. Activated N-Ras signaling regulates arterial-venous specification in zebrafish

    PubMed Central

    2013-01-01

    Background The aberrant activation of Ras signaling is associated with human diseases including hematological malignancies and vascular disorders. So far the pathological roles of activated Ras signaling in hematopoiesis and vasculogenesis are largely unknown. Methods A conditional Cre/loxP transgenic strategy was used to mediate the specific expression of a constitutively active form of human N-Ras in zebrafish endothelial and hematopoietic cells driven by the zebrafish lmo2 promoter. The expression of hematopoietic and endothelial marker genes was analyzed both via whole mount in situ hybridization (WISH) assay and real-time quantitative PCR (qPCR). The embryonic vascular morphogenesis was characterized both by living imaging and immunofluorescence on the sections with a confocal microscopy, and the number of endothelial cells in the embryos was quantified by flow cytometry. The functional analyses of the blood circulation were carried out by fluorescence microangiography assay and morpholino injection. Results In the activated N-Ras transgenic embryos, the primitive hematopoiesis appeared normal, however, the definitive hematopoiesis of these embryos was completely absent. Further analysis of endothelial cell markers confirmed that transcription of arterial marker ephrinB2 was significantly decreased and expression of venous marker flt4 excessively increased, indicating the activated N-Ras signaling promotes the venous development at the expense of arteriogenesis during zebrafish embryogenesis. The activated N-Ras-expressing embryos showed atrophic axial arteries and expansive axial veins, leading to no definitive hematopoietic stem cell formation, the blood circulation failure and subsequently embryonic lethality. Conclusions Our studies revealed for the first time that activated N-Ras signaling during the endothelial differentiation in vertebrates can disrupt the balance of arterial-venous specification, thus providing new insights into the pathogenesis of the

  3. C. elegans EOR-1/PLZF and EOR-2 positively regulate Ras and Wnt signaling and function redundantly with LIN-25 and the SUR-2 Mediator component.

    PubMed

    Howard, Robyn M; Sundaram, Meera V

    2002-07-15

    In Caenorhabditis elegans, Ras/ERK and Wnt/beta-catenin signaling pathways cooperate to induce P12 and vulval cell fates in a Hox-dependent manner. Here we describe eor-1 and eor-2, two new positively acting nuclear components of the Ras and Wnt pathways. eor-1 and eor-2 act downstream or in parallel to ERK and function redundantly with the Mediator complex gene sur-2 and the functionally related gene lin-25, such that removal of both eor-1/eor-2 and sur-2/lin-25 mimics the removal of a main Ras pathway component. Furthermore, the eor-1 and eor-2 mutant backgrounds reveal an essential role for the Elk1-related gene lin-1. eor-1 and eor-2 also act downstream or in parallel to pry-1 Axin and therefore act at the convergence of the Ras and Wnt pathways. eor-1 encodes the ortholog of human PLZF, a BTB/zinc-finger transcription factor that is fused to RARalpha in acute promyelocytic leukemia. eor-2 encodes a novel protein. EOR-1/PLZF and EOR-2 appear to function closely together and cooperate with Hox genes to promote the expression of Ras- and Wnt-responsive genes. Further studies of eor-1 and eor-2 may provide insight into the roles of PLZF in normal development and leukemogenesis.

  4. Signaling pathways involved in MDSC regulation.

    PubMed

    Trikha, Prashant; Carson, William E

    2014-08-01

    The immune system has evolved mechanisms to protect the host from the deleterious effects of inflammation. The generation of immune suppressive cells like myeloid derived suppressor cells (MDSCs) that can counteract T cell responses represents one such strategy. There is an accumulation of immature myeloid cells or MDSCs in bone marrow (BM) and lymphoid organs under pathological conditions such as cancer. MDSCs represent a population of heterogeneous myeloid cells comprising of macrophages, granulocytes and dendritic cells that are at early stages of development. Although, the precise signaling pathways and molecular mechanisms that lead to MDSC generation and expansion in cancer remains to be elucidated. It is widely believed that perturbation of signaling pathways involved during normal hematopoietic and myeloid development under pathological conditions such as tumorogenesis contributes to the development of suppressive myeloid cells. In this review we discuss the role played by key signaling pathways such as PI3K, Ras, Jak/Stat and TGFb during myeloid development and how their deregulation under pathological conditions can lead to the generation of suppressive myeloid cells or MDSCs. Targeting these pathways should help in elucidating mechanisms that lead to the expansion of MDSCs in cancer and point to methods for eliminating these cells from the tumor microenvironment.

  5. A network of PUF proteins and Ras signaling promote mRNA repression and oogenesis in C. elegans

    PubMed Central

    Hubstenberger, Arnaud; Cameron, Cristiana; Shtofman, Rebecca; Gutman, Shiri; Evans, Thomas C.

    2012-01-01

    Cell differentiation requires integration of gene expression controls with dynamic changes in cell morphology, function, and control. Post-transcriptional mRNA regulation and signaling systems are important to this process but their mechanisms and connections are unclear. During C. elegans oogenesis, we find that two groups of PUF RNA binding proteins (RNABPs), PUF-3/11 and PUF-5/6/7, control different specific aspects of oocyte formation. PUF-3/11 limits oocyte growth, while PUF-5/6/7 promotes oocyte organization and formation. These two PUF groups repress mRNA translation through overlapping but distinct sets of 3’ untranslated regions (3’UTRs). Several PUF-dependent mRNAs encode other mRNA regulators suggesting both PUF groups control developmental patterning of mRNA regulation circuits. Furthermore, we find that the Ras-MapKinase/ERK pathway functions with PUF-5/6/7 to repress specific mRNAs and control oocyte organization and growth. These results suggest that diversification of PUF proteins and their integration with Ras-MAPK signaling modulates oocyte differentiation. Together with other studies, these findings suggest positive and negative interactions between the Ras-MAPK system and PUF RNA-binding proteins likely occur at multiple levels. Changes in these interactions over time can influence spatiotemporal patterning of tissue development. PMID:22542599

  6. DRoP: a water analysis program identifies Ras-GTP-specific pathway of communication between membrane-interacting regions and the active site.

    PubMed

    Kearney, Bradley M; Johnson, Christian W; Roberts, Daniel M; Swartz, Paul; Mattos, Carla

    2014-02-06

    Ras GTPase mediates several cellular signal transduction pathways and is found mutated in a large number of cancers. It is active in the GTP-bound state, where it interacts with effector proteins, and at rest in the GDP-bound state. The catalytic domain is tethered to the membrane, with which it interacts in a nucleotide-dependent manner. Here we present the program Detection of Related Solvent Positions (DRoP) for crystallographic water analysis on protein surfaces and use it to study Ras. DRoP reads and superimposes multiple Protein Data Bank coordinates, transfers symmetry-related water molecules to the position closest to the protein surface, and ranks the waters according to how well conserved and tightly clustered they are in the set of structures. Coloring according to this rank allows visualization of the results. The effector-binding region of Ras is hydrated with highly conserved water molecules at the interface between the P-loop, switch I, and switch II, as well as at the Raf-RBD binding pocket. Furthermore, we discovered a new conserved water-mediated H-bonding network present in Ras-GTP, but not in Ras-GDP, that links the nucleotide sensor residues R161 and R164 on helix 5 to the active site. The double mutant RasN85A/N86A, where the final link between helix 5 and the nucleotide is not possible, is a severely impaired enzyme, while the single mutant RasN86A, with partial connection to the active site, has a wild-type hydrolysis rate. DRoP was instrumental in determining the water-mediated connectivity networks that link two lobes of the catalytic domain in Ras. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Sinodielide A exerts thermosensitizing effects and induces apoptosis and G2/M cell cycle arrest in DU145 human prostate cancer cells via the Ras/Raf/MAPK and PI3K/Akt signaling pathways.

    PubMed

    Hatashita, Masanori; Taniguchi, Masahiko; Baba, Kimiye; Koshiba, Ken; Sato, Takefumi; Jujo, Yutaka; Suzuki, Ryuta; Hayashi, Sachiko

    2014-02-01

    Sinodielide A (SA) is a naturally occurring guaianolide, which is isolated from the root of Sinodielsia yunnanensis. This root, commonly found in Yunnan province, is used in traditional Chinese medicine as an antipyretic, analgesic and diaphoretic agent. A number of studies have reported that agents isolated from a species of Umbelliferae (Apiaceae) have antitumor activities. We previously reported, using combined treatments with this medicinal herb and hyperthermia at various temperatures, an enhanced cytotoxicity in the human prostate cancer androgen‑independent cell lines, PC3 and DU145, and analyzed the related mechanisms. In the present study, we investigated the effects of treatment with SA prior to hyperthermia on the thermosensitivity of DU145 cells, and the mechanisms related to the induction of apoptosis and G(2)/M cell cycle arrest via the activation of extracellular-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) signaling pathways, as well as the phosphoinositide 3-kinase (PI3K)/Akt signaling pathways. Cells were exposed to hyperthermia alone (40-44˚C) or hyperthermia in combination with SA. Lethal damage to cells treated with mild hyperthermia (40 or 42˚C) for up to 6 h was slight; however, hyperthermia in combination with SA synergistically enhanced thermosensivity. Lethal damage to cells treated with acute hyperthermia (43 or 44˚C) was more severe, but these effects were also enhanced and were more significant by the combined treatment with SA. The kinetics of apoptosis induction and cell cycle distribution were analyzed by flow cytometry. In addition, the levels of ERK1/2, JNK and Akt were determined by western blot analysis. The incidence of apoptotic cells after treatment with SA (20.0 µM) at 37˚C for 4 h, hyperthermia (44˚C) alone for 30 min, and the combination in sequence were examined. The sub-G1 division (%) in the diagram obtained by flow cytometry was applied to that assay. The

  8. ARIA/HRG regulates AChR epsilon subunit gene expression at the neuromuscular synapse via activation of phosphatidylinositol 3-kinase and Ras/MAPK pathway

    PubMed Central

    1996-01-01

    AChR-inducing activity (ARIA)/heregulin, a ligand for erbB receptor tyrosine kinases (RTKs), is likely to be one nerve-supplied signal that induces expression of acetylcholine receptor (AChR) genes at the developing neuromuscular junction. Since some RTKs act through Ras and phosphatidylinositol 3-kinase (PI3K), we investigated the role of these pathways in ARIA signaling. Expression of activated Ras or Raf mimicked ARIA-induction of AChR epsilon subunit genes in muscle cells; whereas dominant negative Ras or Raf blocked the effect of ARIA. ARIA rapidly activated erk1 and erk2 and inhibition of both erks also abolished the effect of ARIA. ARIA stimulated association of PI3K with erbB3, expression of an activated PI3K led to ARIA-independent AChR epsilon subunit expression, and inhibition of PI3K abolished the action of ARIA. Thus, synaptic induction of AChR genes requires activation of both Ras/MAPK and PI3K signal transduction pathways. PMID:8707830

  9. Alphavirus production is inhibited in neurofibromin 1-deficient cells through activated RAS signalling

    SciTech Connect

    Kolokoltsova, Olga A. Domina, Aaron M. Kolokoltsov, Andrey A. Davey, Robert A. | Weaver, Scott C. || Watowich, Stanley J. ||

    2008-07-20

    Virus-host interactions essential for alphavirus pathogenesis are poorly understood. To address this shortcoming, we coupled retrovirus insertional mutagenesis and a cell survival selection strategy to generate clonal cell lines broadly resistant to Sindbis virus (SINV) and other alphaviruses. Resistant cells had significantly impaired SINV production relative to wild-type (WT) cells, although virus binding and fusion events were similar in both sets of cells. Analysis of the retroviral integration sites identified the neurofibromin 1 (NF1) gene as disrupted in alphavirus-resistant cell lines. Subsequent analysis indicated that expression of NF1 was significantly reduced in alphavirus-resistant cells. Importantly, independent down-regulation of NF1 expression in WT HEK 293 cells decreased virus production and increased cell viability during SINV infection, relative to infected WT cells. Additionally, we observed hyperactive RAS signalling in the resistant HEK 293 cells, which was anticipated because NF1 is a negative regulator of RAS. Expression of constitutively active RAS (HRAS-G12V) in a WT HEK 293 cell line resulted in a marked delay in virus production, compared with infected cells transfected with parental plasmid or dominant-negative RAS (HRAS-S17N). This work highlights novel host cell determinants required for alphavirus pathogenesis and suggests that RAS signalling may play an important role in neuronal susceptibility to SINV infection.

  10. Alphavirus production is inhibited in neurofibromin 1-deficient cells through activated RAS signalling.

    PubMed

    Kolokoltsova, Olga A; Domina, Aaron M; Kolokoltsov, Andrey A; Davey, Robert A; Weaver, Scott C; Watowich, Stanley J

    2008-07-20

    Virus-host interactions essential for alphavirus pathogenesis are poorly understood. To address this shortcoming, we coupled retrovirus insertional mutagenesis and a cell survival selection strategy to generate clonal cell lines broadly resistant to Sindbis virus (SINV) and other alphaviruses. Resistant cells had significantly impaired SINV production relative to wild-type (WT) cells, although virus binding and fusion events were similar in both sets of cells. Analysis of the retroviral integration sites identified the neurofibromin 1 (NF1) gene as disrupted in alphavirus-resistant cell lines. Subsequent analysis indicated that expression of NF1 was significantly reduced in alphavirus-resistant cells. Importantly, independent down-regulation of NF1 expression in WT HEK 293 cells decreased virus production and increased cell viability during SINV infection, relative to infected WT cells. Additionally, we observed hyperactive RAS signalling in the resistant HEK 293 cells, which was anticipated because NF1 is a negative regulator of RAS. Expression of constitutively active RAS (HRAS-G12V) in a WT HEK 293 cell line resulted in a marked delay in virus production, compared with infected cells transfected with parental plasmid or dominant-negative RAS (HRAS-S17N). This work highlights novel host cell determinants required for alphavirus pathogenesis and suggests that RAS signalling may play an important role in neuronal susceptibility to SINV infection.

  11. Relapsed neuroblastomas show frequent RAS-MAPK pathway mutations | Office of Cancer Genomics

    Cancer.gov

    The majority of patients with neuroblastoma have tumors that initially respond to chemotherapy, but a large proportion will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole-genome sequencing of 23 paired diagnostic and relapse neuroblastomas showed clonal evolution from the diagnostic tumor, with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK pathway.

  12. Activation of mutant TERT promoter by RAS-ERK signaling is a key step in malignant progression of BRAF-mutant human melanomas

    PubMed Central

    Li, Yinghui; Cheng, Hui Shan; Chng, Wee Joo; Tergaonkar, Vinay

    2016-01-01

    Although activating BRAF/NRAS mutations are frequently seen in melanomas, they are not sufficient to drive malignant transformation and require additional events. Frequent co-occurrence of mutations in the promoter for telomerase reverse transcriptase (TERT), along with BRAF alterations, has recently been noted and correlated with poorer prognosis, implicating a functional link between BRAF signaling and telomerase reactivation in melanomas. Here, we report that RAS-ERK signaling in BRAF mutant melanomas is critical for regulating active chromatin state and recruitment of RNA polymerase II at mutant TERT promoters. Our study provides evidence that the mutant TERT promoter is a key substrate downstream of the RAS-ERK pathway. Reactivating TERT and hence reconstituting telomerase is an important step in melanoma progression from nonmalignant nevi with BRAF mutations. Hence, combined targeting of RAS-ERK and TERT promoter remodeling is a promising avenue to limit long-term survival of a majority of melanomas that harbor these two mutations. PMID:27911794

  13. Activation of mutant TERT promoter by RAS-ERK signaling is a key step in malignant progression of BRAF-mutant human melanomas.

    PubMed

    Li, Yinghui; Cheng, Hui Shan; Chng, Wee Joo; Tergaonkar, Vinay

    2016-12-13

    Although activating BRAF/NRAS mutations are frequently seen in melanomas, they are not sufficient to drive malignant transformation and require additional events. Frequent co-occurrence of mutations in the promoter for telomerase reverse transcriptase (TERT), along with BRAF alterations, has recently been noted and correlated with poorer prognosis, implicating a functional link between BRAF signaling and telomerase reactivation in melanomas. Here, we report that RAS-ERK signaling in BRAF mutant melanomas is critical for regulating active chromatin state and recruitment of RNA polymerase II at mutant TERT promoters. Our study provides evidence that the mutant TERT promoter is a key substrate downstream of the RAS-ERK pathway. Reactivating TERT and hence reconstituting telomerase is an important step in melanoma progression from nonmalignant nevi with BRAF mutations. Hence, combined targeting of RAS-ERK and TERT promoter remodeling is a promising avenue to limit long-term survival of a majority of melanomas that harbor these two mutations.

  14. Sanguinarine-induced G1-phase arrest of the cell cycle results from increased p27KIP1 expression mediated via activation of the Ras/ERK signaling pathway in vascular smooth muscle cells.

    PubMed

    Lee, Beobyi; Lee, Se-Jung; Park, Sung-Soo; Kim, Si-Kwan; Kim, Sung-Ryong; Jung, Jae-Hyun; Kim, Wun-Jae; Moon, Sung-Kwon

    2008-03-15

    The present study identified a novel mechanism for the effects of sanguinarine in vascular smooth muscle cells (VSMC). Sanguinarine treatment of VSMC resulted in significant growth inhibition as a result of G1-phase cell-cycle arrest mediated by induction of p27KIP1 expression, and resulted in a down-regulation of the expression of cyclins and CDKs in VSMC. Moreover, sanguinarine-induced inhibition of cell growth appeared to be linked to activation of Ras/ERK through p27KIP1-mediated G1-phase cell-cycle arrest. Overall, the unexpected effects of sanguinarine treatment in VSMC provide a theoretical basis for clinical use of therapeutic agents in the treatment of atherosclerosis.

  15. Signaling pathways affecting skeletal health.

    PubMed

    Marie, Pierre J

    2012-09-01

    Skeletal health is dependent on the balance between bone resorption and formation during bone remodeling. Multiple signaling pathways play essential roles in the maintenance of skeletal integrity by positively or negatively regulating bone cells. During the last years, significant advances have been made in our understanding of the essential signaling pathways that regulate bone cell commitment, differentiation and survival. New signaling anabolic pathways triggered by parathyroid hormone, local growth factors, Wnt signaling, and calcium sensing receptor have been identified. Novel signals induced by interactions between bone cells-matrix (integrins), osteoblasts/osteocytes (cadherins, connexins), and osteoblasts/osteoclast (ephrins, Wnt-RhoA, semaphorins) have been discovered. Recent studies revealed the key pathways (MAPK, PI3K/Akt) that critically control bone cells and skeletal mass. This review summarizes the most recent knowledge on the major signaling pathways that control bone cells, and their potential impact on the development of therapeutic strategies to improve human bone health.

  16. Acetate Regulation of Spore Formation Is under the Control of the Ras/Cyclic AMP/Protein Kinase A Pathway and Carbon Dioxide in Saccharomyces cerevisiae

    PubMed Central

    Jungbluth, Marc; Mösch, Hans-Ulrich

    2012-01-01

    In Saccharomyces cerevisiae, the Ras/cyclic AMP (cAMP)/protein kinase A (PKA) pathway is a nutrient-sensitive signaling cascade that regulates vegetative growth, carbohydrate metabolism, and entry into meiosis. How this pathway controls later steps of meiotic development is largely unknown. Here, we have analyzed the role of the Ras/cAMP/PKA pathway in spore formation by the meiosis-specific manipulation of Ras and PKA or by the disturbance of cAMP production. We found that the regulation of spore formation by acetate takes place after commitment to meiosis and depends on PKA and appropriate A kinase activation by Ras/Cyr1 adenylyl cyclase but not by activation through the Gpa2/Gpr1 branch. We further discovered that spore formation is regulated by carbon dioxide/bicarbonate, and an analysis of mutants defective in acetate transport (ady2Δ) or carbonic anhydrase (nce103Δ) provided evidence that these metabolites are involved in connecting the nutritional state of the meiotic cell to spore number control. Finally, we observed that the potential PKA target Ady1 is required for the proper localization of the meiotic plaque proteins Mpc70 and Spo74 at spindle pole bodies and for the ability of these proteins to initiate spore formation. Overall, our investigation suggests that the Ras/cAMP/PKA pathway plays a crucial role in the regulation of spore formation by acetate and indicates that the control of meiotic development by this signaling cascade takes places at several steps and is more complex than previously anticipated. PMID:22660623

  17. Targeting the PI3K/Akt pathway in murine MDS/MPN driven by hyperactive Ras

    PubMed Central

    Akutagawa, Jon; Huang, Tannie Q.; Epstein, Inbal; Chang, Tiffany; Quirindongo-Crespo, Maricel; Cottonham, Charisa L.; Dail, Monique; Slusher, Barbara S.; Friedman, Lori S.; Sampath, Deepak; Braun, Benjamin S.

    2016-01-01

    Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are myelodysplastic/myeloproliferative neoplasia (MDS/MPN) overlap syndromes that respond poorly to conventional treatments. Aberrant Ras activation due to NRAS, KRAS, PTPN11, CBL, and NF1 mutations is common in CMML and JMML. However, no mechanism-based treatments currently exist for cancers with any of these mutations. An alternative therapeutic strategy involves targeting Ras-regulated effector pathways that are aberrantly activated in CMML and JMML, which include the Raf/MEK/ERK and phosphoinositide-3´-OH kinase (PI3K)/Akt cascades. Mx1-Cre, KrasD12 and Mx1-Cre, Nf1flox/− mice accurately model many aspects of CMML and JMML. Treating Mx1-Cre, KrasD12 mice with GDC-0941 (also referred to as pictilisib), an orally bioavailable inhibitor of class I PI3K isoforms, reduced leukocytosis, anemia, and splenomegaly while extending survival. However, GDC-0941 treatment attenuated activation of both PI3K/Akt and Raf/MEK/ERK pathways in primary hematopoietic cells, suggesting it could be acting through suppression of Raf/MEK/ERK signals. To interrogate the importance of the PI3K/Akt pathway specifically, we treated mice with the allosteric Akt inhibitor MK-2206. This compound had no effect on Raf/MEK/ERK signaling, yet it also induced robust hematologic responses in Kras and Nf1 mice with MPN. These data support investigating PI3K/Akt pathway inhibitors as a therapeutic strategy in JMML and CMML patients. PMID:26965285

  18. Advances in the development of cancer therapeutics directed against the RAS-mitogen-activated protein kinase pathway.

    PubMed

    Sebolt-Leopold, Judith S

    2008-06-15

    Among mammalian mitogen-activated protein kinase (MAPK) signaling cascades, the extracellular signal-related kinase (ERK) pathway has received the most attention in the oncology drug discovery arena. By virtue of its central role in promoting proliferation, survival, and metastasis, this pathway directly affects both the formation and progression of human tumors. The identification of non-ATP-competitive inhibitors of the MAPK kinase MAPK/ERK kinase (MEK) resulted in the first demonstration that the ERK pathway could be effectively shut down in a highly selective fashion. Subsequent discovery of the oncogenic nature of B-raf kinase led to the escalation of drug discovery efforts revolving around MEK and RAF. The emergence of multiple drug candidates targeting these downstream kinases provides us with the means for validating the importance of the RAS-RAF-MEK-ERK signaling cascade in human tumors. This article highlights the lessons learned in the clinical evaluation of MAPK pathway inhibitors as anticancer agents and the complexities surrounding optimization of their therapeutic potential in light of the challenges posed by genetic heterogeneity within patient populations.

  19. New genes that interact with lin-35 Rb to negatively regulate the let-60 ras pathway in Caenorhabditis elegans.

    PubMed Central

    Thomas, Jeffrey H; Ceol, Craig J; Schwartz, Hillel T; Horvitz, H Robert

    2003-01-01

    Previous studies have shown that a synthetic multivulva phenotype results from mutations in genes that antagonize the ras-mediated intercellular signaling system responsible for vulval induction in Caenorhabditis elegans. Synthetic multivulva mutations define two classes of genes, A and B, and a mutation in a gene of each class is required to produce the multivulva phenotype. The ectopic vulval tissue in multivulva animals is generated by vulval precursor cells that in the wild type do not generate vulval tissue. One of the class B synthetic multivulva genes, lin-35, encodes a protein similar to the retinoblastoma (Rb) protein. In this article, we describe the isolation and characterization of 50 synthetic multivulva mutations, the identification of new components of both the class A and class B lin-35 Rb pathways, and the cloning of lin-52, a class B gene that may have a conserved role in Rb-mediated signaling. PMID:12750327

  20. CNS germinomas are characterized by global demethylation, chromosomal instability and mutational activation of the Kit-, Ras/Raf/Erk- and Akt-pathways

    PubMed Central

    Schulte, Simone Laura; Waha, Andreas; Steiger, Barbara; Denkhaus, Dorota; Dörner, Evelyn; Calaminus, Gabriele; Leuschner, Ivo; Pietsch, Torsten

    2016-01-01

    CNS germinomas represent a unique germ cell tumor entity characterized by undifferentiated tumor cells and a high response rate to current treatment protocols. Limited information is available on their underlying genomic, epigenetic and biological alterations. We performed a genome-wide analysis of genomic copy number alterations in 49 CNS germinomas by molecular inversion profiling. In addition, CpG dinucleotide methylation was studied by immunohistochemistry for methylated cytosine residues. Mutational analysis was performed by resequencing of candidate genes including KIT and RAS family members. Ras/Erk and Akt pathway activation was analyzed by immunostaining with antibodies against phospho-Erk, phosho-Akt, phospho-mTOR and phospho-S6. All germinomas coexpressed Oct4 and Kit but showed an extensive global DNA demethylation compared to other tumors and normal tissues. Molecular inversion profiling showed predominant genomic instability in all tumors with a high frequency of regional gains and losses including high level gene amplifications. Activating mutations of KIT exons 11, 13, and 17 as well as a case with genomic KIT amplification and activating mutations or amplifications of RAS gene family members including KRAS, NRAS and RRAS2 indicated mutational activation of crucial signaling pathways. Co-activation of Ras/Erk and Akt pathways was present in 83% of germinomas. These data suggest that CNS germinoma cells display a demethylated nuclear DNA similar to primordial germ cells in early development. This finding has a striking coincidence with extensive genomic instability. In addition, mutational activation of Kit-, Ras/Raf/Erk- and Akt- pathways indicate the biological importance of these pathways and their components as potential targets for therapy. PMID:27391150

  1. Pipoxolan Ameliorates Cerebral Ischemia via Inhibition of Neuronal Apoptosis and Intimal Hyperplasia through Attenuation of VSMC Migration and Modulation of Matrix Metalloproteinase-2/9 and Ras/MEK/ERK Signaling Pathways

    PubMed Central

    Chen, Yuh-Fung; Tsai, Huei-Yann; Wu, Kuo-Jen; Siao, Lian-Ru; Wood, W. Gibson

    2013-01-01

    Pipoxolan (PIPO) has anti-spasmodic effects, and it is used clinically to relieve smooth muscle spasms. Cerebrovascular disease is one of the leading causes of disability and death worldwide. The main aim of this study was to investigate the effects of PIPO on cerebral ischemia and vascular smooth muscle cell (VSMC) migration in vivo and in vitro. Cerebral infarction area, ratio of intima to media area (I/M ratio) and PCNA antibody staining of the carotid artery in vivo were measured. Cell viability of A7r5 cells, PDGF-BB-stimulated cell migration, and potential mechanisms of PIPO were evaluated by wound healing, transwell and Western blotting. PIPO (10 and 30 mg/kg p.o.) reduced: the cerebral infarction area; neurological deficit; TUNEL-positive cells; cleaved caspase 3-positive cells; intimal hyperplasia; and inhibited proliferating cell nuclear antigen (PCNA)-positive cells in rodents. PIPO (5, 10 and 15 µM) significantly inhibited PDGF-BB-stimulated VSMC migration and reduced Ras, MEK, and p-ERK levels. Moreover, PIPO decreased levels of matrix metalloproteinases -2 and -9 in PDGF-BB-stimulated A7r5 cells. In summary, PIPO is protective in models of ischemia/reperfusion-induced cerebral infarction, carotid artery ligation-induced intimal hyperplasia and VSMC migration both in vivo and in vitro. PIPO could be potentially efficacious in preventing cerebrovascular and vascular diseases. PMID:24086601

  2. Pipoxolan ameliorates cerebral ischemia via inhibition of neuronal apoptosis and intimal hyperplasia through attenuation of VSMC migration and modulation of matrix metalloproteinase-2/9 and Ras/MEK/ERK signaling pathways.

    PubMed

    Chen, Yuh-Fung; Tsai, Huei-Yann; Wu, Kuo-Jen; Siao, Lian-Ru; Wood, W Gibson

    2013-01-01

    Pipoxolan (PIPO) has anti-spasmodic effects, and it is used clinically to relieve smooth muscle spasms. Cerebrovascular disease is one of the leading causes of disability and death worldwide. The main aim of this study was to investigate the effects of PIPO on cerebral ischemia and vascular smooth muscle cell (VSMC) migration in vivo and in vitro. Cerebral infarction area, ratio of intima to media area (I/M ratio) and PCNA antibody staining of the carotid artery in vivo were measured. Cell viability of A7r5 cells, PDGF-BB-stimulated cell migration, and potential mechanisms of PIPO were evaluated by wound healing, transwell and Western blotting. PIPO (10 and 30 mg/kg p.o.) reduced: the cerebral infarction area; neurological deficit; TUNEL-positive cells; cleaved caspase 3-positive cells; intimal hyperplasia; and inhibited proliferating cell nuclear antigen (PCNA)-positive cells in rodents. PIPO (5, 10 and 15 µM) significantly inhibited PDGF-BB-stimulated VSMC migration and reduced Ras, MEK, and p-ERK levels. Moreover, PIPO decreased levels of matrix metalloproteinases -2 and -9 in PDGF-BB-stimulated A7r5 cells. In summary, PIPO is protective in models of ischemia/reperfusion-induced cerebral infarction, carotid artery ligation-induced intimal hyperplasia and VSMC migration both in vivo and in vitro. PIPO could be potentially efficacious in preventing cerebrovascular and vascular diseases.

  3. Activation of the Src/p21ras/Erk pathway by progesterone receptor via cross-talk with estrogen receptor.

    PubMed Central

    Migliaccio, A; Piccolo, D; Castoria, G; Di Domenico, M; Bilancio, A; Lombardi, M; Gong, W; Beato, M; Auricchio, F

    1998-01-01

    The molecular mechanisms by which ovarian hormones stimulate growth of breast tumors are unclear. It has been reported previously that estrogens activate the signal-transducing Src/p21(ras)/Erk pathway in human breast cancer cells via an interaction of estrogen receptor (ER) with c-Src. We now show that progestins stimulate human breast cancer T47D cell proliferation and induce a similar rapid and transient activation of the pathway which, surprisingly, is blocked not only by anti-progestins but also by anti-estrogens. In Cos-7 cells transfected with the B isoform of progesterone receptor (PRB), progestin activation of the MAP kinase pathway depends on co-transfection of ER. A transcriptionally inactive PRB mutant also activates the signaling pathway, demonstrating that this activity is independent of transcriptional effects. PRB does not interact with c-Src but associates via the N-terminal 168 amino acids with ER. This association is required for the signaling pathway activation by progestins. We propose that ER transmits to the Src/p21(ras)/Erk pathway signals received from the agonist-activated PRB. These findings reveal a hitherto unrecognized cross-talk between ovarian hormones which could be crucial for their growth-promoting effects on cancer cells. PMID:9524123

  4. Regulation of Hxt3 and Hxt7 turnover converges on the Vid30 complex and requires inactivation of the Ras/cAMP/PKA pathway in Saccharomyces cerevisiae.

    PubMed

    Snowdon, Chris; van der Merwe, George

    2012-01-01

    Eukaryotic cells adjust their intracellular protein complement as a mechanism to adapt to changing environmental signals. In Saccharomyces cerevisiae the hexose transporters Hxt3 and Hxt7 are expressed and function on the plasma membrane in high and low glucose abundance, respectively. By contrast, Hxt3 is endocytosed and degraded in the vacuole when cells are starved of glucose and Hxt7 in response to rapamycin treatment or when nitrogen is limiting. Yeast uses several signaling pathways, including the TORC1 and Ras/cAMP/Protein Kinase A (PKA) pathways, to adapt to nutrient changes in the environment. The multi-protein Vid30 complex (Vid30c), an E3 ubiquitin ligase required for the degradation of FBPase, assists in this adaptation process in a mechanism that is poorly understood. Here we show the endocytosis and the subsequent degradation of both Hxt3 and Hxt7, in response to different nutrient signals, is dependent on components of the Vid30c. Additionally, we define the signaling events required for the turnover of Hxt3 and Hxt7 by showing that Hxt3 turnover requires Ras2 and PKA inactivation, whereas Hxt7 turnover requires TORC1 and Ras2 inactivation. Further investigation led us to identify Rim15, a kinase that is inhibited by both the TORC1 and Ras/cAMP/PKA pathways, as a key downstream effector in signaling both turnover events. Finally, we show that the turnover of both Hxt3 and Hxt7 is dependent on the essential E3 ubiquitin ligase, Rsp5, indicating that the role of the Vid30c might be indirect of Hxt ubiquitylation.

  5. Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway

    PubMed Central

    Goodwin, J. Shawn; Drake, Kimberly R.; Rogers, Carl; Wright, Latasha; Lippincott-Schwartz, Jennifer; Philips, Mark R.; Kenworthy, Anne K.

    2005-01-01

    Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes. PMID:16027222

  6. RAS and Hedgehog--partners in crime.

    PubMed

    Lauth, Matthias

    2011-06-01

    Both RAS and Hedgehog (HH) pathway activation can be found in approximately one third of all cancers. In many cases, this activation occurs in the same tumor types, suggesting a positive impact of a simultaneous activation of RAS and HH on tumor development. This review aims to summarize the current knowledge about the molecular and functional crosstalk of RAS and HH signaling in the development of hyperproliferative disease.

  7. Ras-Guanine Nucleotide-Releasing Factor 1 (Ras-GRF1) Controls Activation of Extracellular Signal-Regulated Kinase (ERK) Signaling in the Striatum and Long-Term Behavioral Responses to Cocaine

    PubMed Central

    Fasano, Stefania; D’Antoni, Angela; Orban, Paul C.; Valjent, Emmanuel; Putignano, Elena; Vara, Hugo; Pizzorusso, Tommaso; Giustetto, Maurizio; Yoon, Bongjune; Soloway, Paul; Maldonado, Rafael; Caboche, Jocelyne; Brambilla, Riccardo

    2010-01-01

    Background Ras-extracellular signal-regulated kinase (Ras-ERK) signaling is central to the molecular machinery underlying cognitive functions. In the striatum, ERK1/2 kinases are co-activated by glutamate and dopamine D1/5 receptors, but the mechanisms providing such signaling integration are still unknown. The Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1), a neuronal specific activator of Ras-ERK signaling, is a likely candidate for coupling these neurotransmitter signals to ERK kinases in the striatonigral medium spiny neurons (MSN) and for modulating behavioral responses to drug abuse such as cocaine. Methods We used genetically modified mouse mutants for Ras-GRF1 as a source of primary MSN cultures and organotypic slices, to perform both immunoblot and immunofluorescence studies in response to glutamate and dopamine receptor agonists. Mice were also subjected to behavioral and immunohistochemical investigations upon treatment with cocaine. Results Phosphorylation of ERK1/2 in response to glutamate, dopamine D1 agonist, or both stimuli simultaneously is impaired in Ras-GRF1– deficient striatal cells and organotypic slices of the striatonigral MSN compartment. Consistently, behavioral responses to cocaine are also affected in mice deficient for Ras-GRF1 or overexpressing it. Both locomotor sensitization and conditioned place preference are significantly attenuated in Ras-GRF1– deficient mice, whereas a robust facilitation is observed in overexpressing transgenic animals. Finally, we found corresponding changes in ERK1/2 activation and in accumulation of FosB/ΔFosB, a well-characterized marker for long-term responses to cocaine, in MSN from these animals. Conclusions These results strongly implicate Ras-GRF1 in the integration of the two main neurotransmitter inputs to the striatum and in the maladaptive modulation of striatal networks in response to cocaine. PMID:19446794

  8. Beside P53 and PTEN: Identification of molecular alterations of the RAS/MAPK and PI3K/AKT signaling pathways in high-grade serous ovarian carcinomas to determine potential novel therapeutic targets

    PubMed Central

    Chen, Shuhui; Cavazza, Elisa; Barlier, Catherine; Salleron, Julia; Filhine-Tresarrieu, Pierre; Gavoilles, Céline; Merlin, Jean-Louis; Harlé, Alexandre

    2016-01-01

    Despite great histological and molecular heterogeneity, the clinical management of high-grade ovarian carcinomas remains unspecialized. As a major subgroup, high-grade serous ovarian carcinomas (HGSOCs) require novel therapies. In addition to utilizing conventional histological prognostic markers and performing oncogenetic investigations, the molecular diagnostic method of next generation sequencing (NGS) was performed to identify ‘druggable’ targets that could provide access to innovative therapy. The present study was performed in 45 HGSOC patients (mean age, 59.1 years; range, 25–87 years) with histologically proven HGSOC. Breast cancer 1/2 (BRCA1/2) germline mutations were screened in 17 patients with a familial or personal history of cancer, which was justified by oncogenetic investigations. Tumor protein 53 (P53) and phosphatase and tensin homolog (PTEN) expression were assessed in formalin-fixed paraffin-embedded tissues using immunohistochemistry. Somatic mutations of Kirsten rat sarcoma viral oncogene homolog, neuroblastoma RAS viral oncogene homolog (NRAS), B-Raf proto-oncogene, serine/threonine kinase, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA) and MET proto-oncogene, receptor tyrosine kinase (MET) were screened using NGS on DNA extracts from frozen tumor specimens obtained at diagnosis. With a median follow-up of 38 months (range, 6–93 months), 20 patients are alive, 10 patients are disease-free and 14 patients progressed within 6 months following platinum-based therapy. P53 overexpression was detected in 67% of patients and PTEN loss was detected in 38% of the patients. The overexpression of mutant P53 was found to be associated with a longer progression-free and overall survival. In total, 2 NRAS (exon 3), 3 PIK3CA (exon 5 and 10) and 5 MET mutations (exons 14 and 18) were detected. In HGSOCs, in addition to P53 and PTEN alterations, somatic genetic abnormalities can be detected using NGS and provide molecular

  9. Regulation of Ras Exchange Factors and Cellular Localization of Ras Activation by Lipid Messengers in T Cells

    PubMed Central

    Jun, Jesse E.; Rubio, Ignacio; Roose, Jeroen P.

    2013-01-01

    The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs) catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and Son of Sevenless (SOS)-family GEFs. Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood. One large group of biomolecules critically involved in the control of RasGEFs functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR) stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells. PMID:24027568

  10. From Ras to Rap and Back, a Journey of 35 Years.

    PubMed

    Bos, Johannes L

    2017-08-04

    Our laboratory has studied Ras and Ras-like proteins since the discovery of the Ras oncogene 35 years ago. In this review, I will give an account of what we have done in these 35 years and indicate the main papers that have guided our research. Our efforts started with the early analysis of mutant Ras in human tumors followed by deciphering of the role of Ras in signal transduction pathways. In an attempt to interfere in Ras signaling we turned to Rap proteins. These proteins are the closest relatives of Ras and were initially identified as Ras antagonists. However, our studies revealed that the Rap signaling network primarily is involved in spatiotemporal control of cell adhesion, in part through regulation of the actin cytoskeleton. More recently we returned to Ras, trying to interfere in Ras signaling by combinatorial drug testing using the organoid technology. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

  11. Exercise training attenuates renovascular hypertension partly via RAS- ROS- glutamate pathway in the hypothalamic paraventricular nucleus.

    PubMed

    Zhang, Yan; Yu, Xiao-Jing; Chen, Wen-Sheng; Gao, Hong-Li; Liu, Kai-Li; Shi, Xiao-Lian; Fan, Xiao-Yan; Jia, Lin-Lin; Cui, Wei; Zhu, Guo-Qing; Liu, Jin-Jun; Kang, Yu-Ming

    2016-11-24

    Exercise training (ExT) has been reported to benefit hypertension; however, the exact mechanisms involved are unclear. We hypothesized that ExT attenuates hypertension, in part, through the renin-angiotensin system (RAS), reactive oxygen species (ROS), and glutamate in the paraventricular nucleus (PVN). Two-kidney, one-clip (2K1C) renovascular hypertensive rats were assigned to sedentary (Sed) or treadmill running groups for eight weeks. Dizocilpine (MK801), a glutamate receptor blocker, or losartan (Los), an angiotensin II type1 receptor (AT1-R) blocker, were microinjected into the PVN at the end of the experiment. We found that 2K1C rats had higher mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). These rats also had excessive oxidative stress and overactivated RAS in PVN. Eight weeks of ExT significantly decreased MAP and RSNA in 2K1C hypertensive rats. ExT inhibited angiotensin-converting enzyme (ACE), AT1-R, and glutamate in the PVN, and angiotensin II (ANG II) in the plasma. Moreover, ExT attenuated ROS by augmenting copper/zinc superoxide dismutase (Cu/Zn-SOD) and decreasing p(47phox) and gp(91phox) in the PVN. MK801or Los significantly decreased blood pressure in rats. Together, these findings suggest that the beneficial effects of ExT on renovascular hypertension may be, in part, through the RAS-ROS-glutamate pathway in the PVN.

  12. Exercise training attenuates renovascular hypertension partly via RAS- ROS- glutamate pathway in the hypothalamic paraventricular nucleus

    PubMed Central

    Zhang, Yan; Yu, Xiao-Jing; Chen, Wen-Sheng; Gao, Hong-Li; Liu, Kai-Li; Shi, Xiao-Lian; Fan, Xiao-Yan; Jia, Lin-Lin; Cui, Wei; Zhu, Guo-Qing; Liu, Jin-Jun; Kang, Yu-Ming

    2016-01-01

    Exercise training (ExT) has been reported to benefit hypertension; however, the exact mechanisms involved are unclear. We hypothesized that ExT attenuates hypertension, in part, through the renin-angiotensin system (RAS), reactive oxygen species (ROS), and glutamate in the paraventricular nucleus (PVN). Two-kidney, one-clip (2K1C) renovascular hypertensive rats were assigned to sedentary (Sed) or treadmill running groups for eight weeks. Dizocilpine (MK801), a glutamate receptor blocker, or losartan (Los), an angiotensin II type1 receptor (AT1-R) blocker, were microinjected into the PVN at the end of the experiment. We found that 2K1C rats had higher mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). These rats also had excessive oxidative stress and overactivated RAS in PVN. Eight weeks of ExT significantly decreased MAP and RSNA in 2K1C hypertensive rats. ExT inhibited angiotensin-converting enzyme (ACE), AT1-R, and glutamate in the PVN, and angiotensin II (ANG II) in the plasma. Moreover, ExT attenuated ROS by augmenting copper/zinc superoxide dismutase (Cu/Zn-SOD) and decreasing p47phox and gp91phox in the PVN. MK801or Los significantly decreased blood pressure in rats. Together, these findings suggest that the beneficial effects of ExT on renovascular hypertension may be, in part, through the RAS-ROS-glutamate pathway in the PVN. PMID:27881877

  13. Oncogenic ETS proteins mimic activated RAS/MAPK signaling in prostate cells

    PubMed Central

    Hollenhorst, Peter C.; Ferris, Mary W.; Hull, Megan A.; Chae, Heejoon; Kim, Sun; Graves, Barbara J.

    2011-01-01

    The aberrant expression of an oncogenic ETS transcription factor is implicated in the progression of the majority of prostate cancers, 40% of melanomas, and most cases of gastrointestinal stromal tumor and Ewing's sarcoma. Chromosomal rearrangements in prostate cancer result in overexpression of any one of four ETS transcription factors. How these four oncogenic ETS genes differ from the numerous other ETS genes expressed in normal prostate and contribute to tumor progression is not understood. We report that these oncogenic ETS proteins, but not other ETS factors, enhance prostate cell migration. Genome-wide binding analysis matched this specific biological function to occupancy of a unique set of genomic sites highlighted by the presence of ETS- and AP-1-binding sequences. ETS/AP-1-binding sequences are prototypical RAS-responsive elements, but oncogenic ETS proteins activated a RAS/MAPK transcriptional program in the absence of MAPK activation. Thus, overexpression of oncogenic ETS proteins can replace RAS/MAPK pathway activation in prostate cells. The genomic description of this ETS/AP-1-regulated, RAS-responsive, gene expression program provides a resource for understanding the role of these ETS factors in both an oncogenic setting and the developmental processes where these genes normally function. PMID:22012618

  14. Retroactive Signaling in Short Signaling Pathways

    PubMed Central

    Sepulchre, Jacques-Alexandre; Merajver, Sofía D.; Ventura, Alejandra C.

    2012-01-01

    In biochemical signaling pathways without explicit feedback connections, the core signal transduction is usually described as a one-way communication, going from upstream to downstream in a feedforward chain or network of covalent modification cycles. In this paper we explore the possibility of a new type of signaling called retroactive signaling, offered by the recently demonstrated property of retroactivity in signaling cascades. The possibility of retroactive signaling is analysed in the simplest case of the stationary states of a bicyclic cascade of signaling cycles. In this case, we work out the conditions for which variables of the upstream cycle are affected by a change of the total amount of protein in the downstream cycle, or by a variation of the phosphatase deactivating the same protein. Particularly, we predict the characteristic ranges of the downstream protein, or of the downstream phosphatase, for which a retroactive effect can be observed on the upstream cycle variables. Next, we extend the possibility of retroactive signaling in short but nonlinear signaling pathways involving a few covalent modification cycles. PMID:22848403

  15. C. elegans La-related protein, LARP-1, localizes to germline P bodies and attenuates Ras-MAPK signaling during oogenesis.

    PubMed

    Nykamp, Keith; Lee, Myon-Hee; Kimble, Judith

    2008-07-01

    RNA regulators are critical for animal development, especially in the germ line where gene expression is often modulated by changes in mRNA stability, translation, and localization. In this paper, we focus on Caenorhabditis elegans LARP-1, a representative of one La-related protein (Larp) family found broadly among eukaryotes. LARP-1 possesses a signature La motif, which is an ancient RNA-binding domain, plus a second conserved motif, typical of LARP-1 homologs and therefore dubbed the LARP1 domain. LARP-1 appears to bind RNA in vitro via both the La motif and the LARP1 domain. larp-1 null mutants have an oogenesis defect reminiscent of hyperactive Ras-MAPK signaling; this defect is suppressed or enhanced by down- or up-regulating the Ras-MAPK pathway, respectively. Consistent with a role in down-regulating the Ras-MAPK pathway, larp-1 null mutants have higher than normal levels of selected pathway mRNAs and proteins. LARP-1 protein colocalizes with P bodies, which function in RNA degradation. We suggest that LARP-1 functions in P bodies to attenuate the abundance of conserved Ras-MAPK mRNAs. We also propose that the cluster of LARP-1 homologs may function generally to control the expression of key developmental regulators.

  16. C. elegans La-related protein, LARP-1, localizes to germline P bodies and attenuates Ras-MAPK signaling during oogenesis

    PubMed Central

    Nykamp, Keith; Lee, Myon-Hee; Kimble, Judith

    2008-01-01

    RNA regulators are critical for animal development, especially in the germ line where gene expression is often modulated by changes in mRNA stability, translation, and localization. In this paper, we focus on Caenorhabditis elegans LARP-1, a representative of one La-related protein (Larp) family found broadly among eukaryotes. LARP-1 possesses a signature La motif, which is an ancient RNA-binding domain, plus a second conserved motif, typical of LARP-1 homologs and therefore dubbed the LARP1 domain. LARP-1 appears to bind RNA in vitro via both the La motif and the LARP1 domain. larp-1 null mutants have an oogenesis defect reminiscent of hyperactive Ras-MAPK signaling; this defect is suppressed or enhanced by down- or up-regulating the Ras-MAPK pathway, respectively. Consistent with a role in down-regulating the Ras-MAPK pathway, larp-1 null mutants have higher than normal levels of selected pathway mRNAs and proteins. LARP-1 protein colocalizes with P bodies, which function in RNA degradation. We suggest that LARP-1 functions in P bodies to attenuate the abundance of conserved Ras-MAPK mRNAs. We also propose that the cluster of LARP-1 homologs may function generally to control the expression of key developmental regulators. PMID:18515547

  17. Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae.

    PubMed Central

    Mösch, H U; Roberts, R L; Fink, G R

    1996-01-01

    RAS2val19, a dominant activated form of Saccharomyces cerevisiae Ras2, stimulates both filamentous growth and expression of a transcriptional reporter FG(TyA)::lacZ but does not induce the mating pathway reporter FUS1::lacZ. This induction depends upon elements of the conserved mitogen-activated protein kinase (MAPK) pathway that is required for both filamentous growth and mating, two distinct morphogenetic events. Full induction requires Ste20 (homolog of mammalian p65PAK protein kinases), Ste11 [an MEK kinase (MEKK) or MAPK kinase (MEK) kinase], Ste7 (MEK or MAPK kinase), and the transcription factor Ste12. Moreover, the Rho family protein Cdc42, a conserved morphogenetic G protein, is also a potent regulator of filamentous growth and FG(TyA)::lacZ expression in S. cerevisiae. Stimulation of both filamentous growth and FG(TyA)::lacZ by Cdc42 depends upon Ste20. In addition, dominant negative CDC42Ala118 blocks RAS2val19 activation, placing Cdc42 downstream of Ras2. Our results suggest that filamentous growth in budding yeast is regulated by an evolutionarily conserved signaling pathway that controls cell morphology. Images Fig. 1 Fig. 2 Fig. 3 PMID:8643578

  18. The Hominoid-specific Oncogene TBC1D3 Activates Ras and Modulates Epidermal Growth Factor Receptor Signaling and Trafficking*S⃞

    PubMed Central

    Wainszelbaum, Marisa J.; Charron, Audra J.; Kong, Chen; Kirkpatrick, Donald S.; Srikanth, Priya; Barbieri, M. Alejandro; Gygi, Steven P.; Stahl, Philip D.

    2008-01-01

    Hominoid- and human-specific genes may have evolved to modulate signaling pathways of a higher order of complexity. TBC1D3 is a hominoid-specific oncogene encoded by a cluster of eight paralogs on chromosome 17. Initial work indicates that TBC1D3 is widely expressed in human tissues (Hodzic, D., Kong, C., Wainszelbaum, M. J., Charron, A. J., Su, X., and Stahl, P. D. (2006) Genomics 88,731 -73616863688). In this study, we show that TBC1D3 expression has a powerful effect on cell proliferation that is further enhanced by epidermal growth factor (EGF) in both human and mouse cell lines. EGF activation of the Erk and protein kinase B/Akt pathways is enhanced, both in amplitude and duration, by TBC1D3 expression, whereas RNA interference silencing of TBC1D3 suppresses the activation. Light microscopy and Western blot experiments demonstrate that increased signaling in response to EGF is coupled with a significant delay in EGF receptor (EGFR) trafficking and degradation, which significantly extends the life span of EGFR. Moreover, TBC1D3 suppresses polyubiquitination of the EGFR and the recruitment of c-Cbl. Using the Ras binding domain of Raf1 to monitor GTP-Ras we show that TBC1D3 expression enhances Ras activation in quiescent cells, which is further increased by EGF treatment. We speculate that TBC1D3 may alter Ras GTP loading. We conclude that the expression of TBC1D3 generates a delay in EGFR degradation, a decrease in ubiquitination, and a failure to recruit adapter proteins that ultimately dysregulate EGFR signal transduction and enhance cell proliferation. Altered growth factor receptor trafficking and GTP-Ras turnover may be sites where recently evolved genes such as TBC1D3 selectively modulate signaling in hominoids and humans. PMID:18319245

  19. Regulation of maltose utilization in Saccharomyces cerevisiae by genes of the RAS/protein kinase A pathway.

    PubMed

    Wanke, V; Vavassori, M; Thevelein, J M; Tortora, P; Vanoni, M

    1997-02-03

    In Saccharomyces cerevisiae maltose utilization requires a functional MAL locus, each composed of three genes: MALR (gene 3) encoding a regulatory protein, MALT (gene 1) encoding maltose permease and MALS (gene 2) encoding maltase. We show that constitutive activation of the RAS/protein kinase A pathway severely reduces growth of MAL1 strains on maltose. This may be a consequence of reduction in MALT mRNA, reduced Vmax and increased catabolite inactivation of the MALT-encoded maltose transporter in the MAL1 strain. Mutations in the GGS1/TPS1 gene, which restricts glucose influx and possibly affects signalling, relieve carbon catabolite repression on both maltase and maltose permease and reduce maltose permease inactivation.

  20. Induction of the cholesterol metabolic pathway regulates the farnesylation of RAS in embryonic chick heart cells: a new role for ras in regulating the expression of muscarinic receptors and G proteins.

    PubMed Central

    Gadbut, A P; Wu, L; Tang, D; Papageorge, A; Watson, J A; Galper, J B

    1997-01-01

    We propose a novel mechanism for the regulation of the processing of Ras and demonstrate a new function for Ras in regulating the expression of cardiac autonomic receptors and their associated G proteins. We have demonstrated previously that induction of endogenous cholesterol synthesis in cultured cardiac myocytes resulted in a coordinated increase in expression of muscarinic receptors, the G protein alpha-subunit, G-alphai2, and the inward rectifying K+ channel, GIRK1. These changes in gene expression were associated with a marked increase in the response of heart cells to parasympathetic stimulation. In this study, we demonstrate that the induction of the cholesterol metabolic pathway regulates Ras processing and that Ras regulates expression of G-alphai2. We show that in primary cultured myocytes most of the RAS is localized to the cytoplasm in an unfarnesylated form. Induction of the cholesterol metabolic pathway results in increased farnesylation and membrane association of RAS. Studies of Ras mutants expressed in cultured heart cells demonstrate that activation of Ras by induction of the cholesterol metabolic pathway results in increased expression of G-alphai2 mRNA. Hence farnesylation of Ras is a regulatable process that plays a novel role in the control of second messenger pathways. PMID:9405354

  1. Signaling on the endocytic pathway.

    PubMed

    McPherson, P S; Kay, B K; Hussain, N K

    2001-06-01

    Ligand binding to receptor tyrosine kinases and G-protein-coupled receptors initiates signal transduction events and induces receptor endocytosis via clathrin-coated pits and vesicles. While receptor-mediated endocytosis has been traditionally considered an effective mechanism to attenuate ligand-activated responses, more recent studies demonstrate that signaling continues on the endocytic pathway. In fact, certain signaling events, such as the activation of the extracellular signal-regulated kinases, appear to require endocytosis. Protein components of signal transduction cascades can assemble at clathrin coated pits and remain associated with endocytic vesicles following their dynamin-dependent release from the plasma membrane. Thus, endocytic vesicles can function as a signaling compartment distinct from the plasma membrane. These observations demonstrate that endocytosis plays an important role in the activation and propagation of signaling pathways.

  2. Lipid-Sorting Specificity Encoded in K-Ras Membrane Anchor Regulates Signal Output.

    PubMed

    Zhou, Yong; Prakash, Priyanka; Liang, Hong; Cho, Kwang-Jin; Gorfe, Alemayehu A; Hancock, John F

    2017-01-12

    K-Ras is targeted to the plasma membrane by a C-terminal membrane anchor that comprises a farnesyl-cysteine-methyl-ester and a polybasic domain. We used quantitative spatial imaging and atomistic molecular dynamics simulations to examine molecular details of K-Ras plasma membrane binding. We found that the K-Ras anchor binds selected plasma membrane anionic lipids with defined head groups and lipid side chains. The precise amino acid sequence and prenyl group define a combinatorial code for lipid binding that extends beyond simple electrostatics; within this code lysine and arginine residues are non-equivalent and prenyl chain length modifies nascent polybasic domain lipid preferences. The code is realized by distinct dynamic tertiary structures of the anchor on the plasma membrane that govern amino acid side-chain-lipid interactions. An important consequence of this specificity is the ability of such anchors when aggregated to sort subsets of phospholipids into nanoclusters with defined lipid compositions that determine K-Ras signaling output.

  3. BRAF inhibitors induce metastasis in RAS mutant or inhibitor-resistant melanoma cells by reactivating MEK and ERK signaling.

    PubMed

    Sanchez-Laorden, Berta; Viros, Amaya; Girotti, Maria Romina; Pedersen, Malin; Saturno, Grazia; Zambon, Alfonso; Niculescu-Duvaz, Dan; Turajlic, Samra; Hayes, Andrew; Gore, Martin; Larkin, James; Lorigan, Paul; Cook, Martin; Springer, Caroline; Marais, Richard

    2014-03-25

    Melanoma is a highly metastatic and lethal form of skin cancer. The protein kinase BRAF is mutated in about 40% of melanomas, and BRAF inhibitors improve progression-free and overall survival in these patients. However, after a relatively short period of disease control, most patients develop resistance because of reactivation of the RAF-ERK (extracellular signal-regulated kinase) pathway, mediated in many cases by mutations in RAS. We found that BRAF inhibition induces invasion and metastasis in RAS mutant melanoma cells through a mechanism mediated by the reactivation of the MEK (mitogen-activated protein kinase kinase)-ERK pathway, increased expression and secretion of interleukin 8, and induction of protease-dependent invasion. These events were accompanied by a cell morphology switch from predominantly rounded to predominantly elongated cells. We also observed similar responses in BRAF inhibitor-resistant melanoma cells. These data show that BRAF inhibitors can induce melanoma cell invasion and metastasis in tumors that develop resistance to these drugs.

  4. CrkII signals from epidermal growth factor receptor to Ras.

    PubMed Central

    Kizaka-Kondoh, S; Matsuda, M; Okayama, H

    1996-01-01

    A rat fibroblast mutant defective in oncogenic transformation and signaling from epidermal growth factor receptor to Ras has been isolated. The mutant contains dominant negative-type point mutations in the C-terminal SH3 domain of one crkII gene. Among the adapters tested, the mutant is complemented only by crkII cDNA. Expression of the mutated crkII in parent cells generates the phenotype indistinguishable from the mutant cell. Yet overexpression or reduced expression of Grb2 in the mutant before and after complementation with crkII have little effect on its phenotype. We conclude that adapter molecules are highly specific and that the oncogenic growth signal from epidermal growth factor receptor to Ras is predominantly mediated by CrkII in rat fibroblast. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8901553

  5. EGF RECEPTOR SIGNALING IS ESSENTIAL FOR K-RAS ONCOGENE-DRIVEN PANCREATIC DUCTAL ADENOCARCINOMA

    PubMed Central

    Navas, Carolina; Hernández-Porras, Isabel; Schuhmacher, Alberto J; Sibilia, Maria; Guerra, Carmen; Barbacid, Mariano

    2013-01-01

    Clinical evidence indicates that mutation/activation of EGF receptors (EGFRs) is mutually exclusive with the presence of K-RAS oncogenes in lung and colon tumors. We have validated these observations using genetically engineered mouse models. However, pancreatic ductal adenocarcinomas driven by K-Ras oncogenes are totally dependent on EGFR signaling. Similar results were obtained using human pancreatic tumor cell lines. EGFRs were also essential even in the context of pancreatic injury and absence of p16Ink4a/p19Arf. Only loss of p53 made pancreatic tumors independent of EGFR signaling. Additional inhibition of PI3K and STAT3 effectively prevented proliferation of explants derived from these p53–defective pancreatic tumors. These findings may provide the bases for more rational approaches to treat pancreatic tumors in the clinic. PMID:22975375

  6. Requirement for Ras/Raf/ERK pathway in naringin-induced G1-cell-cycle arrest via p21WAF1 expression.

    PubMed

    Kim, Dong-Il; Lee, Se-Jung; Lee, Soo-Bok; Park, Keerang; Kim, Wun-Jae; Moon, Sung-Kwon

    2008-09-01

    Naringin, an active flavonoid found in citrus fruit extracts, has pharmacological utility. The present study identified a novel mechanism of the anticancer effects of naringin in urinary bladder cancer cells. Naringin treatment resulted in significant dose-dependent growth inhibition together with G(1)-phase cell-cycle arrest at a dose of 100 microM (the half maximal inhibitory concentration) in 5637 cells. In addition, naringin treatment strongly induced p21WAF1 expression, independent of the p53 pathway, and downregulated expression of cyclins and cyclin dependent kinases (CDKs). Moreover, treatment with naringin induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase and c-Jun N-terminal kinase. Among the pathways examined, only PD98059, an ERK-specific inhibitor, blocked naringin-dependent p21WAF1 expression. Consistently, blockade of ERK function reversed naringin-mediated inhibition of cell proliferation and decreased cell-cycle proteins. Furthermore, naringin treatment increased both Ras and Raf activation. Transfection of cells with dominant-negative Ras (RasN17) and Raf (RafS621A) mutant genes suppressed naringin-induced ERK activity and p21WAF1 expression. Finally, the naringin-induced reduction in cell proliferation and cell-cycle proteins also was abolished in the presence of RasN17 and RafS621A mutant genes. These data demonstrate that the Ras/Raf/ERK pathway participates in p21WAF1 induction, subsequently leading to a decrease in the levels of cyclin D1/CDK4 and cyclin E-CDK2 complexes and naringin-dependent inhibition of cell growth. Overall, these unexpected findings concerning the molecular mechanisms of naringin in 5637 cancer cells provide a theoretical basis for the therapeutic use of flavonoids to treat malignancies.

  7. The equilibrium between antagonistic signaling pathways determines the number of synapses in Drosophila

    PubMed Central

    Casas-Tintó, Sergio; Acebes, Ángel

    2017-01-01

    The number of synapses is a major determinant of behavior and many neural diseases exhibit deviations in that number. However, how signaling pathways control this number is still poorly understood. Using the Drosophila larval neuromuscular junction, we show here a PI3K-dependent pathway for synaptogenesis which is functionally connected with other previously known elements including the Wit receptor, its ligand Gbb, and the MAPkinases cascade. Based on epistasis assays, we determined the functional hierarchy within the pathway. Wit seems to trigger signaling through PI3K, and Ras85D also contributes to the initiation of synaptogenesis. However, contrary to other signaling pathways, PI3K does not require Ras85D binding in the context of synaptogenesis. In addition to the MAPK cascade, Bsk/JNK undergoes regulation by Puc and Ras85D which results in a narrow range of activity of this kinase to determine normalcy of synapse number. The transcriptional readout of the synaptogenesis pathway involves the Fos/Jun complex and the repressor Cic. In addition, we identified an antagonistic pathway that uses the transcription factors Mad and Medea and the microRNA bantam to down-regulate key elements of the pro-synaptogenesis pathway. Like its counterpart, the anti-synaptogenesis signaling uses small GTPases and MAPKs including Ras64B, Ras-like-a, p38a and Licorne. Bantam downregulates the pro-synaptogenesis factors PI3K, Hiw, Ras85D and Bsk, but not AKT. AKT, however, can suppress Mad which, in conjunction with the reported suppression of Mad by Hiw, closes the mutual regulation between both pathways. Thus, the number of synapses seems to result from the balanced output from these two pathways. PMID:28892511

  8. The Role of Notch Signaling Pathway in Breast Cancer Pathogenesis

    DTIC Science & Technology

    2004-07-01

    coexpression of a constitutively active form of Notch1 in immortalized breast epithelial HMLE cells expressing low levels of oncogenic Ras rendered them...the Notch-Ras pathway interaction revealed that nuclear localization of Notch1 is essential for this cooperation. Dissection of Ras-pathways using the...activates Raf/MAPK pathway, formed efficient colonies with activated Notch1 . Interestingly, I found that expression of activated Notch1 rendered the

  9. Past, Present, and Future of Targeting Ras for Cancer Therapies.

    PubMed

    Tan, Zhi; Zhang, Shuxing

    2016-01-01

    For decades, mutant Ras (mut-Ras) proteins have been identified as drivers of multiple cancers including pancreatic, lung, and colon cancers. However, targeting this oncogene has been challenging and no Ras inhibitors are on the market to date. Lately several candidates targeting the downstream pathways of Ras signaling, including PI3K and Raf, were approved for cancer treatment. However, they do not present promising therapeutic effects on patients harboring Ras mutations. Recently, a variety of compounds have been reported to impair the activity of Ras, and these exciting discoveries reignite the hope for development of novel drugs targeting mut-Ras. In this article, we will review the progress made in this field and the current state-of-the-art technologies to develop Ras inhibitors. Also we will discuss the future direction of targeting Ras.

  10. Urinary excretion of RAS, BMP, and WNT pathway components in diabetic kidney disease

    PubMed Central

    Afkarian, Maryam; Hirsch, Irl B.; Tuttle, Katherine R.; Greenbaum, Carla; Himmelfarb, Jonathan; de Boer, Ian H.

    2014-01-01

    Abstract The renin–angiotensin system (RAS), bone morphogenetic protein (BMP), and WNT pathways are involved in pathogenesis of diabetic kidney disease (DKD). This study characterized assays for urinary angiotensinogen (AGT), gremlin‐1, and matrix metalloproteinase 7 (MMP‐7), components of the RAS, BMP, and WNT pathways and examined their excretion in DKD. We measured urine AGT, gremlin‐1, and MMP‐7 in individuals with type 1 diabetes and prevalent DKD (n = 20) or longstanding (n = 61) or new‐onset (n = 10) type 1 diabetes without DKD. These urine proteins were also quantified in type 2 DKD (n = 11) before and after treatment with candesartan. The utilized immunoassays had comparable inter‐ and intra‐assay and intraindividual variation to assays used for urine albumin. Median (IQR) urine AGT concentrations were 226.0 (82.1, 550.3) and 13.0 (7.8, 20.0) μg/g creatinine in type 1 diabetes with and without DKD, respectively (P < 0.001). Median (IQR) urine gremlin‐1 concentrations were 48.6 (14.2, 254.1) and 3.6 (1.7, 5.5) μg/g, respectively (P < 0.001). Median (IQR) urine MMP‐7 concentrations were 6.0 (3.8, 10.5) and 1.0 (0.4, 2.9) μg/g creatinine, respectively (P < 0.001). Treatment with candesartan was associated with a reduction in median (IQR) urine AGT/creatinine from 23.5 (1.6, 105.1) to 2.0 (1.4, 13.7) μg/g, which did not reach statistical significance. Urine gremlin‐1 and MMP‐7 excretion did not decrease with candesartan. In conclusion, DKD is characterized by markedly elevated urine AGT, MMP‐7, and gremlin‐1. AGT decreased in response to RAS inhibition, suggesting that this marker reflects therapeutic response. Urinary components of the RAS, BMP, and WNT pathways may identify risk of DKD and aid development of novel therapeutics. PMID:24793984

  11. Urinary excretion of RAS, BMP, and WNT pathway components in diabetic kidney disease.

    PubMed

    Afkarian, Maryam; Hirsch, Irl B; Tuttle, Katherine R; Greenbaum, Carla; Himmelfarb, Jonathan; de Boer, Ian H

    2014-01-01

    Abstract The renin-angiotensin system (RAS), bone morphogenetic protein (BMP), and WNT pathways are involved in pathogenesis of diabetic kidney disease (DKD). This study characterized assays for urinary angiotensinogen (AGT), gremlin-1, and matrix metalloproteinase 7 (MMP-7), components of the RAS, BMP, and WNT pathways and examined their excretion in DKD. We measured urine AGT, gremlin-1, and MMP-7 in individuals with type 1 diabetes and prevalent DKD (n = 20) or longstanding (n = 61) or new-onset (n = 10) type 1 diabetes without DKD. These urine proteins were also quantified in type 2 DKD (n = 11) before and after treatment with candesartan. The utilized immunoassays had comparable inter- and intra-assay and intraindividual variation to assays used for urine albumin. Median (IQR) urine AGT concentrations were 226.0 (82.1, 550.3) and 13.0 (7.8, 20.0) μg/g creatinine in type 1 diabetes with and without DKD, respectively (P < 0.001). Median (IQR) urine gremlin-1 concentrations were 48.6 (14.2, 254.1) and 3.6 (1.7, 5.5) μg/g, respectively (P < 0.001). Median (IQR) urine MMP-7 concentrations were 6.0 (3.8, 10.5) and 1.0 (0.4, 2.9) μg/g creatinine, respectively (P < 0.001). Treatment with candesartan was associated with a reduction in median (IQR) urine AGT/creatinine from 23.5 (1.6, 105.1) to 2.0 (1.4, 13.7) μg/g, which did not reach statistical significance. Urine gremlin-1 and MMP-7 excretion did not decrease with candesartan. In conclusion, DKD is characterized by markedly elevated urine AGT, MMP-7, and gremlin-1. AGT decreased in response to RAS inhibition, suggesting that this marker reflects therapeutic response. Urinary components of the RAS, BMP, and WNT pathways may identify risk of DKD and aid development of novel therapeutics.

  12. Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling

    PubMed Central

    Kauko, Otto; Laajala, Teemu Daniel; Jumppanen, Mikael; Hintsanen, Petteri; Suni, Veronika; Haapaniemi, Pekka; Corthals, Garry; Aittokallio, Tero; Westermarck, Jukka; Imanishi, Susumu Y.

    2015-01-01

    Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how these proteins functionally interact. Here we have analyzed phosphoproteomes regulated by either RAS or PP2A, by phosphopeptide enrichment followed by mass-spectrometry-based label-free quantification. To allow data normalization in situations where depletion of RAS or PP2A inhibitor CIP2A causes a large uni-directional change in the phosphopeptide abundance, we developed a novel normalization strategy, named pairwise normalization. This normalization is based on adjusting phosphopeptide abundances measured before and after the enrichment. The superior performance of the pairwise normalization was verified by various independent methods. Additionally, we demonstrate how the selected normalization method influences the downstream analyses and interpretation of pathway activities. Consequently, bioinformatics analysis of RAS and CIP2A regulated phosphoproteomes revealed a significant overlap in their functional pathways. This is most likely biologically meaningful as we observed a synergistic survival effect between CIP2A and RAS expression as well as KRAS activating mutations in TCGA pan-cancer data set, and synergistic relationship between CIP2A and KRAS depletion in colony growth assays. PMID:26278961

  13. Signaling Pathways in Melanogenesis

    PubMed Central

    D’Mello, Stacey A. N.; Finlay, Graeme J.; Baguley, Bruce C.; Askarian-Amiri, Marjan E.

    2016-01-01

    Melanocytes are melanin-producing cells found in skin, hair follicles, eyes, inner ear, bones, heart and brain of humans. They arise from pluripotent neural crest cells and differentiate in response to a complex network of interacting regulatory pathways. Melanins are pigment molecules that are endogenously synthesized by melanocytes. The light absorption of melanin in skin and hair leads to photoreceptor shielding, thermoregulation, photoprotection, camouflage and display coloring. Melanins are also powerful cation chelators and may act as free radical sinks. Melanin formation is a product of complex biochemical events that starts from amino acid tyrosine and its metabolite, dopa. The types and amounts of melanin produced by melanocytes are determined genetically and are influenced by a variety of extrinsic and intrinsic factors such as hormonal changes, inflammation, age and exposure to UV light. These stimuli affect the different pathways in melanogenesis. In this review we will discuss the regulatory mechanisms involved in melanogenesis and explain how intrinsic and extrinsic factors regulate melanin production. We will also explain the regulatory roles of different proteins involved in melanogenesis. PMID:27428965

  14. Phorbol ester stimulation of RasGRP1 regulates the sodium-chloride cotransporter by a PKC-independent pathway

    PubMed Central

    Ko, Benjamin; Joshi, Leena M.; Cooke, Leslie L.; Vazquez, Norma; Musch, Mark W.; Hebert, Steven C.; Gamba, Gerardo; Hoover, Robert S.

    2007-01-01

    The sodium-chloride cotransporter (NCC) is the principal salt-absorptive pathway in the mammalian distal convoluted tubule (DCT) and is the site of action of one of the most effective classes of antihypertensive medications, thiazide diuretics. We developed a cell model system to assess NCC function in a mammalian cell line that natively expresses NCC, the mouse DCT (mDCT) cell line. We used this system to study the complex regulation of NCC by the phorbol ester (PE) 12-O-tetradecanoylphorbol-13-acetate (TPA), a diacylglycerol (DAG) analog. It has generally been thought that PEs mediate their effects on transporters through the activation of PKC. However, there are at least five other DAG/PE targets. Here we describe how one of those alternate targets of DAG/PE effects, Ras guanyl-releasing protein 1 (RasGRP1), mediates the PE-induced suppression of function and the surface expression of NCC. Functional assessment of NCC by using thiazide-sensitive 22Na+ uptakes revealed that TPA completely suppresses NCC function. Biotinylation experiments demonstrated that this result was primarily because of decreased surface expression of NCC. Although inhibitors of PKC had no effect on this suppression, MAPK inhibitors completely prevented the TPA effect. RasGRP1 activates the MAPK pathway through activation of the small G protein Ras. Gene silencing of RasGRP1 prevented the PE-mediated suppression of NCC activity, the activation of the H-Ras isoform of Ras, and the activation of ERK1/2 MAPK. This finding confirmed the critical role of RasGRP1 in mediating the PE-induced suppression of NCC activity through the stimulation of the MAPK pathway. PMID:18077438

  15. Propiconazole-enhanced hepatic cell proliferation is associated with dysregulation of the cholesterol biosynthesis pathway leading to activation of Erk1/2 through Ras farnesylation

    SciTech Connect

    Murphy, Lynea A.; Moore, Tanya; Nesnow, Stephen

    2012-04-15

    Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic cholesterol metabolites and bile acids, and transcriptomic studies revealed that genes within the cholesterol biosynthesis, cholesterol metabolism and bile acid biosyntheses pathways were up-regulated. Hepatic cell proliferation was also increased by propiconazole. AML12 immortalized hepatocytes were used to study propiconazole's effects on cell proliferation focusing on the dysregulation of cholesterol biosynthesis and resulting effects on Ras farnesylation and Erk1/2 activation as a primary pathway. Mevalonate, a key intermediate in the cholesterol biosynthesis pathway, increases cell proliferation in several cancer cell lines and tumors in vivo and serves as the precursor for isoprenoids (e.g. farnesyl pyrophosphate) which are crucial in the farnesylation of the Ras protein by farnesyl transferase. Farnesylation targets Ras to the cell membrane where it is involved in signal transduction, including the mitogen-activated protein kinase (MAPK) pathway. In our studies, mevalonic acid lactone (MVAL), a source of mevalonic acid, increased cell proliferation in AML12 cells which was reduced by farnesyl transferase inhibitors (L-744,832 or manumycin) or simvastatin, an HMG-CoA reductase inhibitor, indicating that this cell system responded to alterations in the cholesterol biosynthesis pathway. Cell proliferation in AML12 cells was increased by propiconazole which was reversed by co-incubation with L-744,832 or simvastatin. Increasing concentrations of exogenous cholesterol muted the proliferative effects of propiconazole and the inhibitory effects of L-733,832, results ascribed to reduced stimulation of the endogenous cholesterol biosynthesis pathway. Western blot analysis of subcellular

  16. lin-1 has both positive and negative functions in specifying multiple cell fates induced by Ras/MAP kinase signaling in C. elegans.

    PubMed

    Tiensuu, Teresa; Larsen, Morten Krog; Vernersson, Emma; Tuck, Simon

    2005-10-01

    lin-1 encodes an ETS domain transcription factor that functions downstream of a Ras/MAP kinase pathway mediating induction of the 1 degrees cell fate during vulval development in the C. elegans hermaphrodite. Mutants lacking lin-1 activity display a phenotype similar to that caused by mutations that constitutively activate let-60 Ras consistent with a model in which lin-1 is a repressor of the 1 degree fate whose activity is inhibited by phosphorylation by MPK-1 MAP kinase. Here, we show that, contrary the current model, lin-1 is required positively for the proper expression of several genes regulated by the pathway in cells adopting the 1 degrees cell fate. We show that the positive requirement for lin-1 is downstream of let-60 Ras and mpk-1 MAP kinase, and that it has a focus in the vulval precursor cells themselves. lin-1 alleles encoding proteins lacking a docking site for MPK-1 MAP kinase are defective in the positive function. We also show that lin-1 apparently has both positive and negative functions during the specification of the fates of other cells in the worm requiring Ras/MAP kinase signaling.

  17. The RAS/PI3K Pathway is Involved in the Impairment of Long-term Potentiation Induced by Acute Aluminum Treatment in Rats.

    PubMed

    Song, Jing; Liu, Ying; Zhang, Hui Fang; Niu, Qiao

    2016-11-01

    To explore the role of RAS/PI3K pathway in the impairment of long-term potentiation (LTP) induced by acute aluminum (Al) treatment in rats in vivo. First, different dosages of aluminum-maltolate complex [Al(mal)3] were given to rats via acute intracerebroventricular (i.c.v.) injection. Following Al exposure, the RAS activity of rat hippocampus were detected by ELISA assay after the hippocampal LTP recording by field potentiation technique in vivo. Second, the antagonism on the aluminum-induced suppression of hippocampal LTP was observed after the treatment of the RAS activator epidermal growth factor (EGF). Finally, the antagonism on the downstream molecules (PKB activity and the phosphorylation of GluR1 S831 and S845) were tested by ELISA and West-blot assays at the same time. With the increasing aluminum dosage, a gradually decreasing in RAS activity of the rat hippocampus was produced after a gradually suppressing on LTP. The aluminum-induced early suppression of hippocampal LTP was antagonized by the RAS activator epidermal growth factor (EGF). And the EGF treatment produced changes similar to those observed for LTP between the groups on PKB activity as well as the phosphorylation of GluR1 S831 and S845. The RAS→PI3K/PKB→GluR1 S831 and S845 signal transduction pathway may be involved in the inhibition of hippocampal LTP by aluminum exposure in rats. However, the mechanisms underlying this observation need further investigation. Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  18. TRPC6 channel-mediated neurite outgrowth in PC12 cells and hippocampal neurons involves activation of RAS/MEK/ERK, PI3K, and CAMKIV signaling.

    PubMed

    Heiser, Jeanine H; Schuwald, Anita M; Sillani, Giacomo; Ye, Lian; Müller, Walter E; Leuner, Kristina

    2013-11-01

    The non-selective cationic transient receptor canonical 6 (TRPC6) channels are involved in synaptic plasticity changes ranging from dendritic growth, spine morphology changes and increase in excitatory synapses. We previously showed that the TRPC6 activator hyperforin, the active antidepressant component of St. John's wort, induces neuritic outgrowth and spine morphology changes in PC12 cells and hippocampal CA1 neurons. However, the signaling cascade that transmits the hyperforin-induced transient rise in intracellular calcium into neuritic outgrowth is not yet fully understood. Several signaling pathways are involved in calcium transient-mediated changes in synaptic plasticity, ranging from calmodulin-mediated Ras-induced signaling cascades comprising the mitogen-activated protein kinase, PI3K signal transduction pathways as well as Ca(2+) /calmodulin-dependent protein kinase II (CAMKII) and CAMKIV. We show that several mechanisms are involved in TRPC6-mediated synaptic plasticity changes in PC12 cells and primary hippocampal neurons. Influx of calcium via TRPC6 channels activates different pathways including Ras/mitogen-activated protein kinase/extracellular signal-regulated kinases, phosphatidylinositide 3-kinase/protein kinase B, and CAMKIV in both cell types, leading to cAMP-response element binding protein phosphorylation. These findings are interesting not only in terms of the downstream targets of TRPC6 channels but also because of their potential to facilitate further understanding of St. John's wort extract-mediated antidepressant activity. Alterations in synaptic plasticity are considered to play an important role in the pathogenesis of depression. Beside several other proteins, TRPC6 channels regulate synaptic plasticity. This study demonstrates that different pathways including Ras/MEK/ERK, PI3K/Akt, and CAMKIV are involved in the improvement of synaptic plasticity by the TRPC6 activator hyperforin, the antidepressant active constituent of St. John

  19. The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells

    PubMed Central

    Melillo, Rosa Marina; Castellone, Maria Domenica; Guarino, Valentina; De Falco, Valentina; Cirafici, Anna Maria; Salvatore, Giuliana; Caiazzo, Fiorina; Basolo, Fulvio; Giannini, Riccardo; Kruhoffer, Mogens; Orntoft, Torben; Fusco, Alfredo; Santoro, Massimo

    2005-01-01

    In papillary thyroid carcinomas (PTCs), rearrangements of the RET receptor (RET/PTC) and activating mutations in the BRAF or RAS oncogenes are mutually exclusive. Here we show that the 3 proteins function along a linear oncogenic signaling cascade in which RET/PTC induces RAS-dependent BRAF activation and RAS- and BRAF-dependent ERK activation. Adoptive activation of the RET/PTC-RAS-BRAF axis induced cell proliferation and Matrigel invasion of thyroid follicular cells. Gene expression profiling revealed that the 3 oncogenes activate a common transcriptional program in thyroid cells that includes upregulation of the CXCL1 and CXCL10 chemokines, which in turn stimulate proliferation and invasion. Thus, motile and mitogenic properties are intrinsic to transformed thyroid cells and are governed by an epistatic oncogenic signaling cascade. PMID:15761501

  20. Ras history

    PubMed Central

    2010-01-01

    Although the roots of Ras sprouted from the rich history of retrovirus research, it was the discovery of mutationally activated RAS genes in human cancer in 1982 that stimulated an intensive research effort to understand Ras protein structure, biochemistry and biology. While the ultimate goal has been developing anti-Ras drugs for cancer treatment, discoveries from Ras have laid the foundation for three broad areas of science. First, they focused studies on the origins of cancer to the molecular level, with the subsequent discovery of genes mutated in cancer that now number in the thousands. Second, elucidation of the biochemical mechanisms by which Ras facilitates signal transduction established many of our fundamental concepts of how a normal cell orchestrates responses to extracellular cues. Third, Ras proteins are also founding members of a large superfamily of small GTPases that regulate all key cellular processes and established the versatile role of small GTP-binding proteins in biology. We highlight some of the key findings of the last 28 years. PMID:21686117

  1. Signaling Pathways in Cell Polarity

    PubMed Central

    McCaffrey, Luke Martin; Macara, Ian G.

    2012-01-01

    A key function of signal transduction during cell polarization is the creation of spatially segregated regions of the cell cortex that possess different lipid and protein compositions and have distinct functions. Polarity can be initiated spontaneously or in response to signaling inputs from adjacent cells or soluble factors and is stabilized by positive-feedback loops. A conserved group of proteins, the Par proteins, plays a central role in polarity establishment and maintenance in many contexts. These proteins generate and maintain their distinct locations in cells by actively excluding one another from specific regions of the plasma membrane. The Par signaling pathway intersects with multiple other pathways that control cell growth, death, and organization. PMID:22553378

  2. Signaling Pathways Mediating Alcohol Effects

    PubMed Central

    Ron, Dorit

    2013-01-01

    Ethanol’s effects on intracellular signaling pathways contribute to acute effects of ethanol as well as to neuroadaptive responses to repeated ethanol exposure. In this chapter we review recent discoveries that demonstrate how ethanol alters signaling pathways involving several receptor tyrosine kinases and intracellular tyrosine and serine-threonine kinases, with consequences for regulation of cell surface receptor function, gene expression, protein translation, neuronal excitability and animal behavior. We also describe recent work that demonstrates a key role for ethanol in regulating the function of scaffolding proteins that organize signaling complexes into functional units. Finally, we review recent exciting studies demonstrating ethanol modulation of DNA and histone modification and the expression of microRNAs, indicating epigenetic mechanisms by which ethanol regulates neuronal gene expression and addictive behaviors. PMID:21877259

  3. The pore-forming α-toxin from clostridium septicum activates the MAPK pathway in a Ras-c-Raf-dependent and independent manner.

    PubMed

    Chakravorty, Anjana; Awad, Milena M; Cheung, Jackie K; Hiscox, Thomas J; Lyras, Dena; Rood, Julian I

    2015-02-10

    Clostridium septicum is the causative agent of atraumatic gas gangrene, with α-toxin, an extracellular pore-forming toxin, essential for disease. How C. septicum modulates the host's innate immune response is poorly defined, although α-toxin-intoxicated muscle cells undergo cellular oncosis, characterised by mitochondrial dysfunction and release of reactive oxygen species. Nonetheless, the signalling events that occur prior to the initiation of oncosis are poorly characterised. Our aims were to characterise the ability of α-toxin to activate the host mitogen activated protein kinase (MAPK) signalling pathway both in vitro and in vivo. Treatment of Vero cells with purified α-toxin activated the extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 arms of the MAPK pathway and stimulated the release of TNF-α in a dose-dependent manner. Studies using inhibitors of all three MAPK components suggested that activation of ERK occurred in a Ras-c-Raf dependent manner, whereas activation of JNK and p38 occurred by a Ras-independent mechanism. Toxin-mediated activation was dependent on efficient receptor binding and pore formation and on an influx of extracellular calcium ions. In the mouse myonecrosis model we showed that the MAPK pathway was activated in tissues of infected mice, implying that it has an important role in the disease process.

  4. Carcinogen-specific mutations in preferred Ras-Raf pathway oncogenes directed by strand bias.

    PubMed

    Keller, Ross R; Gestl, Shelley A; Lu, Amy Q; Hoke, Alicia; Feith, David J; Gunther, Edward J

    2016-08-01

    Carcinogen exposures inscribe mutation patterns on cancer genomes and sometimes bias the acquisition of driver mutations toward preferred oncogenes, potentially dictating sensitivity to targeted agents. Whether and how carcinogen-specific mutation patterns direct activation of preferred oncogenes remains poorly understood. Here, mouse models of breast cancer were exploited to uncover a mechanistic link between strand-biased mutagenesis and oncogene preference. When chemical carcinogens were employed during Wnt1-initiated mammary tumorigenesis, exposure to either 7,12-dimethylbenz(a)anthracene (DMBA) or N-ethyl-N-nitrosourea (ENU) dramatically accelerated tumor onset. Mammary tumors that followed DMBA exposure nearly always activated the Ras pathway via somatic Hras(CAA61CTA) mutations. Surprisingly, mammary tumors that followed ENU exposure typically lacked Hras mutations, and instead activated the Ras pathway downstream via Braf(GTG636GAG) mutations. Hras(CAA61CTA) mutations involve an A-to-T change on the sense strand, whereas Braf(GTG636GAG) mutations involve an inverse T-to-A change, suggesting that strand-biased mutagenesis may determine oncogene preference. To examine this possibility further, we turned to an alternative Wnt-driven tumor model in which carcinogen exposures augment a latent mammary tumor predisposition in Apc(min) mice. DMBA and ENU each accelerated mammary tumor onset in Apc(min) mice by introducing somatic, "second-hit" Apc mutations. Consistent with our strand bias model, DMBA and ENU generated strikingly distinct Apc mutation patterns, including stringently strand-inverse mutation signatures at A:T sites. Crucially, these contrasting signatures precisely match those proposed to confer bias toward Hras(CAA61CTA) versus Braf(GTG636GAG) mutations in the original tumor sets. Our findings highlight a novel mechanism whereby exposure history acts through strand-biased mutagenesis to specify activation of preferred oncogenes. © The Author 2016

  5. Oncogenic K-ras expression is associated with derangement of the cAMP/PKA pathway and forskolin-reversible alterations of mitochondrial dynamics and respiration.

    PubMed

    Palorini, R; De Rasmo, D; Gaviraghi, M; Sala Danna, L; Signorile, A; Cirulli, C; Chiaradonna, F; Alberghina, L; Papa, S

    2013-01-17

    The Warburg effect in cancer cells has been proposed to involve several mechanisms, including adaptation to hypoxia, oncogenes activation or loss of oncosuppressors and impaired mitochondrial function. In previous papers, it has been shown that K-ras transformed mouse cells are much more sensitive as compared with normal cells to glucose withdrawal (undergoing apoptosis) and present a high glycolytic rate and a strong reduction of mitochondrial complex I. Recent observations suggest that transformed cells have a derangement in the cyclic adenosine monophosphate/cAMP-dependent protein kinase (cAMP/PKA) pathway, which is known to regulate several mitochondrial functions. Herein, the derangement of the cAMP/PKA pathway and its impact on transformation-linked changes of mitochondrial functions is investigated. Exogenous stimulation of PKA activity, achieved by forskolin treatment, protected K-ras-transformed cells from apoptosis induced by glucose deprivation, enhanced complex I activity, intracellular adenosine triphosphate (ATP) levels, mitochondrial fusion and decreased intracellular reactive oxygen species (ROS) levels. Several of these effects were almost completely prevented by inhibiting the PKA activity. Short-time treatment with compounds favoring mitochondrial fusion strongly decreased the cellular ROS levels especially in transformed cells. These findings support the notion that glucose shortage-induced apoptosis, specific of K-ras-transformed cells, is associated to a derangement of PKA signaling that leads to mitochondrial complex I decrease, reduction of ATP formation, prevalence of mitochondrial fission over fusion, and thereby opening new approaches for development of anticancer drugs.

  6. Oog1, an oocyte-specific protein, interacts with Ras and Ras-signaling proteins during early embryogenesis

    SciTech Connect

    Tsukamoto, Satoshi; Ihara, Ryo; Aizawa, Akira; Kishida, Shosei; Kikuchi, Akira; Imai, Hiroshi; Minami, Naojiro . E-mail: naojiro@kais.kyoto-u.ac.jp

    2006-05-19

    We previously identified an oocyte-specific gene, Oogenesin 1 (Oog1), that encodes 326 amino acids containing a leucine zipper structure and a leucine-rich repeat. In the present study, to identify the interacting proteins of Oog1, we performed a yeast two-hybrid screening using a GV-oocyte cDNA library and found that Ral guanine nucleotide dissociation stimulator (RalGDS) is the binding partner of Oog1. Coimmunoprecipitation assay confirmed the interaction between Oog1 and RalGDS proteins. Colocalization experiments provide the evidence that the nuclear localization of RalGDS depends on the expression of Oog1. Interestingly, RalGDS protein localized in the nucleus rather than the cytoplasm between late 1-cell and early 2-cell stages, the time when Oog1 localizes in the nucleus. We also examined the interaction between Oog1 and Ras by GST pull-down assay and revealed that Oog1 interacts with Ras in a GTP-dependent manner. These findings suggest a role of Oog1 as a Ras-binding protein.

  7. NF1 regulation of RAS/ERK signaling is required for appropriate granule neuron progenitor expansion and migration in cerebellar development.

    PubMed

    Sanchez-Ortiz, Efrain; Cho, Woosung; Nazarenko, Inga; Mo, Wei; Chen, Jian; Parada, Luis F

    2014-11-01

    Cerebellar development is regulated by a coordinated spatiotemporal interplay between granule neuron progenitors (GNPs), Purkinje neurons, and glia. Abnormal development can trigger motor deficits, and more recent data indicate important roles in aspects of memory, behavior, and autism spectrum disorders (ASDs). Germline mutation in the NF1 tumor suppressor gene underlies Neurofibromatosis type 1, a complex disease that enhances susceptibility to certain cancers and neurological disorders, including intellectual deficits and ASD. The NF1 gene encodes for neurofibromin, a RAS GTPase-activating protein, and thus negatively regulates the RAS signaling pathway. Here, using mouse models to direct conditional NF1 ablation in either embryonic cerebellar progenitors or neonatal GNPs, we show that neurofibromin is required for appropriate development of cerebellar folia layering and structure. Remarkably, neonatal administration of inhibitors of the ERK pathway reversed the morphological defects. Thus, our findings establish a critical cell-autonomous role for the NF1-RAS-ERK pathway in the appropriate regulation of cerebellar development and provide a basis for using neonatal ERK inhibitor-based therapies to treat NF1-induced cerebellar disorders.

  8. Ifenprodil Attenuates Methamphetamine-Induced Behavioral Sensitization and Activation of Ras-ERK-∆FosB Pathway in the Caudate Putamen.

    PubMed

    Li, Lu; Liu, Xinshe; Qiao, Chuchu; Chen, Gang; Li, Tao

    2016-10-01

    Addiction is a debilitating, chronic psychiatric disorder that is difficult to cure completely owing to the high rate of relapse. Behavioral sensitization is considered to may underlie behavioral changes, such as relapse, caused by chronic abuse of psychomotor stimulants. Thus, its animal models have been widely used to explore the etiology of addiction. Recently, increasing evidence has demonstrated that N-methyl-D-aspartate receptors (NMDARs) play an important role in addiction to psychomotor stimulants. However, the role of GluN2B-containing receptors and their downstream signaling pathway(s) in behavioral sensitization induced by methamphetamine (METH) have not been investigated yet. In this study, we used different doses of ifenprodil (2.5, 5, 10 mg/kg), a selective antagonist of the GluN2B subunit, to investigate the role of GluN2B-containing NMDARs in METH-induced behavioral sensitization. We then examined changes in the levels of Ras, phosphorylated extracellular signal-regulated kinase (pERK)/ERK, and ∆FosB in the caudate putamen (CPu) by western blot. We found that 2.5 or 10 mg/kg ifenprodil significantly attenuated METH-induced behavioral sensitization, whereas the mice treated with a moderate dose of ifenprodil (5 mg/kg) displayed no significant changes. Further results of western blot experiments showed that repeated administration of METH caused the increases in the levels of Ras, pERK/ERK and ∆FosB in the CPu, and these changes were inhibited by only the 2.5 mg/kg dose of ifenprodil. In conclusion, these results demonstrated that 2.5 mg/kg ifenprodil could attenuate METH-induced behavioral sensitization. Moreover, GluN2B-containing NMDARs and their downstream Ras-ERK-∆FosB signaling pathway in the CPu might be involved in METH-induced behavioral sensitization.

  9. An RNAi-Based Dimorphic Genetic Screen Identified the Double Bromodomain Protein BET-1 as a Sumo-Dependent Attenuator of RAS-Mediated Signalling

    PubMed Central

    Gee, Fiona; Fisher, Kate; Klemstein, Ulrike; Poulin, Gino B.

    2013-01-01

    Attenuation of RAS/RAF/MAPK signalling is essential to prevent hyperactivation of this oncogenic pathway. In C. elegans, the sumoylation pathway and a combination of histone tail modifications regulate gene expression to attenuate the LET-60 (RAS) signalling pathway. We hypothesised that a number of chromatin regulators are likely to depend on sumoylation to attenuate the pathway. To reveal these, we designed an RNAi-based dimorphic genetic screen that selects candidates based on their ability to act as enhancers of a sumo mutant phenotype, such interactions would suggest that the candidates may be physically associated with sumoylation. We found 16 enhancers, one of which BET-1, is a conserved double bromodomain containing protein. We further characterised BET-1 and showed that it can physically associate with SMO-1 and UBC-9, and that it can be sumoylated in vitro within the second bromodomain at lysine 252. Previous work has shown that BET-1 can bind acetyl-lysines on histone tails to influence gene expression. In conclusion, our screening approach has identified BET-1 as a Sumo-dependent attenuator of LET-60-mediated signalling and our characterisation suggests that BET-1 can be sumoylated. PMID:24349540

  10. An RNAi-based dimorphic genetic screen identified the double bromodomain protein BET-1 as a sumo-dependent attenuator of RAS-mediated signalling.

    PubMed

    Gee, Fiona; Fisher, Kate; Klemstein, Ulrike; Poulin, Gino B

    2013-01-01

    Attenuation of RAS/RAF/MAPK signalling is essential to prevent hyperactivation of this oncogenic pathway. In C. elegans, the sumoylation pathway and a combination of histone tail modifications regulate gene expression to attenuate the LET-60 (RAS) signalling pathway. We hypothesised that a number of chromatin regulators are likely to depend on sumoylation to attenuate the pathway. To reveal these, we designed an RNAi-based dimorphic genetic screen that selects candidates based on their ability to act as enhancers of a sumo mutant phenotype, such interactions would suggest that the candidates may be physically associated with sumoylation. We found 16 enhancers, one of which BET-1, is a conserved double bromodomain containing protein. We further characterised BET-1 and showed that it can physically associate with SMO-1 and UBC-9, and that it can be sumoylated in vitro within the second bromodomain at lysine 252. Previous work has shown that BET-1 can bind acetyl-lysines on histone tails to influence gene expression. In conclusion, our screening approach has identified BET-1 as a Sumo-dependent attenuator of LET-60-mediated signalling and our characterisation suggests that BET-1 can be sumoylated.

  11. Para-phenylenediamine-induces apoptosis via a pathway dependent on PTK-Ras-Raf-JNK activation but independent of the PI3K/Akt pathway in NRK-52E cells.

    PubMed

    Kasi, Reena A P; Moi, Chye Soi; Kien, Yip Wai; Yian, Koh Rhun; Chin, Ng Wei; Yen, Ng Khuen; Ponnudurai, Gnanajothy; Fong, Seow Heng

    2015-03-01

    para‑Phenylenediamine (p‑PD) is a potential carcinogen, and widely used in marketed hair dye formulations. In the present study, the role of the protein tyrosine kinase (PTK)/Ras/Raf/c‑Jun N‑terminal kinase (JNK) and phosphoinositide 3‑kinase (PI3k)/protein kinase B (Akt) pathways on the growth of NRK‑52E cells was investigated. The results demonstrated that p‑PD reduced cell viability in a dose‑dependent manner. The cell death due to apoptosis was confirmed by cell cycle analysis and an Annexin‑V‑fluorescein isothiocyanate binding assay. Subsequent to staining with 2',7'‑dichlorofluorescin diacetate, the treated cells demonstrated a significant increase in reactive oxygen species (ROS) generation compared with the controls. The effects of p‑PD on the signalling pathways were analysed by western blotting. p‑PD‑treated cells exhibited an upregulated phospho‑stress‑activated protein kinase/JNK protein expression level and downregulated Ras and Raf protein expression levels; however, Akt, Bcl‑2, Bcl‑XL and Bad protein expression levels were not significantly altered compared with the control. In conclusion, p‑PD induced apoptosis by a PTK/Ras/Raf/JNK‑dependent pathway and was independent of the PI3K/Akt pathway in NRK‑52E cells.

  12. Pharmacology of intracellular signalling pathways

    PubMed Central

    Nahorski, Stefan R

    2006-01-01

    This article provides a brief and somewhat personalized review of the dramatic developments that have occurred over the last 45 years in our understanding of intracellular signalling pathways associated with G-protein-coupled receptor activation. Signalling via cyclic AMP, the phosphoinositides and Ca2+ is emphasized and these systems have already been revealed as new pharmacological targets. The therapeutic benefits of most of such targets are, however, yet to be realized, but it is certain that the discipline of pharmacology needs to widen its boundaries to meet these challenges in the future. PMID:16402119

  13. Importance of the type I insulin-like growth factor receptor in HER2, FGFR2 and MET-unamplified gastric cancer with and without Ras pathway activation

    PubMed Central

    Saisana, Marina; Griffin, S. Michael; May, Felicity E.B.

    2016-01-01

    Amplification of seven oncogenes: HER2, EGFR, FGFR1, FGFR2, MET, KRAS and IGF1R has been identified in gastric cancer. The first five are targeted therapeutically in patients with HER2-positivity, FGFR2- or MET-amplification but the majority of patients are triple-negative and require alternative strategies. Our aim was to evaluate the importance of the IGF1R tyrosine kinase in triple-negative gastric cancer with and without oncogenic KRAS, BRAF or PI3K3CA mutations. Cell lines and metastatic tumor cells isolated from patients expressed IGF1R, and insulin-like growth factor-1 (IGF-1) activated the PI3-kinase/Akt and Ras/Raf/MAP-kinase pathways. IGF-1 protected triple-negative cells from caspase-dependent apoptosis and anoikis. Protection was mediated via the PI3-kinase/Akt pathway. Remarkably, IGF-1-dependent cell survival was greater in patient samples. IGF-1 stimulated triple-negative gastric cancer cell growth was prevented by IGF1R knockdown and Ras/Raf/MAP-kinase pathway inhibition. The importance of the receptor in cell line and metastatic tumor cell growth in serum-containing medium was demonstrated by knockdown and pharmacological inhibition with figitumumab. The proportions of cells in S-phase and mitotic-phase, and Ras/Raf/MAP-kinase pathway activity, were reduced concomitantly. KRAS-addicted and BRAF-impaired gastric cancer cells were particularly susceptible. In conclusion, IGF1R and the IGF signal transduction pathway merit consideration as potential therapeutic targets in patients with triple-negative gastric cancer. PMID:27437872

  14. RAS - Screens & Assays

    Cancer.gov

    A primary goal of the RAS Initiative is to develop assays for RAS activity, localization, and signaling and adapt those assays so they can be used for finding new drug candidates. Explore the work leading to highly validated screening protocols.

  15. Signaling Pathways in Cartilage Repair

    PubMed Central

    Mariani, Erminia; Pulsatelli, Lia; Facchini, Andrea

    2014-01-01

    In adult healthy cartilage, chondrocytes are in a quiescent phase characterized by a fine balance between anabolic and catabolic activities. In ageing, degenerative joint diseases and traumatic injuries of cartilage, a loss of homeostatic conditions and an up-regulation of catabolic pathways occur. Since cartilage differentiation and maintenance of homeostasis are finely tuned by a complex network of signaling molecules and biophysical factors, shedding light on these mechanisms appears to be extremely relevant for both the identification of pathogenic key factors, as specific therapeutic targets, and the development of biological approaches for cartilage regeneration. This review will focus on the main signaling pathways that can activate cellular and molecular processes, regulating the functional behavior of cartilage in both physiological and pathological conditions. These networks may be relevant in the crosstalk among joint compartments and increased knowledge in this field may lead to the development of more effective strategies for inducing cartilage repair. PMID:24837833

  16. Hydrogen sulfide in signaling pathways.

    PubMed

    Olas, Beata

    2015-01-15

    For a long time hydrogen sulfide (H₂S) was considered a toxic compound, but recently H₂S (at low concentrations) has been found to play an important function in physiological processes. Hydrogen sulfide, like other well-known compounds - nitric oxide (NO) and carbon monoxide (CO) is a gaseous intracellular signal transducer. It regulates the cell cycle, apoptosis and the oxidative stress. Moreover, its functions include neuromodulation, regulation of cardiovascular system and inflammation. In this review, I focus on the metabolism of hydrogen sulfide (including enzymatic pathways of H₂S synthesis from l- and d-cysteine) and its signaling pathways in the cardiovascular system and the nervous system. I also describe how hydrogen sulfide may be used as therapeutic agent, i.e. in the cardiovascular diseases.

  17. Cyclic-GMP-dependent protein kinase inhibits the Ras/Mitogen-activated protein kinase pathway.

    PubMed

    Suhasini, M; Li, H; Lohmann, S M; Boss, G R; Pilz, R B

    1998-12-01

    Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3', 5'-cyclic monophosphate and 8-bromoguanosine-3',5'-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Ibeta expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser43, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser43 uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser43 was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser43 in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser43 phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by

  18. Alterations in K-ras, APC and p53-multiple genetic pathway in colorectal cancer among Indians.

    PubMed

    Malhotra, Pooja; Anwar, Mumtaz; Nanda, Neha; Kochhar, Rakesh; Wig, Jai Dev; Vaiphei, Kim; Mahmood, Safrun

    2013-06-01

    The incidence of colorectal cancer (CRC) is increasing rapidly in Asian countries during the past few decades, but no comprehensive analysis has been done to find out the exact cause of this disease. In this study, we investigated the frequencies of mutations and expression pattern of K-ras, APC (adenomatosis polyposis coli) and p53 in tumor, adjoining and distant normal mucosa and to correlate these alterations with patients clinicopathological parameters as well as with the survival. Polymerase chain reaction (PCR)-restriction digestion was used to detect mutations in K-ras and PCR-SSCP (Single Strand Conformation Polymorphism) followed by DNA sequencing was used to detect mutations in APC and p53 genes. Immunohistochemistry was used to detect the expression pattern of K-ras, APC and p53 proteins. The frequencies of mutations of K-ras, APC and p53 in 30 tumor tissues samples were 26.7 %, 46.7 % and 20 %, respectively. Only 3.3 % of tumors contained mutations in all the three genes. The most common combination of mutation was APC and p53 whereas mutation in both p53 and K-ras were extremely rare. There was no association between the mutations and expression pattern of K-ras, APC and p53 (p>0.05). In Indians, the frequency of alterations of K-ras and APC is similar as in Westerns, whereas the frequency of p53 mutation is slightly lower. The lack of multiple mutations in tumor specimens suggests that these genetic alterations might have independent influences on CRC development and there could be multiple alternative genetic pathways to CRC in our present study cohort.

  19. Activated Ki-Ras complements erythropoietin signaling in CTLL-2 cells, inducing tyrosine phosphorylation of a 160-kDa protein.

    PubMed Central

    Yamamura, Y; Noda, M; Ikawa, Y

    1994-01-01

    We have previously shown that expression of erythropoietin (EPO) receptor (EPOR) alone failed to confer EPO responsiveness on the interleukin 2-dependent T-cell line CTLL-2, whereas the introduction of the EPOR into interleukin 3-dependent pro-B-cell lines, such as BAF-B03, allowed the cells to proliferate in response to EPO. Here, we report that additional expression of v-Ki-Ras conferred EPO-dependent growth on CTLL-2 cells expressing the EPOR, with additional formation of a high-affinity EPOR. To investigate possible mechanisms of EPOR downstream signaling induced by v-Ki-Ras expression in these CTLL-2-derived cells, we carried out anti-phosphotyrosine immunoblot analysis of the EPOR complex immunoprecipitated with anti-EPOR antibody from lysates of cells with and without cytokine stimulation, revealing two 160-kDa and 130-kDa phosphotyrosyl proteins. An anti-JAK2 antibody did not react with these proteins, suggesting that they may represent cellular components involved in an EPO-EPOR signaling pathway induced by v-Ki-Ras. Similar phosphotyrosyl proteins were present among Friend erythroleukemia cell lines, in which the Friend virus gp55/EPOR complex on the cell surface constitutively sends signals for cell growth. Images PMID:7522324

  20. Regulation of the Notch target gene Hes-1 by TGF{alpha} induced Ras/MAPK signaling in human neuroblastoma cells

    SciTech Connect

    Stockhausen, Marie-Therese; Sjoelund, Jonas; Axelson, Hakan . E-mail: hakan.axelson@molmed.mas.lu.se

    2005-10-15

    Ras and Notch signaling have recently been shown to cooperate in the maintenance of neoplastic transformation. Here, we show that TGF{alpha}, a known activator of Ras signaling, can drive cell proliferation and at the same time induce the expression of the Notch target Hes-1 in the neuroblastoma cell line SK-N-BE(2)c. The up-regulation of Hes-1 occurred both at the transcriptional and protein levels and by use of EGFR and MEK inhibitors we could show that the Hes-1 response was dependent on activation of the MAP kinase ERK. Blocking Notch activation by {gamma}-secretase inhibition did not profoundly affect the Hes-1 levels, neither in untreated nor in TGF{alpha} treated cells. The up-regulation of Hes-1 was associated with down-regulation of its pro-neuronal target gene Hash-1. Taken together, these results show that TGF{alpha} is a potent mitogen of neuroblastoma cells and suggest a connection between activation of ERK and Hes-1, thus providing a link between the Ras and Notch signaling pathways.

  1. Single cell analysis of low-power laser irradiation-induced activation of signaling pathway in cell proliferation

    NASA Astrophysics Data System (ADS)

    Xing, Da; Gao, Xuejuan

    2007-02-01

    Low-power laser irradiation (LPLI) has been shown to promote cell proliferation in various cell types, yet the mechanism of which has not been fully clarified. Investigating the signaling pathways involved in the laser irradiation is important for understanding these processes. The small G protein Ras works as a binary switch in many important intracellular signaling pathways and, therefore, has been one of the focal targets of signal-transduction investigations and drug development. The Ras/Raf/MEK/ERK (extracellular-signal-regulated kinase) signaling pathway is a network that governs proliferation, differentiation and cell survival. Recent studies suggest that Ras/Raf signaling pathway is involved in the LPLI-induced cell proliferation. On the other hand, Protein kinase Cs (PKCs), the Ca 2+ activated, phospholipid-dependent serine/threonine protein kinases, have been recently presumed to be involved in the regulation of cell proliferation induced by LPLI. In this report, to monitor the direct activations of Ras and PKCs after LPLI treatment in living cells in real time, Raichu-Ras reporter and C kinase activity reporter (CKAR) were utilized, both of which were constructed based on fluorescence resonance energy transfer (FRET) technique. The direct activation of Ras is predominantly initiated from the different microdomains of the plasma membrane. The results are monitored during cell proliferation induced by LPLI (0.8 J/cm2) in serum-starved COS-7 cells expressing Raichu-Ras reporter using FRET imaging on laser scanning confocal microscope. Furthermore, the increasing activation of PKCs is also monitored during cell proliferation induced by LPLI (0.8 J/cm2) in serum-starved human lung adenocarcinoma cells (ASTC-a-1) expressing CKAR reporter using the similar way. Taken together, the dynamic increases of H-Ras and PKCs activities are observed during the processes of cell proliferation induced by LPLI.

  2. Di2-ethylhexyl phthalate disrupts thyroid hormone homeostasis through activating the Ras/Akt/TRHr pathway and inducing hepatic enzymes

    PubMed Central

    Ye, Hanfeng; Ha, Mei; Yang, Min; Yue, Ping; Xie, Zhengyuan; Liu, Changjiang

    2017-01-01

    Di(2-ethylhexyl) phthalate (DEHP), as a widespread environmental pollutant and an endocrine disruptor, can disturb the homeostasis of thyroid hormones (THs). In order to elucidate roles of the MAPK and PI3K/Akt pathways and hepatic enzymes in thyroid-disrupting effects of DEHP, Sprague-Dawley rats were dosed with DEHP by gavage for 30 consecutive days; Nthy-ori 3-1 cells were treated with DEHP with NAC, k-Ras siRNA or inhibitors (U0126 and wortmannin). Results showed that DEHP led to histopathologic changes in rat thyroid and liver, such as the decrease in thyroid follicular cavity diameter, hepatocyte edema. Triiodothyronine (T3), thyroxine (T4) and thyrotropin releasing hormone (TRH) were reduced. DEHP caused ROS production, oxidative stress and k-Ras upregulation, thereby activating the ERK and Akt pathways in vivo and in vitro. Moreover, TRH receptor (TRHr) level was elevated after the activation of the Akt pathway and was downregulated after the inhibition of the Akt pathway. However, TRHr was not modulated by the ERK pathway. Additionally, hepatic enzymes, including Ugt1a1, CYP2b1, Sult1e1, and Sult2b1, were significantly induced after DEHP exposure. Taken together, DEHP can perturb TH homeostasis and reduce TH levels. The activated Ras/Akt/TRHr pathway and induced hepatic enzymes play vital roles in thyroid-disrupting effects of DEHP. PMID:28065941

  3. Di2-ethylhexyl phthalate disrupts thyroid hormone homeostasis through activating the Ras/Akt/TRHr pathway and inducing hepatic enzymes.

    PubMed

    Ye, Hanfeng; Ha, Mei; Yang, Min; Yue, Ping; Xie, Zhengyuan; Liu, Changjiang

    2017-01-09

    Di(2-ethylhexyl) phthalate (DEHP), as a widespread environmental pollutant and an endocrine disruptor, can disturb the homeostasis of thyroid hormones (THs). In order to elucidate roles of the MAPK and PI3K/Akt pathways and hepatic enzymes in thyroid-disrupting effects of DEHP, Sprague-Dawley rats were dosed with DEHP by gavage for 30 consecutive days; Nthy-ori 3-1 cells were treated with DEHP with NAC, k-Ras siRNA or inhibitors (U0126 and wortmannin). Results showed that DEHP led to histopathologic changes in rat thyroid and liver, such as the decrease in thyroid follicular cavity diameter, hepatocyte edema. Triiodothyronine (T3), thyroxine (T4) and thyrotropin releasing hormone (TRH) were reduced. DEHP caused ROS production, oxidative stress and k-Ras upregulation, thereby activating the ERK and Akt pathways in vivo and in vitro. Moreover, TRH receptor (TRHr) level was elevated after the activation of the Akt pathway and was downregulated after the inhibition of the Akt pathway. However, TRHr was not modulated by the ERK pathway. Additionally, hepatic enzymes, including Ugt1a1, CYP2b1, Sult1e1, and Sult2b1, were significantly induced after DEHP exposure. Taken together, DEHP can perturb TH homeostasis and reduce TH levels. The activated Ras/Akt/TRHr pathway and induced hepatic enzymes play vital roles in thyroid-disrupting effects of DEHP.

  4. Radicicol suppresses transformation and restores tropomyosin-2 expression in both ras- and MEK-transformed cells without inhibiting the Raf/MEK/ERK signaling cascade.

    PubMed

    Kim, P N; Jonasch, E; Mosterman, B C; Mier, J W; Janssen, R A

    2001-11-01

    The antibiotic radicicol suppresses transformation in a variety of transformed cells. The antineoplastic effects of the drug have been attributed to the degradation of Raf and the inactivation of the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling cascade. Here we demonstrate that radicicol induces cell spreading, suppresses anchorage-independent cell growth, and increases the expression of the high-molecular weight tropomyosin isoform TM-2 in cells stably expressing a constitutively active form of MEK-1 as well as in ras-transformed cells. Furthermore, the reverting effects of the drug are achieved at concentrations below those required to deplete Raf from the cell or to inhibit the phosphorylation of ERK or its substrates Elk and pp90(RSK). In contrast, low concentrations of radicicol significantly inhibited activator protein (AP-1) and serum response factor (SRF)-mediated transcription. The lack of correlation between the effects of radicicol on cell phenotype and on the signaling activities of the Raf/MEK/ERK pathway indicate that Raf depletion or disruption of proximal signaling events in the mitogen-activated protein kinase pathway are not the predominant mechanisms by which the drug suppresses the transformed phenotype. Our observation that low concentrations of radicicol block transcriptional activities mediated by AP-1 and SRF suggests that interference with signaling upstream of these transcription factors may contribute to the reverting effects of the drug.

  5. The UV (Ribotoxic) Stress Response of Human Keratinocytes Involves the Unexpected Uncoupling of the Ras-Extracellular Signal-Regulated Kinase Signaling Cascade from the Activated Epidermal Growth Factor Receptor

    PubMed Central

    Iordanov, Mihail S.; Choi, Remy J.; Ryabinina, Olga P.; Dinh, Thanh-Hoai; Bright, Robert K.; Magun, Bruce E.

    2002-01-01

    In mammals, UVB radiation is of biological relevance primarily for the cells of the epidermis. We report here the existence of a UVB response that is specific for proliferating human epidermal keratinocytes. Unlike other cell types that also display a UVB response, keratinocytes respond to UVB irradiation with a transient but potent downregulation of the Ras-extracellular signal-regulated kinase (ERK) signaling cascade. The downregulation of ERK precedes a profound decrease in the steady-state levels of cyclin D1, a mediator of the proliferative action of ERK. Keratinocytes exhibit high constitutive activity of the Ras-ERK signaling cascade even in culture medium lacking supplemental growth factors. The increased activity of Ras and phosphorylation of ERK in these cells are maintained by the autocrine production of secreted molecules that activate the epidermal growth factor receptor (EGFR). Irradiation of keratinocytes increases the phosphorylation of EGFR on tyrosine residues Y845, Y992, Y1045, Y1068, Y1086, Y1148, and Y1173 above the basal levels and leads to the increased recruitment of the adaptor proteins Grb2 and ShcA and of a p55 form of the regulatory subunit of the phosphatidylinositide 3-kinase to the UVB-activated EGFR. Paradoxically, however, UVB causes, at the same time, the inactivation of Ras and a subsequent dephosphorylation of ERK. By contrast, the signaling pathway leading from the activated EGFR to the phosphorylation of PKB/Akt1 is potentiated by UVB. The UVB response of keratinocytes appeared to be a manifestation of the more general ribotoxic stress response inasmuch as the transduction of the UVB-generated inhibitory signal to Ras and ERK required the presence of active ribosomes at the time of irradiation. PMID:12101233

  6. ERK1 and ERK2 mitogen-activated protein kinases affect Ras-dependent cell signaling differentially

    PubMed Central

    Vantaggiato, Chiara; Formentini, Ivan; Bondanza, Attilio; Bonini, Chiara; Naldini, Luigi; Brambilla, Riccardo

    2006-01-01

    Background The mitogen-activated protein (MAP) kinases p44ERK1 and p42ERK2 are crucial components of the regulatory machinery underlying normal and malignant cell proliferation. A currently accepted model maintains that ERK1 and ERK2 are regulated similarly and contribute to intracellular signaling by phosphorylating a largely common subset of substrates, both in the cytosol and in the nucleus. Results Here, we show that ablation of ERK1 in mouse embryo fibroblasts and NIH 3T3 cells by gene targeting and RNA interference results in an enhancement of ERK2-dependent signaling and in a significant growth advantage. By contrast, knockdown of ERK2 almost completely abolishes normal and Ras-dependent cell proliferation. Ectopic expression of ERK1 but not of ERK2 in NIH 3T3 cells inhibits oncogenic Ras-mediated proliferation and colony formation. These phenotypes are independent of the kinase activity of ERK1, as expression of a catalytically inactive form of ERK1 is equally effective. Finally, ectopic expression of ERK1 but not ERK2 is sufficient to attenuate Ras-dependent tumor formation in nude mice. Conclusion These results reveal an unexpected interplay between ERK1 and ERK2 in transducing Ras-dependent cell signaling and proliferation. Whereas ERK2 seems to have a positive role in controlling normal and Ras-dependent cell proliferation, ERK1 probably affects the overall signaling output of the cell by antagonizing ERK2 activity. PMID:16805921

  7. Classic Ras Proteins Promote Proliferation and Survival Via Distinct Phosphoproteome Alterations in Neurofibromin-Null Malignant Peripheral Nerve Sheath Tumor Cells

    PubMed Central

    Brossier, Nicole M.; Prechtl, Amanda M.; Longo, Jody Fromm; Barnes, Stephen; Wilson, Landon S.; Byer, Stephanie J.; Brosius, Stephanie N.; Carroll, Steven L.

    2015-01-01

    Neurofibromin, the tumor suppressor encoded by the neurofibromatosis type 1 (NF1) gene, potentially suppresses the activation of H-Ras, N-Ras and K-Ras. However, it is not known whether these classic Ras proteins are hyperactivated in NF1-null nerve sheath tumors, how they contribute to tumorigenesis and what signaling pathways mediate their effects. Here we show that H-Ras, N-Ras and K-Ras are coexpressed with their activators, (guanine nucleotide exchange factors), in neurofibromin-null malignant peripheral nerve sheath tumor (MPNST) cells and that all 3 Ras proteins are activated. Dominant negative (DN) H-Ras, a pan-inhibitor of the classic Ras family, inhibited MPNST proliferation and survival, but not migration. However, NF1-null MPNST cells were variably dependent on individual Ras proteins. In some lines, ablation of H-Ras, N-Ras and/or K-Ras inhibited mitogenesis. In others, ablation of a single Ras protein had no effect on proliferation; in these lines, ablation of a single Ras protein resulted in compensatory increases in the activation and/or expression of other Ras proteins. Using mass spectrometry-based phosphoproteomics, we identified 7 signaling networks affecting morphology, proliferation and survival that are regulated by DN H-Ras. Thus, neurofibromin loss activates multiple classic Ras proteins that promote proliferation and survival by regulating several distinct signaling cascades. PMID:25946318

  8. Acylcarnitines activate proinflammatory signaling pathways.

    PubMed

    Rutkowsky, Jennifer M; Knotts, Trina A; Ono-Moore, Kikumi D; McCoin, Colin S; Huang, Shurong; Schneider, Dina; Singh, Shamsher; Adams, Sean H; Hwang, Daniel H

    2014-06-15

    Incomplete β-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM). Previous studies revealed that plasma concentrations of medium- and long-chain acylcarnitines (by-products of incomplete β-oxidation) are elevated in T2DM and insulin resistance. In a previous study, we reported that mixed D,L isomers of C12- or C14-carnitine induced an NF-κB-luciferase reporter gene in RAW 264.7 cells, suggesting potential activation of proinflammatory pathways. Here, we determined whether the physiologically relevant L-acylcarnitines activate classical proinflammatory signaling pathways and if these outcomes involve pattern recognition receptor (PRR)-associated pathways. Acylcarnitines induced the expression of cyclooxygenase-2 in a chain length-dependent manner in RAW 264.7 cells. L-C14 carnitine (5-25 μM), used as a representative acylcarnitine, stimulated the expression and secretion of proinflammatory cytokines in a dose-dependent manner. Furthermore, L-C14 carnitine induced phosphorylation of JNK and ERK, common downstream components of many proinflammatory signaling pathways including PRRs. Knockdown of MyD88, a key cofactor in PRR signaling and inflammation, blunted the proinflammatory effects of acylcarnitine. While these results point to potential involvement of PRRs, L-C14 carnitine promoted IL-8 secretion from human epithelial cells (HCT-116) lacking Toll-like receptors (TLR)2 and -4, and did not activate reporter constructs in TLR overexpression cell models. Thus, acylcarnitines have the potential to activate inflammation, but the specific molecular and tissue target(s) involved remain to be identified.

  9. Acylcarnitines activate proinflammatory signaling pathways

    PubMed Central

    Rutkowsky, Jennifer M.; Knotts, Trina A.; Ono-Moore, Kikumi D.; McCoin, Colin S.; Huang, Shurong; Schneider, Dina; Singh, Shamsher; Hwang, Daniel H.

    2014-01-01

    Incomplete β-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM). Previous studies revealed that plasma concentrations of medium- and long-chain acylcarnitines (by-products of incomplete β-oxidation) are elevated in T2DM and insulin resistance. In a previous study, we reported that mixed d,l isomers of C12- or C14-carnitine induced an NF-κB-luciferase reporter gene in RAW 264.7 cells, suggesting potential activation of proinflammatory pathways. Here, we determined whether the physiologically relevant l-acylcarnitines activate classical proinflammatory signaling pathways and if these outcomes involve pattern recognition receptor (PRR)-associated pathways. Acylcarnitines induced the expression of cyclooxygenase-2 in a chain length-dependent manner in RAW 264.7 cells. l-C14 carnitine (5–25 μM), used as a representative acylcarnitine, stimulated the expression and secretion of proinflammatory cytokines in a dose-dependent manner. Furthermore, l-C14 carnitine induced phosphorylation of JNK and ERK, common downstream components of many proinflammatory signaling pathways including PRRs. Knockdown of MyD88, a key cofactor in PRR signaling and inflammation, blunted the proinflammatory effects of acylcarnitine. While these results point to potential involvement of PRRs, l-C14 carnitine promoted IL-8 secretion from human epithelial cells (HCT-116) lacking Toll-like receptors (TLR)2 and -4, and did not activate reporter constructs in TLR overexpression cell models. Thus, acylcarnitines have the potential to activate inflammation, but the specific molecular and tissue target(s) involved remain to be identified. PMID:24760988

  10. Small G-protein Signaling in Neuronal Plasticity and Memory Formation: the Specific Role of Ras Family Proteins

    PubMed Central

    Ye, Xiaojing; Carew, Thomas J.

    2010-01-01

    Small G-proteins are an extensive family of proteins that bind and hydrolyze GTP. They are ubiquitous inside cells, regulating a wide range of cellular processes. Recently, many studies have examined the role of small G-proteins, particularly the Ras family of G-proteins, in memory formation. Once thought to be primarily involved in the transduction of a variety of extracellular signals during development, it is now clear that Ras family proteins also play critical roles in molecular processing underlying neuronal and behavioral plasticity. We here review a number of recent studies that explore how the signaling of Ras family proteins contributes to memory formation. Understanding these signaling processes is of fundamental importance both from a basic scientific perspective, with the goal of providing mechanistic insights into a critical aspect of cognitive behavior, and from a clinical perspective, with the goal of providing effective therapies for a range of disorders involving cognitive impairments. PMID:21040840

  11. SOS1 over-expression in genital skin fibroblasts from hirsute women: a putative role of the SOS1/RAS pathway in the pathogenesis of hirsutism.

    PubMed

    Minella, D; Wannenes, F; Biancolella, M; Amati, F; Testa, B; Nardone, A; Bueno, S; Fabbri, A; Lauro, D; Novelli, G; Moretti, C

    2011-01-01

    Hirsutism is the development of androgen-dependent terminal body hair in women in places in which terminal hair are normally not found. It is often associated with hyperandrogenemia and/or polycystic ovary syndrome (PCOS), but the existence of uncommom hirsutism forms that are not related to altered androgen plasma levels lead also to the definition of - idiopathic hirsutism. Although the pathophysiology of hirsutism has been linked to increasing 5-alpha reductase (SRD5A) activity and to an alteration of the androgen receptor (AR) transcriptional machinery, many aspects remain unclear. In particular, the relationships between androgens and local factors are poorly understood. In the present paper, we selected for a genital skin biopsy, 8 women affected with severe hirsutism (Ferriman-Gallway score greater than 25) but with normal plasma androgen levels, with the exception of slightly higher serum 3alpha-diol-glucuronide levels, and 6 healthy controls and analyzed their androgen- and insulin-specific transcriptional profile using a specific custom low density microarray (AndroChip 2, GPL9164). We identified the over-expression of the Son of Sevenless-1 (SOS1) gene in all of the hirsute skin fibroblast primary cell cultures compared to control healthy women. Since SOS1 is a guanine nucleotide exchange factor that couples receptor tyrosine kinases to the RAS signaling pathway that controls cell proliferation and differentiation, we further analyzed SOS1 expression, protein level and RAS signaling activation pathway in an in vitro model (NHDF, normal human dermal fibroblast cell line). NHDF treated for 24 h with different concentrations of DHT and T showed an increase in SOS1 levels (both mRNA and protein) and also an activation of the RAS pathway. Our in vivo and in vitro data represent a novel preliminary observation that factors activating SOS1 could act as local proliferative modulators linked to the androgen pathway in the pilosebaceous unit. SOS1 over

  12. The developmental pattern of the RAS/RAF/Erk1/2 pathway in the BTBR autism mouse model.

    PubMed

    Yin, Ailan; Qiu, Yuwen; Jia, Bei; Song, Tianrong; Yu, Yanhong; Alberts, Ian; Zhong, Mei

    2014-12-01

    BTBR mice exhibit several autistic-like behaviors and are currently used as a model for understanding mechanisms that may be responsible for the pathogenesis of autism. Ras/Raf/ERK1/2 signaling has been suggested to play an important role in neural development, learning, memory, and cognition. Two studies reported that a deletion of a locus on chromosome 16 containing the mitogen-activated protein kinase 3 (MAPK3) gene, which encodes ERK1, is associated with autism. In the present study, Ras/Raf/ERK1/2 signaling was found to be up-regulated in BTBR mice relative to matched control B6 mice, to further suggest involvement in the pathogenesis of autism. To further characterize the developmental pattern of Ras/Raf/ERK1/2 signaling, varying stages during development were sampled to reveal an up-regulation in newborn and 2-week old BTBR mice relative to age-matched B6 mice. By the age of 3-week, Ras/Raf/ERK1/2 signaling in the brain of BTBR mice was unaltered relative to B6 mice, with this trend maintained in 6-week samples. These results suggest that the alteration of Ras/Raf/ERK signaling in the early developmental stages in mice could contribute to the noted autistic phenotype. Furthermore, these findings support the value of BTBR mice to serve as a human analog for autistic etiological research and aid in a better understanding of the developmental mechanisms of autism. Copyright © 2014 ISDN. All rights reserved.

  13. Ras proteins control mitochondrial biogenesis and function in Saccharomyces cerevisiae.

    PubMed

    Hlavatá, L; Nyström, T

    2003-01-01

    The evolutionarily conserved Ras proteins function as a point of convergence for different signaling pathways in eukaryotes and have been implicated in both aging and cancer development. In Saccharomyces cerevisiae the plasma membrane proteins Ras1 and Ras2 are sensing the nutritional status of the environments, e.g., the abundance and quality of available carbon sources. The cAMP-protein kinase A pathway is the most explored signaling pathway controlled by Ras proteins; it affects a large number of genes, some of which are important to defend the cell against oxidative stress. In addition, recent analysis has shown that the Ras system of yeast is involved in the development of mitochondria and in regulating their activity. As a sensor of environmental status and an effector of mitochondrial activity, Ras serves as a Rosetta stone of cellular energy transduction. This review summarizes the physical and functional involvement of Ras proteins and Ras-dependent signaling pathways in mitochondrial function in S. cerevisiae. Since mitochondria produce harmful reactive oxygen species as an inevitable byproduct and are partly under control of Ras, illuminating these regulatory interactions may improve our understanding of both cancer and aging.

  14. Epidermal Growth Factor Receptor Cell Proliferation Signaling Pathways

    PubMed Central

    Wee, Ping; Wang, Zhixiang

    2017-01-01

    The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is commonly upregulated in cancers such as in non-small-cell lung cancer, metastatic colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer, and breast cancer. Various mechanisms mediate the upregulation of EGFR activity, including common mutations and truncations to its extracellular domain, such as in the EGFRvIII truncations, as well as to its kinase domain, such as the L858R and T790M mutations, or the exon 19 truncation. These EGFR aberrations over-activate downstream pro-oncogenic signaling pathways, including the RAS-RAF-MEK-ERK MAPK and AKT-PI3K-mTOR pathways. These pathways then activate many biological outputs that are beneficial to cancer cell proliferation, including their chronic initiation and progression through the cell cycle. Here, we review the molecular mechanisms that regulate EGFR signal transduction, including the EGFR structure and its mutations, ligand binding and EGFR dimerization, as well as the signaling pathways that lead to G1 cell cycle progression. We focus on the induction of CYCLIN D expression, CDK4/6 activation, and the repression of cyclin-dependent kinase inhibitor proteins (CDKi) by EGFR signaling pathways. We also discuss the successes and challenges of EGFR-targeted therapies, and the potential for their use in combination with CDK4/6 inhibitors. PMID:28513565

  15. Effects of RAF inhibitors on PI3K/AKT signalling depend on mutational status of the RAS/RAF signalling axis

    PubMed Central

    Fritsche-Guenther, Raphaela; Witzel, Franziska; Kempa, Stefan; Brummer, Tilman; Sers, Christine; Blüthgen, Nils

    2016-01-01

    Targeted therapies within the RAS/RAF/MEK/ERK signalling axis become increasingly popular, yet cross-talk and feedbacks in the signalling network lead to unexpected effects. Here we look systematically into how inhibiting RAF and MEK with clinically relevant inhibitors result in changes in PI3K/AKT activation. We measure the signalling response using a bead-based ELISA, and use a panel of three cell lines, and isogenic cell lines that express mutant forms of the oncogenes KRAS and BRAF to interrogate the effects of the MEK and RAF inhibitors on signalling. We find that treatment with the RAF inhibitors have opposing effects on AKT phosphorylation depending on the mutational status of two important oncogenes, KRAS and BRAF. If these two genes are in wildtype configuration, RAF inhibitors reduce AKT phosphorylation. In contrast, if BRAF or KRAS are mutant, RAF inhibitors will leave AKT phosphorylation unaffected or lead to an increase of AKT phosphorylation. Down-regulation of phospho-AKT by RAF inhibitors also extends to downstream transcription factors, and correlates with apoptosis induction. Our results show that oncogenes rewire signalling such that targeted therapies can have opposing effects on parallel pathways, which depend on the mutational status of the cell. PMID:26799289

  16. Reduction of metastasis, cell invasion, and adhesion in mouse osteosarcoma by YM529/ONO-5920-induced blockade of the Ras/MEK/ERK and Ras/PI3K/Akt pathway

    SciTech Connect

    Tsubaki, Masanobu; Satou, Takao; Itoh, Tatsuki; Imano, Motohiro; Ogaki, Mitsuhiko; Yanae, Masashi; Nishida, Shozo

    2012-03-15

    Osteosarcoma is one of the most common primary malignant bone tumors in children and adolescents. Some patients continue to have a poor prognosis, because of the metastatic disease. YM529/ONO-5920 is a nitrogen-containing bisphosphonate that has been used for the treatment of osteoporosis. YM529/ONO-5920 has recently been reported to induce apoptosis in various tumors including osteosarcoma. However, the mode of metastasis suppression in osteosarcoma by YM529/ONO-5920 is unclear. In the present study, we investigated whether YM529/ONO-5920 inhibited tumor cell migration, invasion, adhesion, or metastasis in the LM8 mouse osteosarcoma cell line. We found that YM529/ONO-5920 significantly inhibited metastasis, cell migration, invasion, and adhesion at concentrations that did not have antiproliferative effects on LM8 cells. YM529/ONO-5920 also inhibited the mRNA expression and protein activities of matrix metalloproteinases (MMPs). In addition, YM529/ONO-5920 suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and the serine/threonine protein kinase B (Akt) by the inhibition of Ras prenylation. Moreover, U0126, a mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, also inhibited LM8 cell migration, invasion, adhesion, and metastasis, as well as the mRNA expression and protein activities of MMP-1, MMP-2, MMP-9, and MT1-MMP. The results indicated that YM529/ONO-5920 suppressed the Ras/MEK/ERK and Ras/PI3K/Akt pathways, thereby inhibiting LM8 cell migration, invasion, adhesion, and metastasis. These findings suggest that YM529/ONO-5920 has potential clinical applications for the treatment of tumor cell metastasis in osteosarcoma. -- Highlights: ► We investigated whether YM529/ONO-5920 inhibited tumor metastasis in osteosarcoma. ► YM529/ONO-5920 inhibited metastasis, cell migration, invasion, and adhesion. ► YM529/ONO-5920 suppressed Ras signalings. ► YM529/ONO-5920

  17. Single-molecule fluorescence imaging of RalGDS on cell surfaces during signal transduction from Ras to Ral.

    PubMed

    Yoshizawa, Ryo; Umeki, Nobuhisa; Yanagawa, Masataka; Murata, Masayuki; Sako, Yasushi

    2017-01-01

    RalGDS is one of the Ras effectors and functions as a guanine nucleotide exchange factor for the small G-protein, Ral, which regulates membrane trafficking and cytoskeletal remodeling. The translocation of RalGDS from the cytoplasm to the plasma membrane is required for Ral activation. In this study, to understand the mechanism of Ras-Ral signaling we performed a single-molecule fluorescence analysis of RalGDS and its functional domains (RBD and REMCDC) on the plasma membranes of living HeLa cells. Increased molecular density of RalGDS and RBD, but not REMCDC, was observed on the plasma membrane after EGF stimulation of the cells to induce Ras activation, suggesting that the translocation of RalGDS involves an interaction between the GTP-bound active form of Ras and the RBD of RalGDS. Whereas the RBD played an important role in increasing the association rate constant between RalGDS and the plasma membrane, the REMCDC domain affected the dissociation rate constant from the membrane, which decreased after Ras activation or the hyperexpression of Ral. The Y64 residue of Ras and clusters of RalGDS molecules were involved in this reduction. From these findings, we infer that Ras activation not merely increases the cell-surface density of RalGDS, but actively stimulates the RalGDS-Ral interaction through a structural change in RalGDS and/or the accumulation of Ral, as well as the GTP-Ras/RalGDS clusters, to induce the full activation of Ral.

  18. FGFR1 signaling in hypertrophic chondrocytes is attenuated by the Ras-GAP neurofibromin during endochondral bone formation

    PubMed Central

    Karolak, Matthew R.; Yang, Xiangli; Elefteriou, Florent

    2015-01-01

    Aberrant fibroblast growth factor receptor 3 (FGFR3) signaling disrupts chondrocyte proliferation and growth plate size and architecture, leading to various chondrodysplasias or bone overgrowth. These observations suggest that the duration, intensity and cellular context of FGFR signaling during growth plate chondrocyte maturation require tight, regulated control for proper bone elongation. However, the machinery fine-tuning FGFR signaling in chondrocytes is incompletely defined. We report here that neurofibromin, a Ras-GAP encoded by Nf1, has an overlapping expression pattern with FGFR1 and FGFR3 in prehypertrophic chondrocytes, and with FGFR1 in hypertrophic chondrocytes during endochondral ossification. Based on previous evidence that neurofibromin inhibits Ras-ERK signaling in chondrocytes and phenotypic analogies between mice with constitutive FGFR1 activation and Nf1 deficiency in Col2a1-positive chondrocytes, we asked whether neurofibromin is required to control FGFR1-Ras-ERK signaling in maturing chondrocytes in vivo. Genetic Nf1 ablation in Fgfr1-deficient chondrocytes reactivated Ras-ERK1/2 signaling in hypertrophic chondrocytes and reversed the expansion of the hypertrophic zone observed in mice lacking Fgfr1 in Col2a1-positive chondrocytes. Histomorphometric and gene expression analyses suggested that neurofibromin, by inhibiting Rankl expression, attenuates pro-osteoclastogenic FGFR1 signaling in hypertrophic chondrocytes. We also provide evidence suggesting that neurofibromin in prehypertrophic chondrocytes, downstream of FGFRs and via an indirect mechanism, is required for normal extension and organization of proliferative columns. Collectively, this study indicates that FGFR signaling provides an important input into the Ras-Raf-MEK-ERK1/2 signaling axis in chondrocytes, and that this input is differentially regulated during chondrocyte maturation by a complex intracellular machinery, of which neurofibromin is a critical component. PMID:25616962

  19. Co-Targeting the PI3K and RAS Pathways for the Treatment of Neuroendocrine Tumors

    PubMed Central

    Valentino, Joseph D.; Li, Jing; Zaytseva, Yekaterina Y.; Mustain, W. Conan; Elliott, Victoria A.; Kim, Ji Tae; Harris, Jennifer W.; Campbell, Katherine; Weiss, Heidi; Wang, Chi; Song, Jun; Anthony, Lowell; Townsend, Courtney M.; Evers, B. Mark

    2014-01-01

    Background The precise involvement of the PI3K/mTOR and RAS/MEK pathways in carcinoid tumors is not well defined. Therefore, the purpose of our study was to evaluate the role these pathways play in carcinoid cell proliferation, apoptosis, and secretion and to determine the effects of combined treatment on carcinoid tumor inhibition. Methods The human neuroendocrine cell lines BON (pancreatic carcinoid), NCI-H727 (lung carcinoid), and QGP-1 (somatostatinoma) were treated with either the pan-PI3K inhibitor, BKM120, or the dual PI3K-mTOR inhibitor, BEZ235, alone or in combination with the MEK inhibitor, PD0325901; proliferation, apoptosis, and protein expression were assessed. Peptide secretion was evaluated in BON and QGP-1 cells. The anti-proliferative effect of BEZ235, alone or combined with PD0325901, was then tested in vivo. Results Both BKM120 and BEZ235 decreased proliferation and increased apoptosis; combination with PD0325901 significantly enhanced the antineoplastic effects of either treatment alone. In contrast, neurotensin (NT) peptide secretion was markedly stimulated with BKM120 treatment, but not BEZ235. The combination of BEZ235 + PD0325901 significantly inhibited the growth of BON xenografts without systemic toxicity. Conclusions Both BKM120 and BEZ235 effectively inhibited NET cell proliferation and stimulated apoptosis. However, inhibition of the PI3K pathway alone with BKM120 significantly stimulated NT peptide secretion; this did not occur with the dual inhibition of both PI3K and mTOR using BEZ235 suggesting that this would be a more effective treatment regimen for NETs. Moreover, the combination of BEZ235 and the MEK inhibitor PD0325901 was a safe and more effective therapy in vivo compared with single agents alone. PMID:24443523

  20. A Model for Direction Sensing in Dictyostelium discoideum: Ras Activity and Symmetry Breaking Driven by a Gβγ-Mediated, Gα2-Ric8 -- Dependent Signal Transduction Network.

    PubMed

    Cheng, Yougan; Othmer, Hans

    2016-05-01

    Chemotaxis is a dynamic cellular process, comprised of direction sensing, polarization and locomotion, that leads to the directed movement of eukaryotic cells along extracellular gradients. As a primary step in the response of an individual cell to a spatial stimulus, direction sensing has attracted numerous theoretical treatments aimed at explaining experimental observations in a variety of cell types. Here we propose a new model of direction sensing based on experiments using Dictyostelium discoideum (Dicty). The model is built around a reaction-diffusion-translocation system that involves three main component processes: a signal detection step based on G-protein-coupled receptors (GPCR) for cyclic AMP (cAMP), a transduction step based on a heterotrimetic G protein Gα2βγ, and an activation step of a monomeric G-protein Ras. The model can predict the experimentally-observed response of cells treated with latrunculin A, which removes feedback from downstream processes, under a variety of stimulus protocols. We show that [Formula: see text] cycling modulated by Ric8, a nonreceptor guanine exchange factor for [Formula: see text] in Dicty, drives multiple phases of Ras activation and leads to direction sensing and signal amplification in cAMP gradients. The model predicts that both [Formula: see text] and Gβγ are essential for direction sensing, in that membrane-localized [Formula: see text], the activated GTP-bearing form of [Formula: see text], leads to asymmetrical recruitment of RasGEF and Ric8, while globally-diffusing Gβγ mediates their activation. We show that the predicted response at the level of Ras activation encodes sufficient 'memory' to eliminate the 'back-of-the wave' problem, and the effects of diffusion and cell shape on direction sensing are also investigated. In contrast with existing LEGI models of chemotaxis, the results do not require a disparity between the diffusion coefficients of the Ras activator GEF and the Ras inhibitor GAP. Since

  1. A Model for Direction Sensing in Dictyostelium discoideum: Ras Activity and Symmetry Breaking Driven by a Gβγ-Mediated, Gα2-Ric8 -- Dependent Signal Transduction Network

    PubMed Central

    Cheng, Yougan; Othmer, Hans

    2016-01-01

    Chemotaxis is a dynamic cellular process, comprised of direction sensing, polarization and locomotion, that leads to the directed movement of eukaryotic cells along extracellular gradients. As a primary step in the response of an individual cell to a spatial stimulus, direction sensing has attracted numerous theoretical treatments aimed at explaining experimental observations in a variety of cell types. Here we propose a new model of direction sensing based on experiments using Dictyostelium discoideum (Dicty). The model is built around a reaction-diffusion-translocation system that involves three main component processes: a signal detection step based on G-protein-coupled receptors (GPCR) for cyclic AMP (cAMP), a transduction step based on a heterotrimetic G protein Gα2βγ, and an activation step of a monomeric G-protein Ras. The model can predict the experimentally-observed response of cells treated with latrunculin A, which removes feedback from downstream processes, under a variety of stimulus protocols. We show that Gα2βγ cycling modulated by Ric8, a nonreceptor guanine exchange factor for Gα2 in Dicty, drives multiple phases of Ras activation and leads to direction sensing and signal amplification in cAMP gradients. The model predicts that both Gα2 and Gβγ are essential for direction sensing, in that membrane-localized Gα2*, the activated GTP-bearing form of Gα2, leads to asymmetrical recruitment of RasGEF and Ric8, while globally-diffusing Gβγ mediates their activation. We show that the predicted response at the level of Ras activation encodes sufficient ‘memory’ to eliminate the ‘back-of-the wave’ problem, and the effects of diffusion and cell shape on direction sensing are also investigated. In contrast with existing LEGI models of chemotaxis, the results do not require a disparity between the diffusion coefficients of the Ras activator GEF and the Ras inhibitor GAP. Since the signal pathways we study are highly conserved between Dicty

  2. Signalling pathways in pemphigus vulgaris.

    PubMed

    Li, Xiaoguang; Ishii, Norito; Ohata, Chika; Furumura, Minao; Hashimoto, Takashi

    2014-03-01

    Acantholysis in pemphigus vulgaris is induced by binding of autoantibodies to desmoglein 3 (Dsg3). The roles of signalling pathways on development of acantholysis have recently been extensively studied. In the study by Sayar et al., recently published in Exp Dermatol, epidermal growth factor receptor (EGFR) signalling was activated in both in vivo and in vitro pemphigus vulgaris experimental models. However, while EGFR inhibitors suppressed activity of p38 mitogen-activated protein kinase (p38MAPK) linearly, they suppressed activity of c-Myc and acantholysis in a non-linear, V-shaped relationship. These findings indicated complicated interactions among EGFR, p38MAPK and c-Myc in pemphigus vulgaris pathology.

  3. Suppression of sorbitol dependence in a strain bearing a mutation in the SRB1/PSA1/VIG9 gene encoding GDP-mannose pyrophosphorylase by PDE2 overexpression suggests a role for the Ras/cAMP signal-transduction pathway in the control of yeast cell-wall biogenesis.

    PubMed

    Tomlin, G C; Hamilton, G E; Gardner, D C; Walmsley, R M; Stateva, L I; Oliver, S G

    2000-09-01

    Complementation studies and allele replacement in Saccharomyces cerevisiae revealed that PSA1/VIG9, an essential gene that encodes GDP-mannose pyrophosphorylase, is the wild-type SRB1 gene. Cloning and sequencing of the srb1-1 allele showed that it determines a single amino acid change from glycine to aspartic acid at residue 276 (srb1(D276)). Genetic evidence is presented showing that at least one further mutation is required for the sorbitol dependence of srb1(D276). A previously reported complementing gene, which this study has now identified as PDE2, is a multi-copy suppressor of sorbitol dependence and is not, as was previously suggested, the SRB1 gene. srb and pde2 mutants share a number of phenotypes, including lysis upon hypotonic shock and enhanced transformability. These data are consistent with the idea that the Ras/cAMP pathway might modulate cell-wall construction.

  4. Human iPS Cell-Derived Neurons Uncover the Impact of Increased Ras Signaling in Costello Syndrome

    PubMed Central

    Rooney, Gemma E.; Goodwin, Alice F.; Depeille, Philippe; Sharir, Amnon; Schofield, Claude M.; Yeh, Erika; Roose, Jeroen P.; Klein, Ophir D.; Rauen, Katherine A.; Weiss, Lauren A.

    2016-01-01

    Increasing evidence implicates abnormal Ras signaling as a major contributor in neurodevelopmental disorders, yet how such signaling causes cortical pathogenesis is unknown. We examined the consequences of aberrant Ras signaling in the developing mouse brain and uncovered several critical phenotypes, including increased production of cortical neurons and morphological deficits. To determine whether these phenotypes are recapitulated in humans, we generated induced pluripotent stem (iPS) cell lines from patients with Costello syndrome (CS), a developmental disorder caused by abnormal Ras signaling and characterized by neurodevelopmental abnormalities, such as cognitive impairment and autism. Directed differentiation toward a neuroectodermal fate revealed an extended progenitor phase and subsequent increased production of cortical neurons. Morphological analysis of mature neurons revealed significantly altered neurite length and soma size in CS patients. This study demonstrates the synergy between mouse and human models and validates the use of iPS cells as a platform to study the underlying cellular pathologies resulting from signaling deficits. SIGNIFICANCE STATEMENT Increasing evidence implicates Ras signaling dysfunction as a major contributor in psychiatric and neurodevelopmental disorders, such as cognitive impairment and autism, but the underlying cortical cellular pathogenesis remains unclear. This study is the first to reveal human neuronal pathogenesis resulting from abnormal Ras signaling and provides insights into how these phenotypic abnormalities likely contribute to neurodevelopmental disorders. We also demonstrate the synergy between mouse and human models, thereby validating the use of iPS cells as a platform to study underlying cellular pathologies resulting from signaling deficits. Recapitulating human cellular pathologies in vitro facilitates the future high throughput screening of potential therapeutic agents that may reverse phenotypic and

  5. Superoxide Inhibits Guanine Nucleotide Exchange Factor (GEF) Action on Ras, but not on Rho, through Desensitization of Ras to GEF

    PubMed Central

    2015-01-01

    Ras and Rho GTPases are molecular switches for various vital cellular signaling pathways. Overactivation of these GTPases often causes development of cancer. Guanine nucleotide exchange factors (GEFs) and oxidants function to upregulate these GTPases through facilitation of guanine nucleotide exchange (GNE) of these GTPases. However, the effect of oxidants on GEF functions, or vice versa, has not been known. We show that, via targeting Ras Cys51, an oxidant inhibits the catalytic action of Cdc25—the catalytic domain of RasGEFs—on Ras. However, the enhancement of Ras GNE by an oxidant continues regardless of the presence of Cdc25. Limiting RasGEF action by an oxidant may function to prevent the pathophysiological overactivation of Ras in the presence of both RasGEFs and oxidants. The continuous exposure of Ras to nitric oxide and its derivatives can form S-nitrosated Ras (Ras-SNO). This study also shows that an oxidant not only inhibits the catalytic action of Cdc25 on Ras-SNO but also fails to enhance Ras-SNO GNE. This lack of enhancement then populates the biologically inactive Ras-SNO in cells, which may function to prevent the continued redox signaling of the Ras pathophysiological response. Finally, this study also demonstrates that, unlike the case with RasGEFs, an oxidant does not inhibit the catalytic action of RhoGEF—Vav or Dbs—on Rho GTPases such as Rac1, RhoA, RhoC, and Cdc42. This result explains the results of the previous study in which, despite the presence of an oxidant, the catalytic action of Dbs in cells continued to enhance RhoC GNE. PMID:24422478

  6. Human Papillomavirus Type 6b Virus-Like Particles Are Able To Activate the Ras-MAP Kinase Pathway and Induce Cell Proliferation

    PubMed Central

    Payne, Elizabeth; Bowles, Mark R.; Don, Alistair; Hancock, John F.; McMillan, Nigel A. J.

    2001-01-01

    The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the α6 integrin. The α6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. Here we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in cell proliferation, suggesting involvement of the Ras-MAP kinase pathway. Indeed, VLP binding results in rapid phosphorylation of the β4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein Shc to β4. Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c-myc mRNA was observed at 60 min. These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells. PMID:11287564

  7. BRAF and RAS oncogenes regulate Rho GTPase pathways to mediate migration and invasion properties in human colon cancer cells: a comparative study

    PubMed Central

    2011-01-01

    Background Colorectal cancer is a common disease that involves genetic alterations, such as inactivation of tumour suppressor genes and activation of oncogenes. Among them are RAS and BRAF mutations, which rarely coexist in the same tumour. Individual members of the Rho (Ras homology) GTPases contribute with distinct roles in tumour cell morphology, invasion and metastasis. The aim of this study is to dissect cell migration and invasion pathways that are utilised by BRAFV600E as compared to KRASG12V and HRASG12V oncoproteins. In particular, the role of RhoA (Ras homolog gene family, member A), Rac1 (Ras-related C3 botulinum toxin substrate 1) and Cdc42 (cell division cycle 42) in cancer progression induced by each of the three oncogenes is described. Methods Colon adenocarcinoma cells with endogenous as well as ectopically expressed or silenced oncogenic mutations of BRAFV600E, KRASG12V and HRASG12V were employed. Signalling pathways and Rho GTPases were inhibited with specific kinase inhibitors and siRNAs. Cell motility and invasion properties were correlated with cytoskeletal properties and Rho GTPase activities. Results Evidence presented here indicate that BRAFV600E significantly induces cell migration and invasion properties in vitro in colon cancer cells, at least in part through activation of RhoA GTPase. The relationship established between BRAFV600E and RhoA activation is mediated by the MEK-ERK pathway. In parallel, KRASG12V enhances the ability of colon adenocarcinoma cells Caco-2 to migrate and invade through filopodia formation and PI3K-dependent Cdc42 activation. Ultimately increased cell migration and invasion, mediated by Rac1, along with the mesenchymal morphology obtained through the Epithelial-Mesenchymal Transition (EMT) were the main characteristics rendered by HRASG12V in Caco-2 cells. Moreover, BRAF and KRAS oncogenes are shown to cooperate with the TGFβ-1 pathway to provide cells with additional transforming properties. Conclusion This

  8. CD99 triggering induces methuosis of Ewing sarcoma cells through IGF-1R/RAS/Rac1 signaling

    PubMed Central

    Manara, Maria Cristina; Terracciano, Mario; Mancarella, Caterina; Sciandra, Marika; Guerzoni, Clara; Pasello, Michela; Grilli, Andrea; Zini, Nicoletta; Picci, Piero; Colombo, Mario P.; Morrione, Andrea; Scotlandi, Katia

    2016-01-01

    CD99 is a cell surface molecule that has emerged as a novel target for Ewing sarcoma (EWS), an aggressive pediatric bone cancer. This report provides the first evidence of methuosis in EWS, a non-apoptotic form of cell death induced by an antibody directed against the CD99 molecule. Upon mAb triggering, CD99 induces an IGF-1R/RAS/Rac1 complex, which is internalized into RAB5-positive endocytic vacuoles. This complex is then dissociated, with the IGF-1R recycling to the cell membrane while CD99 and RAS/Rac1 are sorted into immature LAMP-1-positive vacuoles, whose excessive accumulation provokes methuosis. This process, which is not detected in CD99-expressing normal mesenchymal cells, is inhibited by disruption of the IGF-1R signaling, whereas enhanced by IGF-1 stimulation. Induction of IGF-1R/RAS/Rac1 was also observed in the EWS xenografts that respond to anti-CD99 mAb, further supporting the role of the IGF/RAS/Rac1 axis in the hyperstimulation of macropinocytosis and selective death of EWS cells. Thus, we describe a vulnerability of EWS cells, including those resistant to standard chemotherapy, to a treatment with anti-CD99 mAb, which requires IGF-1R/RAS signaling but bypasses the need for their direct targeting. Overall, we propose CD99 targeting as new opportunity to treat EWS patients resistant to canonical apoptosis-inducing agents. PMID:27835596

  9. The third international meeting on genetic disorders in the RAS/MAPK pathway: towards a therapeutic approach.

    PubMed

    Korf, Bruce; Ahmadian, Reza; Allanson, Judith; Aoki, Yoko; Bakker, Annette; Wright, Emma Burkitt; Denger, Brian; Elgersma, Ype; Gelb, Bruce D; Gripp, Karen W; Kerr, Bronwyn; Kontaridis, Maria; Lazaro, Conxi; Linardic, Corinne; Lozano, Reymundo; MacRae, Calum A; Messiaen, Ludwine; Mulero-Navarro, Sonia; Neel, Benjamin; Plotkin, Scott; Rauen, Katherine A; Roberts, Amy; Silva, Alcino J; Sittampalam, Sitta G; Zhang, Chao; Schoyer, Lisa

    2015-08-01

    "The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Towards a Therapeutic Approach" was held at the Renaissance Orlando at SeaWorld Hotel (August 2-4, 2013). Seventy-one physicians and scientists attended the meeting, and parallel meetings were held by patient advocacy groups (CFC International, Costello Syndrome Family Network, NF Network and Noonan Syndrome Foundation). Parent and patient advocates opened the meeting with a panel discussion to set the stage regarding their hopes and expectations for therapeutic advances. In keeping with the theme on therapeutic development, the sessions followed a progression from description of the phenotype and definition of therapeutic endpoints, to definition of genomic changes, to identification of therapeutic targets in the RAS/MAPK pathway, to preclinical drug development and testing, to clinical trials. These proceedings will review the major points of discussion.

  10. The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Toward a Therapeutic Approach

    PubMed Central

    Korf, Bruce; Ahmadian, Reza; Allanson, Judith; Aoki, Yoko; Bakker, Annette; Wright, Emma Burkitt; Denger, Brian; Elgersma, Ype; Gelb, Bruce D.; Gripp, Karen W.; Kerr, Bronwyn; Kontaridis, Maria; Lazaro, Conxi; Linardic, Corinne; Lozano, Reymundo; MacRae, Calum A.; Messiaen, Ludwine; Mulero-Navarro, Sonia; Neel, Benjamin; Plotkin, Scott; Rauen, Katherine A.; Roberts, Amy; Silva, Alcino J.; Sittampalam, Sitta G.; Zhang, Chao; Schoyer, Lisa

    2015-01-01

    “The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Towards a Therapeutic Approach” was held at the Renaissance Orlando at SeaWorld Hotel (August 2–4, 2013). Seventy-one physicians and scientists attended the meeting, and parallel meetings were held by patient advocacy groups (CFC International, Costello Syndrome Family Network, NF Network and Noonan Syndrome Foundation). Parent and patient advocates opened the meeting with a panel discussion to set the stage regarding their hopes and expectations for therapeutic advances. In keeping with the theme on therapeutic development, the sessions followed a progression from description of the phenotype and definition of therapeutic endpoints, to definition of genomic changes, to identification of therapeutic targets in the RAS/MAPK pathway, to preclinical drug development and testing, to clinical trials. These proceedings will review the major points of discussion. PMID:25900621

  11. Neurofibromatosis-Noonan syndrome: case report and clinicopathogenic review of the Neurofibromatosis-Noonan syndrome and RAS-MAPK pathway.

    PubMed

    Reig, Irela; Boixeda, Pablo; Fleta, Beatriz; Morenoc, Carmen; Gámez, Lucía; Truchuelo, Mayte

    2011-04-15

    Neurofibromatosis-Noonan syndrome is an entity that combines both features of Noonan syndrome and Neurofibromatosis type 1. This phenotypic overlap can be explained by the involvement of the RAS-MAPK pathway (mitogen-activated protein kinase) in both disorders. We report the case of a 17-year-old boy with Neurofibromatosis 1 with Noonan-like features, who complained of the progressive appearance of blue-gray lesions on his back.

  12. Novel Gbeta Mimic Kelch Proteins (Gpb1 and Gpb2 Connect G-Protein Signaling to Ras via Yeast Neurofibromin Homologs Ira1 and Ira2: A Model for Human NF1

    DTIC Science & Technology

    2008-03-01

    the level of pigmentation produced following 2 or 4 days incubation compared to the isogenic wild type strains and mutants with defects in the enzyme...coupling, Gpb1/2 serve as potent molecular brakes to constrain signaling via the PKA signaling pathway during both vegetative growth and dimorphic...In ad- dition to regulating Ras activity, neurofibromin also gov- erns G protein-mediated adenylyl cyclase activity in the fruit fly Drosophila

  13. Key cancer cell signal transduction pathways as therapeutic targets.

    PubMed

    Bianco, Roberto; Melisi, Davide; Ciardiello, Fortunato; Tortora, Giampaolo

    2006-02-01

    Growth factor signals are propagated from the cell surface, through the action of transmembrane receptors, to intracellular effectors that control critical functions in human cancer cells, such as differentiation, growth, angiogenesis, and inhibition of cell death and apoptosis. Several kinases are involved in transduction pathways via sequential signalling activation. These kinases include transmembrane receptor kinases (e.g., epidermal growth factor receptor EGFR); or cytoplasmic kinases (e.g., PI3 kinase). In cancer cells, these signalling pathways are often altered and results in a phenotype characterized by uncontrolled growth and increased capability to invade surrounding tissue. Therefore, these crucial transduction molecules represent attractive targets for cancer therapy. This review will summarize current knowledge of key signal transduction pathways, that are altered in cancer cells, as therapeutic targets for novel selective inhibitors. The most advanced targeted agents currently under development interfere with function and expression of several signalling molecules, including the EGFR family; the vascular endothelial growth factor and its receptors; and cytoplasmic kinases such as Ras, PI3K and mTOR.

  14. Differential Expression of IL-17, 22 and 23 in the Progression of Colorectal Cancer in Patients with K-ras Mutation: Ras Signal Inhibition and Crosstalk with GM-CSF and IFN-γ

    PubMed Central

    Petanidis, Savvas; Anestakis, Doxakis; Argyraki, Maria; Hadzopoulou-Cladaras, Margarita; Salifoglou, Athanasios

    2013-01-01

    Recent studies have suggested that aberrant K-ras signaling is responsible for triggering immunological responses and inflammation-driven tumorigenesis. Interleukins IL-17, IL-22, and IL-23 have been reported in various types of malignancies, but the exact mechanistic role of these molecules remains to be elucidated. Given the role of K-ras and the involvement of interleukins in colorectal tumorigenesis, research efforts are reported for the first time, showing that differentially expressed interleukin IL-17, IL-22, and IL-23 levels are associated with K-ras in a stage-specific fashion along colorectal cancer progression. Specifically, a) the effect of K-ras signaling was investigated in the overall expression of interleukins in patients with colorectal cancer and healthy controls, and b) an association was established between mutant K-ras and cytokines GM-CSF and IFN-γ. The results indicate that specific interleukins are differentially expressed in K-ras positive patients and the use of K-ras inhibitor Manumycin A decreases both interleukin levels and apoptosis in Caco-2 cells by inhibiting cell viability. Finally, inflammation-driven GM-CSF and IFN-γ levels are modulated through interleukin expression in tumor patients, with interleukin expression in the intestinal lumen and cancerous tissue mediated by aberrant K-ras signaling. Collectively, the findings a) indicate that interleukin expression is influenced by ras signaling and specific interleukins play an oncogenic promoter role in colorectal cancer, highlighting the molecular link between inflammation and tumorigenesis, and b) accentuate the interwoven molecular correlations as leads to new therapeutic approaches in the future. PMID:24040001

  15. Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence*

    PubMed Central

    Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L.; Embrey, Kevin J.; Golovanov, Alexander P.

    2016-01-01

    The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly 15N-labeled Ras as well as [13C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions. PMID:26565026

  16. Single-molecule fluorescence imaging of RalGDS on cell surfaces during signal transduction from Ras to Ral

    PubMed Central

    Yoshizawa, Ryo; Umeki, Nobuhisa; Yanagawa, Masataka; Murata, Masayuki; Sako, Yasushi

    2017-01-01

    RalGDS is one of the Ras effectors and functions as a guanine nucleotide exchange factor for the small G-protein, Ral, which regulates membrane trafficking and cytoskeletal remodeling. The translocation of RalGDS from the cytoplasm to the plasma membrane is required for Ral activation. In this study, to understand the mechanism of Ras–Ral signaling we performed a single-molecule fluorescence analysis of RalGDS and its functional domains (RBD and REMCDC) on the plasma membranes of living HeLa cells. Increased molecular density of RalGDS and RBD, but not REMCDC, was observed on the plasma membrane after EGF stimulation of the cells to induce Ras activation, suggesting that the translocation of RalGDS involves an interaction between the GTP-bound active form of Ras and the RBD of RalGDS. Whereas the RBD played an important role in increasing the association rate constant between RalGDS and the plasma membrane, the REMCDC domain affected the dissociation rate constant from the membrane, which decreased after Ras activation or the hyperexpression of Ral. The Y64 residue of Ras and clusters of RalGDS molecules were involved in this reduction. From these findings, we infer that Ras activation not merely increases the cell-surface density of RalGDS, but actively stimulates the RalGDS–Ral interaction through a structural change in RalGDS and/or the accumulation of Ral, as well as the GTP–Ras/RalGDS clusters, to induce the full activation of Ral. PMID:28744424

  17. Gankyrin plays an essential role in Ras-induced tumorigenesis through regulation of the RhoA/ROCK pathway in mammalian cells

    PubMed Central

    Man, Jiang-Hong; Liang, Bing; Gu, Yue-Xi; Zhou, Tao; Li, Ai-Ling; Li, Tao; Jin, Bao-Feng; Bai, Bing; Zhang, Hai-Ying; Zhang, Wei-Na; Li, Wei-Hua; Gong, Wei-Li; Li, Hui-Yan; Zhang, Xue-Min

    2010-01-01

    Activating mutations in Ras proteins are present in about 30% of human cancers. Despite tremendous progress in the study of Ras oncogenes, many aspects of the molecular mechanisms underlying Ras-induced tumorigenesis remain unknown. Through proteomics analysis, we previously found that the protein Gankyrin, a known oncoprotein in hepatocellular carcinoma, was upregulated during Ras-mediated transformation, although the functional consequences of this were not clear. Here we present evidence that Gankyrin plays an essential role in Ras-initiated tumorigenesis in mouse and human cells. We found that the increased Gankyrin present following Ras activation increased the interaction between the RhoA GTPase and its GDP dissociation inhibitor RhoGDI, which resulted in inhibition of the RhoA effector kinase Rho-associated coiled coil–containing protein kinase (ROCK). Importantly, Gankyrin-mediated ROCK inhibition led to prolonged Akt activation, a critical step in activated Ras–induced transformation and tumorigenesis. In addition, we found that Gankyrin is highly expressed in human lung cancers that have Ras mutations and that increased Gankyrin expression is required for the constitutive activation of Akt and tumorigenesis in these lung cancers. Our findings suggest that Gankyrin is a key regulator of Ras-mediated activation of Akt through inhibition of the downstream RhoA/ROCK pathway and thus plays an essential role in Ras-induced tumorigenesis. PMID:20628200

  18. Gain-of-Function Mutations in RIT1 Cause Noonan Syndrome, a RAS/MAPK Pathway Syndrome

    PubMed Central

    Aoki, Yoko; Niihori, Tetsuya; Banjo, Toshihiro; Okamoto, Nobuhiko; Mizuno, Seiji; Kurosawa, Kenji; Ogata, Tsutomu; Takada, Fumio; Yano, Michihiro; Ando, Toru; Hoshika, Tadataka; Barnett, Christopher; Ohashi, Hirofumi; Kawame, Hiroshi; Hasegawa, Tomonobu; Okutani, Takahiro; Nagashima, Tatsuo; Hasegawa, Satoshi; Funayama, Ryo; Nagashima, Takeshi; Nakayama, Keiko; Inoue, Shin-ichi; Watanabe, Yusuke; Ogura, Toshihiko; Matsubara, Yoichi

    2013-01-01

    RAS GTPases mediate a wide variety of cellular functions, including cell proliferation, survival, and differentiation. Recent studies have revealed that germline mutations and mosaicism for classical RAS mutations, including those in HRAS, KRAS, and NRAS, cause a wide spectrum of genetic disorders. These include Noonan syndrome and related disorders (RAS/mitogen-activated protein kinase [RAS/MAPK] pathway syndromes, or RASopathies), nevus sebaceous, and Schimmelpenning syndrome. In the present study, we identified a total of nine missense, nonsynonymous mutations in RIT1, encoding a member of the RAS subfamily, in 17 of 180 individuals (9%) with Noonan syndrome or a related condition but with no detectable mutations in known Noonan-related genes. Clinical manifestations in the RIT1-mutation-positive individuals are consistent with those of Noonan syndrome, which is characterized by distinctive facial features, short stature, and congenital heart defects. Seventy percent of mutation-positive individuals presented with hypertrophic cardiomyopathy; this frequency is high relative to the overall 20% incidence in individuals with Noonan syndrome. Luciferase assays in NIH 3T3 cells showed that five RIT1 alterations identified in children with Noonan syndrome enhanced ELK1 transactivation. The introduction of mRNAs of mutant RIT1 into 1-cell-stage zebrafish embryos was found to result in a significant increase of embryos with craniofacial abnormalities, incomplete looping, a hypoplastic chamber in the heart, and an elongated yolk sac. These results demonstrate that gain-of-function mutations in RIT1 cause Noonan syndrome and show a similar biological effect to mutations in other RASopathy-related genes. PMID:23791108

  19. Gain-of-function mutations in RIT1 cause Noonan syndrome, a RAS/MAPK pathway syndrome.

    PubMed

    Aoki, Yoko; Niihori, Tetsuya; Banjo, Toshihiro; Okamoto, Nobuhiko; Mizuno, Seiji; Kurosawa, Kenji; Ogata, Tsutomu; Takada, Fumio; Yano, Michihiro; Ando, Toru; Hoshika, Tadataka; Barnett, Christopher; Ohashi, Hirofumi; Kawame, Hiroshi; Hasegawa, Tomonobu; Okutani, Takahiro; Nagashima, Tatsuo; Hasegawa, Satoshi; Funayama, Ryo; Nagashima, Takeshi; Nakayama, Keiko; Inoue, Shin-Ichi; Watanabe, Yusuke; Ogura, Toshihiko; Matsubara, Yoichi

    2013-07-11

    RAS GTPases mediate a wide variety of cellular functions, including cell proliferation, survival, and differentiation. Recent studies have revealed that germline mutations and mosaicism for classical RAS mutations, including those in HRAS, KRAS, and NRAS, cause a wide spectrum of genetic disorders. These include Noonan syndrome and related disorders (RAS/mitogen-activated protein kinase [RAS/MAPK] pathway syndromes, or RASopathies), nevus sebaceous, and Schimmelpenning syndrome. In the present study, we identified a total of nine missense, nonsynonymous mutations in RIT1, encoding a member of the RAS subfamily, in 17 of 180 individuals (9%) with Noonan syndrome or a related condition but with no detectable mutations in known Noonan-related genes. Clinical manifestations in the RIT1-mutation-positive individuals are consistent with those of Noonan syndrome, which is characterized by distinctive facial features, short stature, and congenital heart defects. Seventy percent of mutation-positive individuals presented with hypertrophic cardiomyopathy; this frequency is high relative to the overall 20% incidence in individuals with Noonan syndrome. Luciferase assays in NIH 3T3 cells showed that five RIT1 alterations identified in children with Noonan syndrome enhanced ELK1 transactivation. The introduction of mRNAs of mutant RIT1 into 1-cell-stage zebrafish embryos was found to result in a significant increase of embryos with craniofacial abnormalities, incomplete looping, a hypoplastic chamber in the heart, and an elongated yolk sac. These results demonstrate that gain-of-function mutations in RIT1 cause Noonan syndrome and show a similar biological effect to mutations in other RASopathy-related genes.

  20. HIV-1/Cocaine Induced Oxidative Stress Disrupts Tight Junction Protein-1 in Human Pulmonary Microvascular Endothelial Cells: Role of Ras/ERK1/2 Pathway

    PubMed Central

    Mermis, Joel; Zeng, Ruoxi; Sanderson, Miles; Johnson, Sara; Dai, Yuqiao; Sharma, Garima; Ladner, Amy O’Brien; Dhillon, Navneet K.

    2014-01-01

    Intravenous drug use (IVDU) is the major risk factor in the development of HIV-related pulmonary arterial hypertension (HRPAH); however, the pathogenesis of HRPAH in association with IVDU has yet to be characterized. Endothelial injury is considered to be an initiating factor for pulmonary vascular remodeling in animal models of PAH. Our previous study shows that simultaneous exposure to HIV-Trans-activator of transcription (Tat) and cocaine exacerbates both disruption of tight junction proteins and permeability of human pulmonary artery endothelial cells compared with either treatment alone. We here now demonstrate that this HIV-Tat and cocaine mediated endothelial dysfunction accompanies with increase in hydrogen peroxide and superoxide radicals generation and involves redox sensitive signaling pathway. Pretreatment with antioxidant cocktail attenuated the cocaine and Tat mediated disassembly of Zonula Occludens (ZO)-1 and enhancement of endothelial monolayer permeability. Furthermore, inhibition of NADPH oxidase by apocynin or siRNA-mediated knockdown of gp-91phox abolished the Tat/cocaine-induced reactive oxygen species (ROS) production, suggesting the NADPH oxidase mediated generation of oxidative radicals. In addition, ROS dependent activation of Ras and ERK1/2 Kinase was observed to be mediating the TJP-1 disassembly, and endothelial dysfunction in response to cocaine and Tat exposure. In conclusion, our findings demonstrate that Tat/cocaine -mediated production of ROS activate Ras/Raf/ERK1/2 pathway that contributes to disruption of tight junction protein leading to pulmonary endothelial dysfunction associated with pulmonary vascular remodeling. PMID:24409324

  1. The Ras-MAPK pathway downregulates Caveolin-1 in rodent fibroblast but not in human fibroblasts: implications in the resistance to oncogene-mediated transformation.

    PubMed

    Sasai, K; Kakumoto, K; Hanafusa, H; Akagi, T

    2007-01-18

    Normal human diploid fibroblasts (HDFs) are refractory to oncogene-mediated transformations in vitro, compared with rodent fibroblasts. As successful oncogene-mediated transformations of normal HDFs have been reported using the human telomerase catalytic subunit, it has been considered that telomerase activity contributes to the species-specific transformability. However, these transformed HDFs are much less malignant compared with those of rodent cells, suggesting the existence of undefined mechanisms that render HDFs resistant to malignant transformation. Here, cDNA microarray analysis identified caveolin-1 as one of the possible cellular factors involved in such mechanisms. The mitogen-activated protein kinases (MAPK) pathway downregulates Caveolin-1 in rodent fibroblasts, transformed by coexpression of the SV40 early region and activated H-Ras. In contrast, the coexpression of these two oncogenes in HDFs failed to reduce the expression level of Caveolin-1. These results strongly suggest the presence of critical differences in events following the phosphorylation of ERK during the activation process of the MAPK signaling pathway between human and rodent cells, as the ERK protein was similarly phosphorylated in both systems. Furthermore, the small interfering RNA-mediated suppression of Caveolin-1 facilitated the oncogene-mediated transformation of normal HDFs, clearly indicating that the differences in the transformability between human and rodent cells are due, at least in part, to the mechanism responsible for the resistance to Ras-induced Caveolin-1 downregulation in HDFs.

  2. Rck1 up-regulates pseudohyphal growth by activating the Ras2 and MAP kinase pathways independently in Saccharomyces cerevisiae.

    PubMed

    Chang, Miwha; Kang, Chang-Min; Park, Yong-Sung; Yun, Cheol-Won

    2014-02-21

    Previously, we reported that Rck1 regulates Hog1 and Slt2 activities and affects MAP kinase activity in Saccharomyces cerevisiae. Recently, we found that Rck1 up-regulates phospho-Kss1 and phospho-Fus3. Kss1 has been known as a component in the pseudohyphal growth pathway, and we attempted to identify the function of Rck1 in pseudohyphal growth. Rck1 up-regulated Ras2 at the protein level, not the transcriptional level. Additionally, FLO11 transcription was up-regulated by RCK1 over-expression. RCK1 expression was up-regulated during growth on SLAD+1% butanol medium. On nitrogen starvation agar plates, RCK1 over-expression induced pseudohyphal growth of colonies, and cells over-expressing RCK1 showed a filamentous morphology when grown in SLAD medium. Furthermore, 1-butanol greatly induced filamentous growth when RCK1 was over-expressed. Moreover, invasive growth was activated in haploid cells when RCK1 was over-expressed. The growth defect of cells observed on 1-butanol medium was recovered when RCK1 was over-expressed. Interestingly, Ras2 and phospho-Kss1 were up-regulated by Rck1 independently. Together, these results suggest that Rck1 promotes pseudohyphal growth by activating Ras2 and Kss1 via independent pathways in S. cerevisiae. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Proceedings from the 2009 Genetic Syndromes of the Ras/MAPK Pathway: From Bedside to Bench and Back

    PubMed Central

    Rauen, Katherine A.; Schoyer, Lisa; McCormick, Frank; Lin, Angela E.; Allanson, Judith E.; Stevenson, David A.; Gripp, Karen W.; Neri, Giovanni; Carey, John C.; Legius, Eric; Tartaglia, Marco; Schubbert, Suzanne; Roberts, Amy E.; Gelb, Bruce D.; Shannon, Kevin; Gutmann, David H.; McMahon, Martin; Guerra, Carmen; Fagin, James A.; Yu, Benjamin; Aoki, Yoko; Neel, Ben G.; Balmain, Allan; Drake, Richard R.; Nolan, Garry P.; Zenker, Martin; Bollag, Gideon; Sebolt-Leopold, Judith; Gibbs, Jackson B.; Silva, Alcino J.; Patton, E. Elizabeth; Viskochil, David H.; Kieran, Mark W.; Korf, Bruce R.; Hagerman, Randi J.; Packer, Roger J.; Melese, Teri

    2012-01-01

    The RASopathies are a group of genetic syndromes caused by germline mutations in genes that encode components of the Ras/mitogen-activated protein kinase (MAPK) pathway. Some of these syndromes are neurofibromatosis type 1, Noonan syndrome, Costello syndrome, cardio-facio-cutaneous syndrome, LEOPARD syndrome and Legius syndrome. Their common underlying pathogenetic mechanism brings about significant overlap in phenotypic features and includes craniofacial dysmorphology, cardiac, cutaneous, musculoskeletal, GI and ocular abnormalities, and a predisposition to cancer. The proceedings from the symposium “Genetic Syndromes of the Ras/MAPK Pathway: From Bedside to Bench and Back” chronicle the timely and typical research symposium which brought together clinicians, basic scientists, physician-scientists, advocate leaders, trainees, students and individuals with Ras syndromes and their families. The goals, to discuss basic science and clinical issues, to set forth a solid framework for future research, to direct translational applications towards therapy and to set forth best practices for individuals with RASopathies was successfully meet with a commitment to begin to move towards clinical trials. PMID:20014119

  4. Stress and radiation-induced activation of multiple intracellular signaling pathways.

    PubMed

    Dent, Paul; Yacoub, Adly; Contessa, Joseph; Caron, Ruben; Amorino, George; Valerie, Kristoffer; Hagan, Michael P; Grant, Steven; Schmidt-Ullrich, Rupert

    2003-03-01

    Exposure of cells to a variety of stresses induces compensatory activations of multiple intracellular signaling pathways. These activations can play critical roles in controlling cell survival and repopulation effects in a stress-specific and cell type-dependent manner. Some stress-induced signaling pathways are those normally activated by mitogens such as the EGFR/RAS/PI3K-MAPK pathway. Other pathways activated by stresses such as ionizing radiation include those downstream of death receptors, including pro-caspases and the transcription factor NFKB. This review will attempt to describe some of the complex network of signals induced by ionizing radiation and other cellular stresses in animal cells, with particular attention to signaling by growth factor and death receptors. This includes radiation-induced signaling via the EGFR and IGFI-R to the PI3K, MAPK, JNK, and p38 pathways as well as FAS-R and TNF-R signaling to pro-caspases and NFKB. The roles of autocrine ligands in the responses of cells and bystander cells to radiation and cellular stresses will also be discussed. Based on the data currently available, it appears that radiation can simultaneously activate multiple signaling pathways in cells. Reactive oxygen and nitrogen species may play an important role in this process by inhibiting protein tyrosine phosphatase activity. The ability of radiation to activate signaling pathways may depend on the expression of growth factor receptors, autocrine factors, RAS mutation, and PTEN expression. In other words, just because pathway X is activated by radiation in one cell type does not mean that pathway X will be activated in a different cell type. Radiation-induced signaling through growth factor receptors such as the EGFR may provide radioprotective signals through multiple downstream pathways. In some cell types, enhanced basal signaling by proto-oncogenes such as RAS may provide a radioprotective signal. In many cell types, this may be through PI3K, in others

  5. The canonical Wnt signaling pathway in autism.

    PubMed

    Zhang, Yinghua; Yuan, Xiangshan; Wang, Zhongping; Li, Ruixi

    2014-01-01

    Mounting attention is being focused on the canonical Wnt signaling pathway which has been implicated in the pathogenesis of autism in some our and other recent studies. The canonical Wnt pathway is involved in cell proliferation, differentiation and migration, especially during nervous system development. Given its various functions, dysfunction of the canonical Wnt pathway may exert adverse effects on neurodevelopment and therefore leads to the pathogenesis of autism. Here, we review human and animal studies that implicate the canonical Wnt signal transduction pathway in the pathogenesis of autism. We also describe the crosstalk between the canonical Wnt pathway and the Notch signaling pathway in several types of autism spectrum disorders, including Asperger syndrome and Fragile X. Further research on the crosstalk between the canonical Wnt signaling pathway and other signaling cascades in autism may be an efficient avenue to understand the etiology of autism and ultimately lead to alternative medications for autism-like phenotypes.

  6. Regulation of Caenorhabditis elegans p53/CEP-1–Dependent Germ Cell Apoptosis by Ras/MAPK Signaling

    PubMed Central

    Rutkowski, Rachael; Dickinson, Robin; Stewart, Graeme; Craig, Ashley; Schimpl, Marianne; Keyse, Stephen M.; Gartner, Anton

    2011-01-01

    Maintaining genome stability in the germline is thought to be an evolutionarily ancient role of the p53 family. The sole Caenorhabditis elegans p53 family member CEP-1 is required for apoptosis induction in meiotic, late-stage pachytene germ cells in response to DNA damage and meiotic recombination failure. In an unbiased genetic screen for negative regulators of CEP-1, we found that increased activation of the C. elegans ERK orthologue MPK-1, resulting from either loss of the lip-1 phosphatase or activation of let-60 Ras, results in enhanced cep-1–dependent DNA damage induced apoptosis. We further show that MPK-1 is required for DNA damage–induced germ cell apoptosis. We provide evidence that MPK-1 signaling regulates the apoptotic competency of germ cells by restricting CEP-1 protein expression to cells in late pachytene. Restricting CEP-1 expression to cells in late pachytene is thought to ensure that apoptosis doesn't occur in earlier-stage cells where meiotic recombination occurs. MPK-1 signaling regulates CEP-1 expression in part by regulating the levels of GLD-1, a translational repressor of CEP-1, but also via a GLD-1–independent mechanism. In addition, we show that MPK-1 is phosphorylated and activated upon ionising radiation (IR) in late pachytene germ cells and that MPK-1–dependent CEP-1 activation may be in part direct, as these two proteins interact in a yeast two-hybrid assay. In summary, we report our novel finding that MAP kinase signaling controls CEP-1–dependent apoptosis by several different pathways that converge on CEP-1. Since apoptosis is also restricted to pachytene stage cells in mammalian germlines, analogous mechanisms regulating p53 family members are likely to be conserved throughout evolution. PMID:21901106

  7. Regulation of Caenorhabditis elegans p53/CEP-1-dependent germ cell apoptosis by Ras/MAPK signaling.

    PubMed

    Rutkowski, Rachael; Dickinson, Robin; Stewart, Graeme; Craig, Ashley; Schimpl, Marianne; Keyse, Stephen M; Gartner, Anton

    2011-08-01

    Maintaining genome stability in the germline is thought to be an evolutionarily ancient role of the p53 family. The sole Caenorhabditis elegans p53 family member CEP-1 is required for apoptosis induction in meiotic, late-stage pachytene germ cells in response to DNA damage and meiotic recombination failure. In an unbiased genetic screen for negative regulators of CEP-1, we found that increased activation of the C. elegans ERK orthologue MPK-1, resulting from either loss of the lip-1 phosphatase or activation of let-60 Ras, results in enhanced cep-1-dependent DNA damage induced apoptosis. We further show that MPK-1 is required for DNA damage-induced germ cell apoptosis. We provide evidence that MPK-1 signaling regulates the apoptotic competency of germ cells by restricting CEP-1 protein expression to cells in late pachytene. Restricting CEP-1 expression to cells in late pachytene is thought to ensure that apoptosis doesn't occur in earlier-stage cells where meiotic recombination occurs. MPK-1 signaling regulates CEP-1 expression in part by regulating the levels of GLD-1, a translational repressor of CEP-1, but also via a GLD-1-independent mechanism. In addition, we show that MPK-1 is phosphorylated and activated upon ionising radiation (IR) in late pachytene germ cells and that MPK-1-dependent CEP-1 activation may be in part direct, as these two proteins interact in a yeast two-hybrid assay. In summary, we report our novel finding that MAP kinase signaling controls CEP-1-dependent apoptosis by several different pathways that converge on CEP-1. Since apoptosis is also restricted to pachytene stage cells in mammalian germlines, analogous mechanisms regulating p53 family members are likely to be conserved throughout evolution.

  8. DA-Raf-Mediated Suppression of the Ras--ERK Pathway Is Essential for TGF-β1-Induced Epithelial-Mesenchymal Transition in Alveolar Epithelial Type 2 Cells.

    PubMed

    Watanabe-Takano, Haruko; Takano, Kazunori; Hatano, Masahiko; Tokuhisa, Takeshi; Endo, Takeshi

    2015-01-01

    Myofibroblasts play critical roles in the development of idiopathic pulmonary fibrosis by depositing components of extracellular matrix. One source of lung myofibroblasts is thought to be alveolar epithelial type 2 cells that undergo epithelial-mesenchymal transition (EMT). Rat RLE-6TN alveolar epithelial type 2 cells treated with transforming growth factor-β1 (TGF-β1) are converted into myofibroblasts through EMT. TGF-β induces both canonical Smad signaling and non-canonical signaling, including the Ras-induced ERK pathway (Raf-MEK-ERK). However, the signaling mechanisms regulating TGF-β1-induced EMT are not fully understood. Here, we show that the Ras-ERK pathway negatively regulates TGF-β1-induced EMT in RLE-6TN cells and that DA-Raf1 (DA-Raf), a splicing isoform of A-Raf and a dominant-negative antagonist of the Ras-ERK pathway, plays an essential role in EMT. Stimulation of the cells with fibroblast growth factor 2 (FGF2), which activated the ERK pathway, prominently suppressed TGF-β1-induced EMT. An inhibitor of MEK, but not an inhibitor of phosphatidylinositol 3-kinase, rescued the TGF-β1-treated cells from the suppression of EMT by FGF2. Overexpression of a constitutively active mutant of a component of the Ras-ERK pathway, i.e., H-Ras, B-Raf, or MEK1, interfered with EMT. Knockdown of DA-Raf expression with siRNAs facilitated the activity of MEK and ERK, which were only weakly and transiently activated by TGF-β1. Although DA-Raf knockdown abrogated TGF-β1-induced EMT, the abrogation of EMT was reversed by the addition of the MEK inhibitor. Furthermore, DA-Raf knockdown impaired the TGF-β1-induced nuclear translocation of Smad2, which mediates the transcription required for EMT. These results imply that intrinsic DA-Raf exerts essential functions for EMT by antagonizing the TGF-β1-induced Ras-ERK pathway in RLE-6TN cells.

  9. Role of H-Ras/ERK signaling in carbon nanotube-induced neoplastic-like transformation of human mesothelial cells

    PubMed Central

    Lohcharoenkal, Warangkana; Wang, Liying; Stueckle, Todd A.; Park, Jino; Tse, William; Dinu, Cerasela-Zoica; Rojanasakul, Yon

    2014-01-01

    Rapid development and deployment of engineered nanomaterials such as carbon nanotubes (CNTs) in various commercial and biomedical applications have raised concerns about their potential adverse health effects, especially their long-term effects which have not been well addressed. We demonstrated here that prolonged exposure of human mesothelial cells to single-walled CNT (SWCNT) induced neoplastic-like transformation as indicated by anchorage-independent cell growth and increased cell invasiveness. Such transformation was associated with an up-regulation of H-Ras and activation of ERK1/2. Downregulation of H-Ras by siRNA or inactivation of ERK by chemical inhibitor effectively inhibited the aggressive phenotype of SWCNT-exposed cells. Integrin alpha V and cortactin, but not epithelial-mesenchymal transition (EMT) transcriptional regulators, were up-regulated in the SWCNT-exposed cells, suggesting their role in the aggressive phenotype. Cortactin expression was shown to be controlled by the H-Ras/ERK signaling. Thus, our results indicate a novel role of H-Ras/ERK signaling and cortactin in the aggressive transformation of human mesothelial cells by SWCNT. PMID:24971065

  10. The Rho-GTPase Rnd1 Suppresses Mammary Tumorigenesis and EMT by Restraining Ras-MAPK signaling

    PubMed Central

    Okada, Tomoyo; Sinha, Surajit; Esposito, Ilaria; Schiavon, Gaia; López-Lago, Miguel A.; Su, Wenjing; Pratilas, Christine A.; Abele, Cristina; Hernandez, Jonathan M.; Ohara, Masahiro; Okada, Morihito; Viale, Agnes; Heguy, Adriana; Socci, Nicholas D.; Sapino, Anna; Seshan, Venkatraman E.; Long, Stephen; Inghirami, Giorgio; Rosen, Neal; Giancotti, Filippo G.

    2015-01-01

    SUMMARY We identified the Rho-GTPase Rnd1 as a candidate metastasis suppressor through bioinformatics analysis and showed that its depletion disrupt epithelial adhesion and polarity, induced Epithelial-to-Mesenchymal Transition (EMT), and cooperated with deregulated expression of c-Myc or loss of p53 to cause neoplastic conversion. Mechanistic studies revealed that Rnd1 suppresses Ras signalling by activating the GAP domain of Plexin B1, which inhibits Rap1. Rap1 inhibition in turn led to derepression of p120-RasGAP, which was able to inhibit Ras. Inactivation of Rnd1 in mammary epithelial cells induced highly undifferentiated and invasive tumors in mice. Conversely, Rnd1 expression inhibited spontaneous and experimental lung colonization in mouse models of metastasis. Genomic studies indicated that gene deletion in combination with epigenetic silencing or, more rarely, point mutation inactivates RND1 in human breast cancer. These results reveal a previously unappreciated mechanism through which Rnd1 restrains activation of Ras-MAPK signaling and breast tumor initiation and progression. PMID:25531777

  11. Qingfei Xiaoyan Wan, a traditional Chinese medicine formula, ameliorates Pseudomonas aeruginosa–induced acute lung inflammation by regulation of PI3K/AKT and Ras/MAPK pathways

    PubMed Central

    Hou, Yuanyuan; Nie, Yan; Cheng, Binfeng; Tao, Jin; Ma, Xiaoyao; Jiang, Min; Gao, Jie; Bai, Gang

    2016-01-01

    Gram-negative pathogen–induced nosocomial infections and resistance are a most serious menace to global public health. Qingfei Xiaoyan Wan (QF), a traditional Chinese medicine (TCM) formula, has been used clinically in China for the treatment of upper respiratory tract infections, acute or chronic bronchitis and pulmonary infection. In this study, the effects of QF on Pseudomonas aeruginosa–induced acute pneumonia in mice were evaluated. The mechanisms by which four typical anti-inflammatory ingredients from QF, arctigenin (ATG), cholic acid (CLA), chlorogenic acid (CGA) and sinapic acid (SPA), regulate anti-inflammatory signaling pathways and related targets were investigated using molecular biology and molecular docking techniques. The results showed that pretreatment with QF significantly inhibits the release of cytokines (TNF-α and IL-6) and chemokines (IL-8 and RANTES), reduces leukocytes recruitment into inflamed tissues and ameliorates pulmonary edema and necrosis. In addition, ATG was identified as the primary anti-inflammatory agent with action on the PI3K/AKT and Ras/MAPK pathways. CLA and CGA enhanced the actions of ATG and exhibited synergistic NF-κB inactivation effects possibly via the Ras/MAPK signaling pathway. Moreover, CLA is speculated to target FGFR and MEK firstly. Overall, QF regulated the PI3K/AKT and Ras/MAPK pathways to inhibit pathogenic bacterial infections effectively. PMID:27175332

  12. The Natural Product Honokiol Inhibits Calcineurin Inhibitor-induced and Ras-mediated Tumor Promoting Pathways

    PubMed Central

    Banerjee, Pallavi; Basu, Aninda; Arbiser, Jack L.; Pal, Soumitro

    2013-01-01

    Although calcineurin inhibitors (CNIs) are very useful in preventing allograft rejection, they can mediate a rapid progression of post-transplantation malignancies. The CNI cyclosporine A (CsA) can promote renal tumor growth through activation of the proto-oncogene ras and over-expression of the angiogenic cytokine VEGF; the ras activation also induces over-expression of the cytoprotective enzyme HO-1, which promotes survival of renal cancer cells. Here, we show that the natural product honokiol significantly inhibited CsA-induced and Ras-mediated survival of renal cancer cells through the down-regulations of VEGF and HO-1. Thus, honokiol treatment may help to prevent tumor-promoting effects of CsA in transplant patients. PMID:23752066

  13. The natural product honokiol inhibits calcineurin inhibitor-induced and Ras-mediated tumor promoting pathways.

    PubMed

    Banerjee, Pallavi; Basu, Aninda; Arbiser, Jack L; Pal, Soumitro

    2013-09-28

    Although calcineurin inhibitors (CNIs) are very useful in preventing allograft rejection, they can mediate a rapid progression of post-transplantation malignancies. The CNI cyclosporine A (CsA) can promote renal tumor growth through activation of the proto-oncogene ras and over-expression of the angiogenic cytokine VEGF; the ras activation also induces over-expression of the cytoprotective enzyme HO-1, which promotes survival of renal cancer cells. Here, we show that the natural product honokiol significantly inhibited CsA-induced and Ras-mediated survival of renal cancer cells through the down-regulations of VEGF and HO-1. Thus, honokiol treatment may help to prevent tumor-promoting effects of CsA in transplant patients. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  14. Reactivation of Mitogen-activated Protein Kinase (MAPK) Pathway by FGF Receptor 3 (FGFR3)/Ras Mediates Resistance to Vemurafenib in Human B-RAF V600E Mutant Melanoma*

    PubMed Central

    Yadav, Vipin; Zhang, Xiaoyi; Liu, Jiangang; Estrem, Shawn; Li, Shuyu; Gong, Xue-Qian; Buchanan, Sean; Henry, James R.; Starling, James J.; Peng, Sheng-Bin

    2012-01-01

    Oncogenic B-RAF V600E mutation is found in 50% of melanomas and drives MEK/ERK pathway and cancer progression. Recently, a selective B-RAF inhibitor, vemurafenib (PLX4032), received clinical approval for treatment of melanoma with B-RAF V600E mutation. However, patients on vemurafenib eventually develop resistance to the drug and demonstrate tumor progression within an average of 7 months. Recent reports indicated that multiple complex and context-dependent mechanisms may confer resistance to B-RAF inhibition. In the study described herein, we generated B-RAF V600E melanoma cell lines of acquired-resistance to vemurafenib, and investigated the underlying mechanism(s) of resistance. Biochemical analysis revealed that MEK/ERK reactivation through Ras is the key resistance mechanism in these cells. Further analysis of total gene expression by microarray confirmed a significant increase of Ras and RTK gene signatures in the vemurafenib-resistant cells. Mechanistically, we found that the enhanced activation of fibroblast growth factor receptor 3 (FGFR3) is linked to Ras and MAPK activation, therefore conferring vemurafenib resistance. Pharmacological or genetic inhibition of the FGFR3/Ras axis restored the sensitivity of vemurafenib-resistant cells to vemurafenib. Additionally, activation of FGFR3 sufficiently reactivated Ras/MAPK signaling and conferred resistance to vemurafenib in the parental B-RAF V600E melanoma cells. Finally, we demonstrated that vemurafenib-resistant cells maintain their addiction to the MAPK pathway, and inhibition of MEK or pan-RAF activities is an effective therapeutic strategy to overcome acquired-resistance to vemurafenib. Together, we describe a novel FGFR3/Ras mediated mechanism for acquired-resistance to B-RAF inhibition. Our results have implications for the development of new therapeutic strategies to improve the outcome of patients with B-RAF V600E melanoma. PMID:22730329

  15. Src promotes GTPase activity of Ras via tyrosine 32 phosphorylation

    PubMed Central

    Bunda, Severa; Heir, Pardeep; Srikumar, Tharan; Cook, Jonathan D.; Burrell, Kelly; Kano, Yoshihito; Lee, Jeffrey E.; Zadeh, Gelareh; Raught, Brian; Ohh, Michael

    2014-01-01

    Mutations in Ras GTPase and various other components of the Ras signaling pathways are among the most common genetic alterations in human cancers and also have been identified in several familial developmental syndromes. Over the past few decades it has become clear that the activity or the oncogenic potential of Ras is dependent on the nonreceptor tyrosine kinase Src to promote the Ras/Raf/MAPK pathway essential for proliferation, differentiation, and survival of eukaryotic cells. However, no direct relationship between Ras and Src has been established. We show here that Src binds to and phosphorylates GTP-, but not GDP-, loaded Ras on a conserved Y32 residue within the switch I region in vitro and that in vivo, Ras-Y32 phosphorylation markedly reduces the binding to effector Raf and concomitantly increases binding to GTPase-activating proteins and the rate of GTP hydrolysis. These results suggest that, in the context of predetermined crystallographic structures, Ras-Y32 serves as an Src-dependent keystone regulatory residue that modulates Ras GTPase activity and ensures unidirectionality to the Ras GTPase cycle. PMID:25157176

  16. Lovastatin inhibits the extracellular-signal-regulated kinase pathway in immortalized rat brain neuroblasts

    PubMed Central

    Cerezo-Guisado, Maria Isabel; GarcíA-Román, Natalia; García-MaríN, Luis Jesús; Álvarez-Barrientos, Alberto; Bragado, Maria Julia; Lorenzo, Maria Jesús

    2006-01-01

    We have shown previously that lovastatin, a 3-hydroxy-3-methyl- glutaryl coenzyme A reductase inhibitor, induces apoptosis in spontaneously immortalized rat brain neuroblasts. In the present study, we analysed the intracellular signal transduction pathways by which lovastatin induces neuroblast apoptosis. We showed that lovastatin efficiently inhibited Ras activation, which was associ-ated with a significant decrease in ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Lovastatin also decreased CREB phosphorylation and CREB-mediated gene expression. The effects of lovastatin on the Ras/ERK1/2/CREB pathway were time- and concentration-dependent and fully prevented by meva-lonate. In addition, we showed that two MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] inhibitors, PD98059 and PD184352, were poor inducers of apoptosis in serum-treated neuroblasts. However, these inhibitors significantly increased apop-tosis induced by lovastatin treatment. Furthermore, we showed that pharmacological inhibition of both MEK and phosphoinos-itide 3-kinase activities was able to induce neuroblast apoptosis with similar efficacy as lovastatin. Our results suggest that lovast-atin triggers neuroblast apoptosis by regulating several signalling pathways, including the Ras/ERK1/2 pathway. These findings might also contribute to elucidate the intracellular mechanisms involved in the central nervous system side effects associated with statin therapy. PMID:16952276

  17. Inhibitory effect of peroxiredoxin II (Prx II) on Ras-ERK-NFkappaB pathway in mouse embryonic fibroblast (MEF) senescence.

    PubMed

    Han, Ying-Hao; Kwon, Jeong-Hoon; Yu, Dae-Yeul; Moon, Eun-Yi

    2006-11-01

    Intracellular reactive oxygen species (ROS) were attenuated by the expression of peroxiredoxin II (Prx II). Cellular senescence as judged by senescence-associated (SA)-beta-galactosidase (Gal) positive cell formation was increased in Prx II-deficient mouse embryonic fibroblast (MEF). Ras expression was increased following passages. The level of Ras expression was higher in Prx II-/- MEF than wild type MEF. ERK activity was also augmented by the deletion of Prx II. SA-beta-Gal-positive cell formation was reduced by PD98059, ERK inhibitor. Activated nuclear transcription factor, nuclear factor-kappaB (NFkappaB) by the deletion of Prx II was inhibited by the treatment with PD98059. In contrast, no changes in SA-beta-Gal-positive cell formation were detected by NFkappaB inhibitor, N-alpha-tosyl-L-phenylalanyl chloromethyl ketone (TPCK). Collectively, results suggest that Prx II deletion activate Ras-ERK-NFkappaB pathways and cellular senescence in Prx II-/- MEF cells was mediated by ERK activation but not by NFkappaB activation.

  18. YES oncogenic activity is specified by its SH4 domain and regulates RAS/MAPK signaling in colon carcinoma cells.

    PubMed

    Dubois, Fanny; Leroy, Cédric; Simon, Valérie; Benistant, Christine; Roche, Serge

    2015-01-01

    Members of the SRC family of tyrosine kinases (SFK) display important functions in human cancer, but their specific role in tumorigenesis remains unclear. We previously demonstrated that YES regulates a unique oncogenic signaling important for colorectal cancer (CRC) progression that is not shared with SRC. Here, we addressed the underlying mechanism involved in this process. We show that YES oncogenic signaling relies on palmitoylation of its SH4 domain that controls YES localization in cholesterol-enriched membrane micro-domains. Specifically, deletion of the palmitoylation site compromised YES transforming activity, while addition of a palmitoylation site in the SH4 domain of SRC was sufficient for SRC to restore the transforming properties of cells in which YES had been silenced. Subsequently, SILAC phosphoproteomic analysis revealed that micro-domain-associated cell adhesive components and receptor tyrosine kinases are major YES substrates. YES also phosphorylates upstream regulators of RAS/MAPK signaling, including EGFR, SHC and SHP2, which were not targeted by SRC due to the absence of palmitoylation. Accordingly, EGFR-induced MAPK activity was attenuated by YES down-regulation, while increased RAS activity significantly restored cell transformation that was lost upon YES silencing. Collectively, these results uncover a critical role for the SH4 domain in the specification of SFK oncogenic activity and a selective role for YES in the induction of RAS/MAPK signaling in CRC cells.

  19. YES oncogenic activity is specified by its SH4 domain and regulates RAS/MAPK signaling in colon carcinoma cells

    PubMed Central

    Dubois, Fanny; Leroy, Cédric; Simon, Valérie; Benistant, Christine; Roche, Serge

    2015-01-01

    Members of the SRC family of tyrosine kinases (SFK) display important functions in human cancer, but their specific role in tumorigenesis remains unclear. We previously demonstrated that YES regulates a unique oncogenic signaling important for colorectal cancer (CRC) progression that is not shared with SRC. Here, we addressed the underlying mechanism involved in this process. We show that YES oncogenic signaling relies on palmitoylation of its SH4 domain that controls YES localization in cholesterol-enriched membrane micro-domains. Specifically, deletion of the palmitoylation site compromised YES transforming activity, while addition of a palmitoylation site in the SH4 domain of SRC was sufficient for SRC to restore the transforming properties of cells in which YES had been silenced. Subsequently, SILAC phosphoproteomic analysis revealed that micro-domain-associated cell adhesive components and receptor tyrosine kinases are major YES substrates. YES also phosphorylates upstream regulators of RAS/MAPK signaling, including EGFR, SHC and SHP2, which were not targeted by SRC due to the absence of palmitoylation. Accordingly, EGFR-induced MAPK activity was attenuated by YES down-regulation, while increased RAS activity significantly restored cell transformation that was lost upon YES silencing. Collectively, these results uncover a critical role for the SH4 domain in the specification of SFK oncogenic activity and a selective role for YES in the induction of RAS/MAPK signaling in CRC cells. PMID:26269757

  20. K-RAS(V12) Induces Autocrine Production of EGFR Ligands and Mediates Radioresistance Through EGFR-Dependent Akt Signaling and Activation of DNA-PKcs

    SciTech Connect

    Minjgee, Minjmaa; Toulany, Mahmoud; Kehlbach, Rainer; Giehl, Klaudia; Rodemann, H. Peter

    2011-12-01

    Purpose: It is known that postirradiation survival of tumor cells presenting mutated K-RAS is mediated through autocrine activation of epidermal growth factor receptor (EGFR). In this study the molecular mechanism of radioresistance of cells overexpressing mutated K-RAS(V12) was investigated. Methods and Materials: Head-and-neck cancer cells (FaDu) presenting wild-type K-RAS were transfected with empty vector or vector expressing mutated K-RAS(V12). The effect of K-RAS(V12) on autocrine production of EGFR ligands, activation of EGFR downstream pathways, DNA damage repair, and postirradiation survival was analyzed. Results: Conditioned medium collected from K-RAS(V12)-transfected cells enhanced activation of the phosphatidylinositol-3-kinase-Akt pathway and increased postirradiation survival of wild-type K-RAS parental cells when compared with controls. These effects were reversed by amphiregulin (AREG)-neutralizing antibody. In addition, secretion of the EGFR ligands AREG and transforming growth factor {alpha} was significantly increased upon overexpression of K-RAS(V12). Expression of mutated K-RAS(V12) resulted in an increase in radiation-induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation at S2056. This increase was accompanied by increased repair of DNA double-strand breaks. Abrogation of DNA-PKcs phosphorylation by serum depletion or AREG-neutralizing antibody underscored the role of autocrine production of EGFR ligands, namely, AREG, in regulating DNA-PKcs activation in K-RAS mutated cells. Conclusions: These data indicate that radioresistance of K-RAS mutated tumor cells is at least in part due to constitutive production of EGFR ligands, which mediate enhanced repair of DNA double-strand breaks through the EGFR-phosphatidylinositol-3-kinase-Akt cascade.

  1. VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking.

    PubMed

    Zhou, Mo; Wiener, Heidi; Su, Wenjuan; Zhou, Yong; Liot, Caroline; Ahearn, Ian; Hancock, John F; Philips, Mark R

    2016-08-15

    Ras guanosine triphosphatases (GTPases) regulate signaling pathways only when associated with cellular membranes through their C-terminal prenylated regions. Ras proteins move between membrane compartments in part via diffusion-limited, fluid phase transfer through the cytosol, suggesting that chaperones sequester the polyisoprene lipid from the aqueous environment. In this study, we analyze the nature of the pool of endogenous Ras proteins found in the cytosol. The majority of the pool consists of farnesylated, but not palmitoylated, N-Ras that is associated with a high molecular weight (HMW) complex. Affinity purification and mass spectrographic identification revealed that among the proteins found in the HMW fraction is VPS35, a latent cytosolic component of the retromer coat. VPS35 bound to N-Ras in a farnesyl-dependent, but neither palmitoyl- nor guanosine triphosphate (GTP)-dependent, fashion. Silencing VPS35 increased N-Ras's association with cytoplasmic vesicles, diminished GTP loading of Ras, and inhibited mitogen-activated protein kinase signaling and growth of N-Ras-dependent melanoma cells.

  2. Leptin signalling pathways in hypothalamic neurons.

    PubMed

    Kwon, Obin; Kim, Ki Woo; Kim, Min-Seon

    2016-04-01

    Leptin is the most critical hormone in the homeostatic regulation of energy balance among those so far discovered. Leptin primarily acts on the neurons of the mediobasal part of hypothalamus to regulate food intake, thermogenesis, and the blood glucose level. In the hypothalamic neurons, leptin binding to the long form leptin receptors on the plasma membrane initiates multiple signaling cascades. The signaling pathways known to mediate the actions of leptin include JAK-STAT signaling, PI3K-Akt-FoxO1 signaling, SHP2-ERK signaling, AMPK signaling, and mTOR-S6K signaling. Recent evidence suggests that leptin signaling in hypothalamic neurons is also linked to primary cilia function. On the other hand, signaling molecules/pathways mitigating leptin actions in hypothalamic neurons have been extensively investigated in an effort to treat leptin resistance observed in obesity. These include SOCS3, tyrosine phosphatase PTP1B, and inflammatory signaling pathways such as IKK-NFκB and JNK signaling, and ER stress-mitochondrial signaling. In this review, we discuss leptin signaling pathways in the hypothalamus, with a particular focus on the most recently discovered pathways.

  3. Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics

    PubMed Central

    Lee, Hong-Won; Kyung, Taeyoon; Yoo, Janghyun; Kim, Tackhoon; Chung, Chaeuk; Ryu, Ji Young; Lee, Hanki; Park, Kihyun; Lee, Sangkyu; Jones, Walton D.; Lim, Dae-Sik; Hyeon, Changbong; Do Heo, Won; Yoon, Tae-Young

    2013-01-01

    Co-immunoprecipitation (co-IP) has become a standard technique, but its protein-band output provides only static, qualitative information about protein–protein interactions. Here we demonstrate a real-time single-molecule co-IP technique that generates real-time videos of individual protein–protein interactions as they occur in unpurified cell extracts. By analysing single Ras–Raf interactions with a 50-ms time resolution, we have observed transient intermediates of the protein–protein interaction and determined all the essential kinetic rates. Using this technique, we have quantified the active fraction of native Ras proteins in xenograft tumours, normal tissue and cancer cell lines. We demonstrate that the oncogenic Ras mutations selectively increase the active-Ras fraction by one order of magnitude, without affecting total Ras levels or single-molecule signalling kinetics. Our approach allows us to probe the previously hidden, dynamic aspects of weak protein–protein interactions. It also suggests a path forward towards precision molecular diagnostics at the protein–protein interaction level. PMID:23422673

  4. Unbiased RNAi screen for hepcidin regulators links hepcidin suppression to proliferative Ras/RAF and nutrient-dependent mTOR signaling.

    PubMed

    Mleczko-Sanecka, Katarzyna; Roche, Franziska; da Silva, Ana Rita; Call, Debora; D'Alessio, Flavia; Ragab, Anan; Lapinski, Philip E; Ummanni, Ramesh; Korf, Ulrike; Oakes, Christopher; Damm, Georg; D'Alessandro, Lorenza A; Klingmüller, Ursula; King, Philip D; Boutros, Michael; Hentze, Matthias W; Muckenthaler, Martina U

    2014-03-06

    The hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the "iron-regulated" bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP- and interleukin-6-triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism.

  5. Influenza virus and cell signaling pathways

    PubMed Central

    Gaur, Pratibha; Munjal, Ashok; Lal, Sunil K.

    2011-01-01

    Summary Influenza viruses comprise a major class of human respiratory pathogens, responsible for causing morbidity and mortality worldwide. Influenza A virus, due to its segmented RNA genome, is highly subject to mutation, resulting in rapid formation of variants. During influenza infection, viral proteins interact with host proteins and exploit a variety of cellular pathways for their own benefit. Influenza virus inhibits the synthesis of these cellular proteins and facilitates expression of its own proteins for viral transcription and replication. Infected cell pathways are hijacked by an array of intracellular signaling cascades such as NF-κB signaling, PI3K/Akt pathway, MAPK pathway, PKC/PKR signaling and TLR/RIG-I signaling cascades. This review presents a research update on the subject and discusses the impact of influenza viral infection on these cell signaling pathways. PMID:21629204

  6. Multitarget network strategies to influence memory and forgetting: the Ras/MAPK pathway as a novel option.

    PubMed

    Gyurkó, Márton Dávid; Steták, Attila; Sőti, Csaba; Csermely, Péter

    2015-01-01

    The Ras/mitogen activated protein kinase (MAPK) pathway has key importance in development, cell differentiation and senescence, tumorigenesis, learning and memory. The clinical manifestations associated with this highly conserved pathway are called RASopathies. Phenotypic features are diverse and overlapping, but cognitive impairment is a common symptom. Here, we propose an approach based on molecular networks that link learning, memory and forgetting to the RASopathies and various neurodegenerative and neurodevelopmental diseases such as Alzheimer's disease, Parkinson's disease and autism spectrum disorders. We demonstrate the cross-talks of the molecular pathways in RASopathies and memory and the role of compartmentalization in these processes. The approved drugs are also overviewed, and C. elegans is proposed as a viable model system for experimental exploration and compound target prediction.n.

  7. Cancer stem cell drugs target K-ras signaling in a stemness context

    PubMed Central

    Najumudeen, A K; Jaiswal, A; Lectez, B; Oetken-Lindholm, C; Guzmán, C; Siljamäki, E; Posada, I M D; Lacey, E; Aittokallio, T; Abankwa, D

    2016-01-01

    Cancer stem cells (CSCs) are considered to be responsible for treatment relapse and have therefore become a major target in cancer research. Salinomycin is the most established CSC inhibitor. However, its primary mechanistic target is still unclear, impeding the discovery of compounds with similar anti-CSC activity. Here, we show that salinomycin very specifically interferes with the activity of K-ras4B, but not H-ras, by disrupting its nanoscale membrane organization. We found that caveolae negatively regulate the sensitivity to this drug. On the basis of this novel mechanistic insight, we defined a K-ras-associated and stem cell-derived gene expression signature that predicts the drug response of cancer cells to salinomycin. Consistent with therapy resistance of CSC, 8% of tumor samples in the TCGA-database displayed our signature and were associated with a significantly higher mortality. Using our K-ras-specific screening platform, we identified several new candidate CSC drugs. Two of these, ophiobolin A and conglobatin A, possessed a similar or higher potency than salinomycin. Finally, we established that the most potent compound, ophiobolin A, exerts its K-ras4B-specific activity through inactivation of calmodulin. Our data suggest that specific interference with the K-ras4B/calmodulin interaction selectively inhibits CSC. PMID:26973241

  8. dpl-1 DP and efl-1 E2F act with lin-35 Rb to antagonize Ras signaling in C. elegans vulval development.

    PubMed

    Ceol, C J; Horvitz, H R

    2001-03-01

    The synthetic multivulva (synMuv) genes define two functionally redundant pathways that antagonize RTK/Ras signaling during Caenorhabditis elegans vulval induction. The synMuv gene lin-35 encodes a protein similar to the mammalian tumor suppressor pRB and has been proposed to act as a transcriptional repressor. Studies using mammalian cells have shown that pRB can prevent cell cycle progression by inhibiting DP/E2F-mediated transcriptional activation. We identified C. elegans genes that encode proteins similar to DP or E2F. Loss-of-function mutations in two of these genes, dpl-1 DP and efl-1 E2F, caused the same vulval abnormalities as do lin-35 Rb loss-of-function mutations. We propose that rather than being inhibited by lin-35 Rb, dpl-1 DP and efl-1 E2F act with lin-35 Rb in transcriptional repression to antagonize RTK/Ras signaling during vulval development.

  9. The Drosophila rolled locus encodes a MAP kinase required in the sevenless signal transduction pathway.

    PubMed Central

    Biggs, W H; Zavitz, K H; Dickson, B; van der Straten, A; Brunner, D; Hafen, E; Zipursky, S L

    1994-01-01

    Mitogen-activated protein (MAP) kinases have been proposed to play a critical role in receptor tyrosine kinase (RTK)-mediated signal transduction pathways. Although genetic and biochemical studies of RTK pathways in Caenorhabditis elegans, Drosophila melanogaster and mammals have revealed remarkable similarities, a genetic requirement for MAP kinases in RTK signaling has not been established. During retinal development in Drosophila, the sevenless (Sev) RTK is required for development of the R7 photoreceptor cell. Components of the signal transduction pathway activated by Sev in the R7 precursor include proteins encoded by the gap1, drk, Sos, ras1 and raf loci. In this report we present evidence that a Drosophila MAP kinase, ERK-A, is encoded by the rolled locus and is required downstream of raf in the Sev signal transduction pathway. Images PMID:8157002

  10. Involvement of H- and N-Ras isoforms in transforming growth factor-{beta}1-induced proliferation and in collagen and fibronectin synthesis

    SciTech Connect

    Martinez-Salgado, Carlos . E-mail: carloms@usal.es; Fuentes-Calvo, Isabel; Garcia-Cenador, Begona; Santos, Eugenio; Lopez-Novoa, Jose M.

    2006-07-01

    Transforming growth factor {beta}1 (TGF-{beta}1) has a relevant role in the origin and maintenance of glomerulosclerosis and tubule-interstitial fibrosis. TGF-{beta} and Ras signaling pathways are closely related: TGF-{beta}1 overcomes Ras mitogenic effects and Ras counteracts TGF-{beta} signaling. Tubule-interstitial fibrosis is associated to increases in Ras, Erk, and Akt activation in a renal fibrosis model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors Erk and Akt, in TGF-{beta}1-mediated extracellular matrix (ECM) synthesis and proliferation, using embrionary fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras {sup -/-}/N-ras {sup -/-}) isoforms and from heterozygote mice (H-ras {sup +/-}/N-ras {sup +/-}). ECM synthesis is increased in basal conditions in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts, this increase being higher after stimulation with TGF-{beta}1. TGF-{beta}1-induced fibroblast proliferation is smaller in H-ras {sup -/-}/N-ras {sup -/-} than in H-ras {sup +/-}/N-ras {sup +/-} fibroblasts. Erk activation is decreased in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts; inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM synthesis. We suggest that H- and N-Ras isoforms downregulate ECM synthesis, and mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras.

  11. Ras-dva1 small GTPase regulates telencephalon development in Xenopus laevis embryos by controlling Fgf8 and Agr signaling at the anterior border of the neural plate.

    PubMed

    Tereshina, Maria B; Ermakova, Galina V; Ivanova, Anastasiya S; Zaraisky, Andrey G

    2014-03-15

    We previously found that the small GTPase Ras-dva1 is essential for the telencephalic development in Xenopus laevis because Ras-dva1 controls the Fgf8-mediated induction of FoxG1 expression, a key telencephalic regulator. In this report, we show, however, that Ras-dva1 and FoxG1 are expressed in different groups of cells; whereas Ras-dva1 is expressed in the outer layer of the anterior neural fold, FoxG1 and Fgf8 are activated in the inner layer from which the telencephalon is derived. We resolve this paradox by demonstrating that Ras-dva1 is involved in the transduction of Fgf8 signal received by cells in the outer layer, which in turn send a feedback signal that stimulates FoxG1 expression in the inner layer. We show that this feedback signal is transmitted by secreted Agr proteins, the expression of which is activated in the outer layer by mediation of Ras-dva1 and the homeodomain transcription factor Otx2. In turn, Agrs are essential for maintaining Fgf8 and FoxG1 expression in cells at the anterior neural plate border. Our finding reveals a novel feedback loop mechanism based on the exchange of Fgf8 and Agr signaling between neural and non-neural compartments at the anterior margin of the neural plate and demonstrates a key role of Ras-dva1 in this mechanism.

  12. A Central Role for Ras1 in Morphogenesis of the Basidiomycete Schizophyllum commune

    PubMed Central

    Knabe, Nicole; Jung, Elke-Martina; Freihorst, Daniela; Hennicke, Florian; Horton, J. Stephen

    2013-01-01

    Fungi have been used as model systems to define general processes in eukaryotes, for example, the one gene-one enzyme hypothesis, as well as to study polar growth or pathogenesis. Here, we show a central role for the regulator protein Ras in a mushroom-forming, filamentous basidiomycete linking growth, pheromone signaling, sexual development, and meiosis to different signal transduction pathways. ras1 and Ras-specific gap1 mutants were generated and used to modify the intracellular activation state of the Ras module. Transformants containing constitutive ras1 alleles (ras1G12V and ras1Q61L), as well as their compatible mating interactions, did show strong phenotypes for growth (associated with Cdc42 signaling) and mating (associated with mitogen-activated protein kinase signaling). Normal fruiting bodies with abnormal spores exhibiting a reduced germination rate were produced by outcrossing of these mutant strains. Homozygous Δgap1 primordia, expected to experience increased Ras signaling, showed overlapping phenotypes with a block in basidium development and meiosis. Investigation of cyclic AMP (cAMP)-dependent protein kinase A indicated that constitutively active ras1, as well as Δgap1 mutant strains, exhibit a strong increase in Tpk activity. Ras1-dependent, cAMP-mediated signal transduction is, in addition to the known signaling pathways, involved in fruiting body formation in Schizophyllum commune. To integrate these analyses of Ras signaling, microarray studies were performed. Mutant strains containing constitutively active Ras1, deletion of RasGap1, or constitutively active Cdc42 were characterized and compared. At the transcriptome level, specific regulation highlighting the phenotypic differences of the mutants is clearly visible. PMID:23606288

  13. Adaptor protein Nck1 interacts with p120 Ras GTPase-activating protein and regulates its activity.

    PubMed

    Ger, Marija; Zitkus, Zigmantas; Valius, Mindaugas

    2011-10-01

    Adaptor protein Nck1 binds a number of intracellular proteins and influences various signaling pathways. Here we show that Nck1 directly binds and activates the GTPase-activating protein of Ras (RasGAP), which is responsible for the down-regulation of Ras. The first and the third SH3 domains of Nck1 and the NH(2)-terminal proline-rich sequence of RasGAP contribute most to the complex formation causing direct molecular interaction between the two proteins. Cell adhesion to the substrate is obligatory for the Nck1 and RasGAP association, as cell detachment makes RasGAP incapable of associating with Nck1. This leads to the complex dissipation, decrease of RasGAP activity and the increase of H-Ras-GTP level in the detached cells. Our findings reveal unexpected feature of adaptor protein Nck1 as the regulator of RasGAP activity.

  14. Ras-MEK-ERK signaling cascade regulates androgen receptor element-inducible gene transcription and DNA synthesis in prostate cancer cells.

    PubMed

    Carey, Anne-Marie; Pramanik, Rashida; Nicholson, Linda J; Dew, Tracy K; Martin, Francis L; Muir, Gordon H; Morris, Jonathan D H

    2007-08-01

    Treatment of prostate cancer (CaP) patients frequently involves androgen ablation, but resistance often develops and androgen-insensitive tumors emerge. The molecular basis for the development of refractory CaP that grows in an androgen-independent manner is poorly understood, but alterations in growth factor signaling pathways are likely to be involved. We examined the growth factor modulation of androgen-receptor element (ARE)-inducible luciferase reporter gene activity and consequent DNA synthesis as a measure of proliferative growth in androgen-dependent LNCaP or androgen-independent PC3 or DU145 CaP cells. The synthetic androgen R1881 stimulated ARE-inducible reporter gene activity and prostate-specific antigen expression in LNCaP cells and the MEK/ERK inhibitor U0126 or the anti-androgen bicalutamide (casodex) prevented both of these responses. Activated V12-Ha-Ras expression in LNCaP cells also stimulated ARE-inducible gene transcription, and U0126 or the farnesyltransferase inhibitor FTI-277 but not bicalutamide blocked this. ARE-inducible reporter gene activity was elevated already in PC3 cells, and ERK was constitutively activated in serum-starved LNCaP or DU145 cells. U0126 inhibited each of these responses and also inhibited DNA synthesis in all 3 CaP cell lines. These results demonstrate that chronic stimulation of the Ras-MEK-ERK signaling pathway can sustain ARE-inducible gene transcription and growth of CaP cells, and suggests that components of this pathway may offer targets for cancer therapy. Copyright (c) 2007 Wiley-Liss, Inc.

  15. MicroRNA-132/212 family enhances arteriogenesis after hindlimb ischaemia through modulation of the Ras-MAPK pathway.

    PubMed

    Lei, Zhiyong; van Mil, Alain; Brandt, Maarten M; Grundmann, Sebastian; Hoefer, Imo; Smits, Michiel; El Azzouzi, Hamid; Fukao, Taro; Cheng, Caroline; Doevendans, Pieter A; Sluijter, Joost P G

    2015-08-01

    Arteriogenesis is a complicated process induced by increased local shear-and radial wall-stress, leading to an increase in arterial diameter. This process is enhanced by growth factors secreted by both inflammatory and endothelial cells in response to physical stress. Although therapeutic promotion of arteriogenesis is of great interest for ischaemic diseases, little is known about the modulation of the signalling cascades via microRNAs. We observed that miR-132/212 expression was significantly upregulated after occlusion of the femoral artery. miR-132/212 knockout (KO) mice display a slower perfusion recovery after hind-limb ischaemia compared to wildtype (WT) mice. Immunohistochemical analysis demonstrates a clear trend towards smaller collateral arteries in KO mice. Although Ex vivo aortic ring assays score similar number of branches in miR-132/212 KO mice compared to WT, it can be stimulated with exogenous miR-132, a dominant member of the miR-132/212 family. Moreover, in in vitro pericyte-endothelial co-culture cell assays, overexpression of miR-132 and mir-212 in endothelial cells results in enhanced vascularization, as shown by an increase in tubular structures and junctions. Our results suggested that miR-132/212 may exert their effects by enhancing the Ras-Mitogen-activated protein kinases MAPK signalling pathway through direct inhibition of Rasa1, and Spred1. The miR-132/212 cluster promotes arteriogenesis by modulating Ras-MAPK signalling via direct targeting of its inhibitors Rasa1 and Spred1.

  16. Nuclear Ras2-GTP controls invasive growth in Saccharomyces cerevisiae.

    PubMed

    Broggi, Serena; Martegani, Enzo; Colombo, Sonia

    2013-01-01

    Using an eGFP-RBD3 probe, which specifically binds Ras-GTP, we recently showed that the fluorescent probe was localized to the plasma membrane and to the nucleus in wild type cells growing exponentially on glucose medium, indicating the presence of active Ras in these cellular compartments. To investigate the nuclear function of Ras-GTP, we generated a strain where Ras2 is fused to the nuclear export signal (NES) from the HIV virus, in order to exclude this protein from the nucleus. Our results show that nuclear active Ras2 is required for invasive growth development in haploid yeast, while the expression of the NES-Ras2 protein does not cause growth defects either on fermentable or non-fermentable carbon sources and does not influence protein kinase A (PKA) activity related phenotypes analysed. Moreover, we show that the cAMP/PKA pathway controls invasive growth influencing the localization of active Ras. In particular, we show that PKA activity plays a role in the localization of active Ras and influences the ability of the cells to invade the agar: high PKA activity leads to a predominant nuclear accumulation of active Ras and induces invasive growth, while low PKA activity leads to plasma membrane localization of active Ras and to a defective invasive growth phenotype.

  17. Nuclear Ras2-GTP Controls Invasive Growth in Saccharomyces cerevisiae

    PubMed Central

    Broggi, Serena; Martegani, Enzo; Colombo, Sonia

    2013-01-01

    Using an eGFP-RBD3 probe, which specifically binds Ras-GTP, we recently showed that the fluorescent probe was localized to the plasma membrane and to the nucleus in wild type cells growing exponentially on glucose medium, indicating the presence of active Ras in these cellular compartments. To investigate the nuclear function of Ras-GTP, we generated a strain where Ras2 is fused to the nuclear export signal (NES) from the HIV virus, in order to exclude this protein from the nucleus. Our results show that nuclear active Ras2 is required for invasive growth development in haploid yeast, while the expression of the NES-Ras2 protein does not cause growth defects either on fermentable or non-fermentable carbon sources and does not influence protein kinase A (PKA) activity related phenotypes analysed. Moreover, we show that the cAMP/PKA pathway controls invasive growth influencing the localization of active Ras. In particular, we show that PKA activity plays a role in the localization of active Ras and influences the ability of the cells to invade the agar: high PKA activity leads to a predominant nuclear accumulation of active Ras and induces invasive growth, while low PKA activity leads to plasma membrane localization of active Ras and to a defective invasive growth phenotype. PMID:24244466

  18. Simvastatin inhibits glucose-stimulated vascular smooth muscle cell migration involving increased expression of RhoB and a block of Ras/Akt signal.

    PubMed

    Chan, Kuei-Chuan; Wu, Cheng-Hsun; Huang, Chien-Ning; Lan, Kuang-Ping; Chang, Wen Chun; Wang, Chau-Jong

    2012-04-01

    Diabetic patients are at high risk to develop atherosclerotic cardiovascular disease and have a higher restenotic rate after percutaneous coronary intervention (PCI). Statins improve cardiovascular outcome and reduce restenosis after PCI by inhibiting proliferation and migration of vascular smooth muscle cells (VSMCs). But the effect of statins on diabetes without dyslipidemia was still not fully understood. Our previous study has demonstrated that simvastatin inhibits VSMC proliferation in high glucose status without dyslipidemia, inducing a G0/G1 phase cell cycle growth arrest by acting on multiple steps upstream of pRb, including inhibition of CDK2/4 expression and upregulation of p53, p21, p16, and p27. Following our previous study, we investigated the mechanism of simvastatin inhibition of VSMC migration in a diabetes-like model (A7r5 cells under high glucose conditions without dyslipidemia). Under high glucose conditions, simvastatin dose-dependently inhibited VSMC migration, decreased PI3K/Akt pathway activity, reduced c-Raf and Ras expression, increased RhoB but not RhoA, Rac1, and Cdc2 expression, dose-dependently inhibited MMP-2, but not MMP-9, activity, and dose-dependently inhibited NF-κB activity. The inhibition of VSMC migration under high glucose conditions was via two different pathways. The first pathway is mevalonate-related but not RhoA protein-related and involves suppression of Ras and PI3K/Akt signals. The second pathway is not mevalonate-related and involves increasing RhoB expression directly. © 2010 Blackwell Publishing Ltd.

  19. What makes Ras an efficient molecular switch: a computational, biophysical, and structural study of Ras-GDP interactions with mutants of Raf.

    PubMed

    Filchtinski, Daniel; Sharabi, Oz; Rüppel, Alma; Vetter, Ingrid R; Herrmann, Christian; Shifman, Julia M

    2010-06-11

    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 (Ras(GTP)) with its effectors has been studied intensively. However, very little is known about the much weaker interaction between the GDP-bound Ras (Ras(GDP)) 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 Ras(GDP). Most of our designed mutations narrow the gap between the affinity of Raf for Ras(GTP) and Ras(GDP), producing the desired shift in binding specificity towards Ras(GDP). A combination of our best designed mutation, N71R, with another mutation, A85K, yielded a Raf mutant with a 100-fold improvement in affinity towards Ras(GDP). The Raf A85K and Raf N71R/A85K mutants were used to obtain the first high-resolution structures of Ras(GDP) bound to its effector. Surprisingly, these structures reveal that the loop on Ras previously termed the switch I region in the Ras(GDP).Raf mutant complex is found in a conformation similar to that of Ras(GTP) and not Ras(GDP). Moreover, the structures indicate an increased mobility of the switch I region. This greater flexibility compared to the same loop in Ras(GTP) is likely to explain the natural low affinity of Raf and other Ras effectors to Ras(GDP). 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. Copyright 2010 Elsevier Ltd

  20. Metabolic Dependencies in RAS-Driven Cancers.

    PubMed

    Kimmelman, Alec C

    2015-04-15

    The ability to inhibit the RAS oncogene has been the holy grail of oncology because of the critical role of this gene in a multitude of tumor types. In addition, RAS-mutant tumors are among the most aggressive and refractory to treatment. Although directly targeting the RAS oncogene has proven challenging, an alternative approach for treating RAS-driven cancers is to inhibit critical downstream events that are required for tumor maintenance. Indeed, much focus has been put on inhibiting signaling cascades downstream of RAS. Recent studies have shown that oncogenic RAS promotes a metabolic reprogramming of tumor cells, shifting them toward an anabolic metabolism necessary to produce biomass to support unconstrained proliferation. These cancers also use a diverse set of fuel sources to meet their metabolic needs and have even developed a variety of mechanisms to act as metabolic scavengers to obtain necessary metabolic substrates from both extracellular and intracellular sources. Collectively, these adaptations can create "metabolic bottlenecks" whereby tumor cells rely on particular pathways or rate-limiting metabolites. In this regard, inhibiting individual or combinations of these metabolic pathways can attenuate growth in preclinical models. Because these dependencies are tumor selective and downstream of oncogenic RAS, there is the opportunity for therapeutic intervention. Although targeting tumor metabolism is still in the early days of translation to patients, our continued advances in understanding critical metabolic adaptations in RAS-driven cancers, as well as the ability to study this altered metabolism in relevant tumor models, will accelerate the development of new therapeutic approaches. Clin Cancer Res; 21(8); 1828-34. ©2015 AACR. See all articles in this CCR Focus section, "Targeting RAS-Driven Cancers."

  1. The cyclopentenone 15-deoxy-Δ12,14-prostaglandin J2 binds to and activates H-Ras

    PubMed Central

    Oliva, José Luis; Pérez-Sala, Dolores; Castrillo, Antonio; Martínez, Natalia; Cañada, F. Javier; Boscá, Lisardo; Rojas, José M.

    2003-01-01

    The cyclopentenone 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) induces cell proliferation and mitogen-activated protein kinase activation. Here, we describe that these effects are mediated by 15d-PGJ2-elicited H-Ras activation. We demonstrate that this pathway is specific for H-Ras through the formation of a covalent adduct of 15d-PGJ2 with Cys-184 of H-Ras, but not with N-Ras or K-Ras. Mutation of C184 inhibited H-Ras modification and activation by 15d-PGJ2, whereas serum-elicited stimulation was not affected. These results describe a mechanism for the activation of the Ras signaling pathway, which results from the chemical modification of H-Ras by formation of a covalent adduct with cyclopentenone prostaglandins. PMID:12684535

  2. Analysis of Ras/ERK Compartmentalization by Subcellular Fractionation.

    PubMed

    Agudo-Ibañez, Lorena; Crespo, Piero; Casar, Berta

    2017-01-01

    A vast number of stimuli use the Ras/Raf/MEK/ERK signaling cascade to transmit signals from their cognate receptors, in order to regulate multiple cellular functions, including key processes such as proliferation, cell cycle progression, differentiation, and survival. The duration, intensity and specificity of the responses are, in part, controlled by the compartmentalization/subcellular localization of the signaling intermediaries. Ras proteins are found in different plasma membrane microdomains and endomembranes. At these localizations, Ras is subject to site-specific regulatory mechanisms, distinctively engaging effector pathways and switching-on diverse genetic programs to generate a multitude of biological responses. The Ras effector pathway leading to ERKs activation is also subject to space-related regulatory processes. About half of ERK1/2 substrates are found in the nucleus and function mainly as transcription factors. The other half resides in the cytosol and other cellular organelles. Such subcellular distribution enhances the complexity of the Ras/ERK cascade and constitutes an essential mechanism to endow variability to its signals, which enables their participation in the regulation of a broad variety of functions. Thus, analyzing the subcellular compartmentalization of the members of the Ras/ERK cascade constitutes an important factor to be taken into account when studying specific biological responses evoked by Ras/ERK signals. Herein, we describe methods for such purpose.

  3. LXR signaling pathways and atherosclerosis

    PubMed Central

    Calkin, Anna; Tontonoz, Peter

    2010-01-01

    First discovered as orphan receptors, liver X receptors (LXRs) were subsequently identified as the nuclear receptor target of the cholesterol metabolites, oxysterols.1 There are 2 LXR receptors encoded by distinct genes: LXRα is most highly expressed in the liver, adipose, kidney, adrenal tissues and macrophages, and LXRβ is ubiquitously expressed. Despite differential tissue distribution, these isoforms have 78% homology in their ligand-binding domain and appear to respond to the same endogenous ligands. Work over the past 10 years has shown that the LXR pathway regulates lipid metabolism and inflammation via both the induction and repression of target genes. Given the importance of cholesterol regulation and inflammation in the development of cardiovascular disease, it is not surprising that activation of the LXR pathway attenuates various mechanisms underlying atherosclerotic plaque development.2 In this minireview we will discuss the impact of the LXR pathway on both cholesterol metabolism and atherosclerosis. PMID:20631351

  4. Calcium in plant defence-signalling pathways.

    PubMed

    Lecourieux, David; Ranjeva, Raoul; Pugin, Alain

    2006-01-01

    In plant cells, the calcium ion is a ubiquitous intracellular second messenger involved in numerous signalling pathways. Variations in the cytosolic concentration of Ca2+ ([Ca2+]cyt) couple a large array of signals and responses. Here we concentrate on calcium signalling in plant defence responses, particularly on the generation of the calcium signal and downstream calcium-dependent events participating in the establishment of defence responses with special reference to calcium-binding proteins.

  5. The cannabinoid delta(9)-tetrahydrocannabinol inhibits RAS-MAPK and PI3K-AKT survival signalling and induces BAD-mediated apoptosis in colorectal cancer cells.

    PubMed

    Greenhough, Alexander; Patsos, Helena A; Williams, Ann C; Paraskeva, Christos

    2007-11-15

    Deregulation of cell survival pathways and resistance to apoptosis are widely accepted to be fundamental aspects of tumorigenesis. As in many tumours, the aberrant growth and survival of colorectal tumour cells is dependent upon a small number of highly activated signalling pathways, the inhibition of which elicits potent growth inhibitory or apoptotic responses in tumour cells. Accordingly, there is considerable interest in therapeutics that can modulate survival signalling pathways and target cancer cells for death. There is emerging evidence that cannabinoids, especially Delta(9)-tetrahydrocannabinol (THC), may represent novel anticancer agents, due to their ability to regulate signalling pathways critical for cell growth and survival. Here, we report that CB1 and CB2 cannabinoid receptors are expressed in human colorectal adenoma and carcinoma cells, and show for the first time that THC induces apoptosis in colorectal cancer cells. THC-induced apoptosis was rescued by pharmacological blockade of the CB1, but not CB2, cannabinoid receptor. Importantly, THC treatment resulted in CB1-mediated inhibition of both RAS-MAPK/ERK and PI3K-AKT survival signalling cascades; two key cell survival pathways frequently deregulated in colorectal tumours. The inhibition of ERK and AKT activity by THC was accompanied by activation of the proapoptotic BCL-2 family member BAD. Reduction of BAD protein expression by RNA interference rescued colorectal cancer cells from THC-induced apoptosis. These data suggest an important role for CB1 receptors and BAD in the regulation of apoptosis in colorectal cancer cells. The use of THC, or selective targeting of the CB1 receptor, may represent a novel strategy for colorectal cancer therapy.

  6. Protein Kinase A-independent Ras Protein Activation Cooperates with Rap1 Protein to Mediate Activation of the Extracellular Signal-regulated Kinases (ERK) by cAMP.

    PubMed

    Li, Yanping; Dillon, Tara J; Takahashi, Maho; Earley, Keith T; Stork, Philip J S

    2016-10-07

    Cyclic adenosine monophosphate (cAMP) is an important mediator of hormonal stimulation of cell growth and differentiation through its activation of the extracellular signal-regulated kinase (ERK) cascade. Two small G proteins, Ras and Rap1, have been proposed to mediate this activation, with either Ras or Rap1 acting in distinct cell types. Using Hek293 cells, we show that both Ras and Rap1 are required for cAMP signaling to ERKs. The roles of Ras and Rap1 were distinguished by their mechanism of activation, dependence on the cAMP-dependent protein kinase (PKA), and the magnitude and kinetics of their effects on ERKs. Ras was required for the early portion of ERK activation by cAMP and was activated independently of PKA. Ras activation required the Ras/Rap guanine nucleotide exchange factor (GEF) PDZ-GEF1. Importantly, this action of PDZ-GEF1 was disrupted by mutation within its putative cyclic nucleotide-binding domain within PDZ-GEF1. Compared with Ras, Rap1 activation of ERKs was of longer duration. Rap1 activation was dependent on PKA and required Src family kinases and the Rap1 exchanger C3G. This is the first report of a mechanism for the cooperative actions of Ras and Rap1 in cAMP activation of ERKs. One physiological role for the sustained activation of ERKs is the transcription and stabilization of a range of transcription factors, including c-FOS. We show that the induction of c-FOS by cAMP required both the early and sustained phases of ERK activation, requiring Ras and Rap1, as well as for each of the Raf isoforms, B-Raf and C-Raf.

  7. NF2 loss promotes oncogenic RAS-induced thyroid cancers via YAP-dependent transactivation of RAS proteins and sensitizes them to MEK inhibition

    PubMed Central

    Garcia-Rendueles, Maria E.R.; Ricarte-Filho, Julio C.; Untch, Brian R.; Landa, Iňigo; Knauf, Jeffrey A.; Voza, Francesca; Smith, Vicki E.; Ganly, Ian; Taylor, Barry S.; Persaud, Yogindra; Oler, Gisele; Fang, Yuqiang; Jhanwar, Suresh C.; Viale, Agnes; Heguy, Adriana; Huberman, Kety H.; Giancotti, Filippo; Ghossein, Ronald; Fagin, James A.

    2015-01-01

    Ch22q LOH is preferentially associated with RAS mutations in papillary and in poorly differentiated thyroid cancer (PDTC). The 22q tumor suppressor NF2, encoding merlin, is implicated in this interaction because of its frequent loss of function in human thyroid cancer cell lines. Nf2 deletion or Hras mutation are insufficient for transformation, whereas their combined disruption leads to murine PDTC with increased MAPK signaling. Merlin loss induces RAS signaling in part through inactivation of Hippo, which activates a YAP-TEAD transcriptional program. We find that the three RAS genes are themselves YAP-TEAD1 transcriptional targets, providing a novel mechanism of promotion of RAS-induced tumorigenesis. Moreover, pharmacological disruption of YAP-TEAD with verteporfin blocks RAS transcription and signaling, and inhibits cell growth. The increased MAPK output generated by NF2 loss in RAS-mutant cancers may inform therapeutic strategies, as it generates greater dependency on the MAPK pathway for viability. PMID:26359368

  8. TNF and MAP kinase signaling pathways

    PubMed Central

    Sabio, Guadalupe; Davis, Roger J.

    2014-01-01

    The binding of tumor necrosis factor α (TNFα) to cell surface receptors engages multiple signal transduction pathways, including three groups of mitogen-activated protein (MAP) kinases: extracellular-signal-regulated kinases (ERKs); the cJun NH2-terminal kinases (JNKs); and the p38 MAP kinases. These MAP kinase signalling pathways induce a secondary response by increasing the expression of several inflammatory cytokines (including TNFα) that contribute to the biological activity of TNFα. MAP kinases therefore function both upstream and down-stream of signalling by TNFα receptors. Here we review mechanisms that mediate these actions of MAP kinases during the response to TNFα. PMID:24647229

  9. Modularized TGFbeta-Smad Signaling Pathway

    NASA Technical Reports Server (NTRS)

    Li, Yongfeng; Wang, M.; Carra, C.; Cucinotta, F. A.

    2011-01-01

    The Transforming Growth Factor beta (TGFbeta) signaling pathway is a prominent regulatory signaling pathway controlling various important cellular processes. It can be induced by several factors, including ionizing radiation. It is regulated by Smads in a negative feedback loop through promoting increases in the regulatory Smads in the cell nucleus, and subsequent expression of inhibitory Smad, Smad7 to form a ubiquitin ligase with Smurf targeting active TGF receptors for degradation. In this work, we proposed a mathematical model to study the radiation-induced Smad-regulated TGF signaling pathway. By modularization, we are able to analyze each module (subsystem) and recover the nonlinear dynamics of the entire network system. Meanwhile the excitability, a common feature observed in the biological systems, along the TGF signaling pathway is discussed by mathematical analysis and numerical simulation.

  10. The NM23-H1/H2 homolog NDK-1 is required for full activation of Ras signaling in C. elegans.

    PubMed

    Masoudi, Neda; Fancsalszky, Luca; Pourkarimi, Ehsan; Vellai, Tibor; Alexa, Anita; Reményi, Attila; Gartner, Anton; Mehta, Anil; Takács-Vellai, Krisztina

    2013-08-01

    The group I members of the Nm23 (non-metastatic) gene family encode nucleoside diphosphate kinases (NDPKs) that have been implicated in the regulation of cell migration, proliferation and differentiation. Despite their developmental and medical significance, the molecular functions of these NDPKs remain ill defined. To minimize confounding effects of functional compensation between closely related Nm23 family members, we studied ndk-1, the sole Caenorhabditis elegans ortholog of group I NDPKs, and focused on its role in Ras/mitogen-activated protein kinase (MAPK)-mediated signaling events during development. ndk-1 inactivation leads to a protruding vulva phenotype and affects vulval cell fate specification through the Ras/MAPK cascade. ndk-1 mutant worms show severe reduction of activated, diphosphorylated MAPK in somatic tissues, indicative of compromised Ras/MAPK signaling. A genetic epistasis analysis using the vulval induction system revealed that NDK-1 acts downstream of LIN-45/Raf, but upstream of MPK-1/MAPK, at the level of the kinase suppressors of ras (KSR-1/2). KSR proteins act as scaffolds facilitating Ras signaling events by tethering signaling components, and we suggest that NDK-1 modulates KSR activity through direct physical interaction. Our study reveals that C. elegans NDK-1/Nm23 influences differentiation by enhancing the level of Ras/MAPK signaling. These results might help to better understand how dysregulated Nm23 in humans contributes to tumorigenesis.

  11. The NM23-H1/H2 homolog NDK-1 is required for full activation of Ras signaling in C. elegans

    PubMed Central

    Masoudi, Neda; Fancsalszky, Luca; Pourkarimi, Ehsan; Vellai, Tibor; Alexa, Anita; Reményi, Attila; Gartner, Anton; Mehta, Anil; Takács-Vellai, Krisztina

    2013-01-01

    The group I members of the Nm23 (non-metastatic) gene family encode nucleoside diphosphate kinases (NDPKs) that have been implicated in the regulation of cell migration, proliferation and differentiation. Despite their developmental and medical significance, the molecular functions of these NDPKs remain ill defined. To minimize confounding effects of functional compensation between closely related Nm23 family members, we studied ndk-1, the sole Caenorhabditis elegans ortholog of group I NDPKs, and focused on its role in Ras/mitogen-activated protein kinase (MAPK)-mediated signaling events during development. ndk-1 inactivation leads to a protruding vulva phenotype and affects vulval cell fate specification through the Ras/MAPK cascade. ndk-1 mutant worms show severe reduction of activated, diphosphorylated MAPK in somatic tissues, indicative of compromised Ras/MAPK signaling. A genetic epistasis analysis using the vulval induction system revealed that NDK-1 acts downstream of LIN-45/Raf, but upstream of MPK-1/MAPK, at the level of the kinase suppressors of ras (KSR-1/2). KSR proteins act as scaffolds facilitating Ras signaling events by tethering signaling components, and we suggest that NDK-1 modulates KSR activity through direct physical interaction. Our study reveals that C. elegans NDK-1/Nm23 influences differentiation by enhancing the level of Ras/MAPK signaling. These results might help to better understand how dysregulated Nm23 in humans contributes to tumorigenesis. PMID:23900546

  12. Fungal communication requires the MAK-2 pathway elements STE-20 and RAS-2, the NRC-1 adapter STE-50 and the MAP kinase scaffold HAM-5.

    PubMed

    Dettmann, Anne; Heilig, Yvonne; Valerius, Oliver; Ludwig, Sarah; Seiler, Stephan

    2014-11-01

    Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell-cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell-cell communication in fungi and higher eukaryotes.

  13. K-RasG12D expression induces hyperproliferation and aberrant signaling in primary hematopoietic stem/progenitor cells

    PubMed Central

    Van Meter, Margaret E. M.; Díaz-Flores, Ernesto; Archard, Joehleen A.; Passegué, Emmanuelle; Irish, Jonathan M.; Kotecha, Nikesh; Nolan, Garry P.

    2007-01-01

    Defining how cancer-associated mutations perturb signaling networks in stem/progenitor populations that are integral to tumor formation and maintenance is a fundamental problem with biologic and clinical implications. Point mutations in RAS genes contribute to many cancers, including myeloid malignancies. We investigated the effects of an oncogenic KrasG12D allele on phosphorylated signaling molecules in primary c-kit+ lin−/low hematopoietic stem/progenitor cells. Comparison of wild-type and KrasG12D c-kit+ lin−/low cells shows that K-RasG12D expression causes hyperproliferation in vivo and results in abnormal levels of phosphorylated STAT5, ERK, and S6 under basal and stimulated conditions. Whereas KrasG12D cells demonstrate hyperactive signaling after exposure to granulocyte-macrophage colony-stimulating factor, we unexpectedly observe a paradoxical attenuation of ERK and S6 phosphorylation in response to stem cell factor. These studies provide direct biochemical evidence that cancer stem/progenitor cells remodel signaling networks in response to oncogenic stress and demonstrate that multi-parameter flow cytometry can be used to monitor the effects of targeted therapeutics in vivo. This strategy has broad implications for defining the architecture of signaling networks in primary cancer cells and for implementing stem cell–targeted interventions. PMID:17192389

  14. Evaluation of Signaling Pathways Involved in γ-Globin Gene Induction Using Fetal Hemoglobin Inducer Drugs.

    PubMed

    Rahim, Fakher; Allahmoradi, Hossein; Salari, Fatemeh; Shahjahani, Mohammad; Fard, Ali Dehghani; Hosseini, Seyed Ahmad; Mousakhani, Hadi

    2013-01-01

    Potent induction of fetal hemoglobin (HbF) production results in alleviating the complications of β-thalassemia and sickle cell disease (SCD). HbF inducer agents can trigger several molecular signaling pathways critical for erythropoiesis. Janus kinase/Signal transducer and activator of transcription (JAK/STAT), mitogen activated protein kinas (MAPK) and Phosphoinositide 3-kinase (PI3K) are considered as main signaling pathways, which may play a significant role in HbF induction. All these signaling pathways are triggered by erythropoietin (EPO) as the main growth factor inducing erythroid differentiation, when it binds to its cell surface receptor, erythropoietin receptor (EPO-R) HbF inducer agents have been shown to upregulate HbF production level by triggering certain signaling pathways. As a result, understanding the pivotal signaling pathways influencing HbF induction leads to effective upregulation of HbF. In this mini review article, we try to consider the correlation between HbF inducer agents and their molecular mechanisms of γ-globin upregulation. Several studies suggest that activating P38 MAPK, RAS and STAT5 signaling pathways result in efficient HbF induction. Nevertheless, the role of other erythroid signaling pathways in HbF induction seems to be indispensible and should be emphasized.

  15. [Arnold-Chiari malformation in Noonan syndrome and other syndromes of the RAS/MAPK pathway].

    PubMed

    Ejarque, Ismael; Millán-Salvador, José M; Oltra, Silvestre; Pesudo-Martínez, José V; Beneyto, Magdalena; Pérez-Aytés, Antonio

    2015-05-01

    Introduccion. El sindrome de Noonan (SN) y otros sindromes con fenotipo similar, como LEOPARD, cardiofaciocutaneo, Costello y Legius, estan asociados a mutaciones en genes incluidos en la via RAS/MAPK (rasopatias), una importante via de señalizacion relacionada con la proliferacion celular. El descenso de las amigdalas cerebelares dentro del canal medular cervical, conocido como malformacion de Arnold-Chiari (MAC), se ha descrito en pacientes afectos de SN, lo que ha llevado a sugerir que la MAC podria formar parte del espectro fenotipico del SN. Presentamos dos casos con SN y MAC. Casos clinicos. Caso 1: mujer de 29 años con fenotipo de Noonan. Fue intervenida a los 9 años de estenosis valvular pulmonar. A los 27 años, presento MAC sintomatica que preciso descompresion quirurgica. Presentaba mutacion c.922A>G (N308D) en el gen PTPN perteneciente a la via RAS/MAPK. Caso 2: niña de 10 años con fenotipo de Noonan y MAC asintomatica detectada en resonancia magnetica cerebral. Era portadora de la mutacion c.923A>G (N308S) en el gen PTPN11. Conclusiones. Hemos encontrado en la bibliografia seis pacientes con esta asociacion, cuatro con fenotipo Noonan y dos con LEOPARD. Nuestros dos pacientes aportan evidencia suplementaria a la hipotesis de que la MAC formaria parte del espectro fenotipico del SN. El escaso numero de pacientes publicados con esta asociacion no permite extraer recomendaciones sobre el momento y la frecuencia de estudio de neuroimagen; no obstante, una exploracion neurologica cuidadosa deberia incluirse en la guia anticipatoria de salud en los sindromes de la via RAS/MAPK.

  16. The renaissance of Ras.

    PubMed

    Milroy, Lech-Gustav; Ottmann, Christian

    2014-11-21

    Increased signaling by the small G protein Ras is found in many human cancers and is often caused by direct mutation of this protein. Hence, small-molecule attenuation of pathological Ras activity is of utmost interest in oncology. However, despite nearly three decades of intense drug discovery efforts, no clinically viable option for Ras inhibition has been developed. Very recently, reports of a number of new approaches of addressing Ras activity have led to the revival of this molecular target with the prospect of finally fulfilling the therapy promises associated with this important protein.

  17. The role of feedback control mechanisms on the establishment of oscillatory regimes in the Ras/cAMP/PKA pathway in S. cerevisiae

    PubMed Central

    2012-01-01

    In the yeast Saccharomyces cerevisiae, the Ras/cAMP/PKA pathway is involved in the regulation of cell growth and proliferation in response to nutritional sensing and stress conditions. The pathway is tightly regulated by multiple feedback loops, exerted by the protein kinase A (PKA) on a few pivotal components of the pathway. In this article, we investigate the dynamics of the second messenger cAMP by performing stochastic simulations and parameter sweep analysis of a mechanistic model of the Ras/cAMP/PKA pathway, to determine the effects that the modulation of these feedback mechanisms has on the establishment of stable oscillatory regimes. In particular, we start by studying the role of phosphodiesterases, the enzymes that catalyze the degradation of cAMP, which represent the major negative feedback in this pathway. Then, we show the results on cAMP oscillations when perturbing the amount of protein Cdc25 coupled with the alteration of the intracellular ratio of the guanine nucleotides (GTP/GDP), which are known to regulate the switch of the GTPase Ras protein. This multi-level regulation of the amplitude and frequency of oscillations in the Ras/cAMP/PKA pathway might act as a fine tuning mechanism for the downstream targets of PKA, as also recently evidenced by some experimental investigations on the nucleocytoplasmic shuttling of the transcription factor Msn2 in yeast cells. PMID:22818197

  18. A Novel Ras Effector Pathway Found to Play Significant Role in Tumor Suppression | Poster

    Cancer.gov

    By Nancy Parrish, Staff Writer; photo by Richard Frederickson, Staff Photographer Normal cells have mechanisms to prevent the development of cancer. Among these is a type of tumor suppressor mechanism known as oncogene-induced senescence, or OIS, which halts the uncontrolled growth of cells caused by mutations in oncogenes. The oncogene Ras plays a crucial role in inducing OIS through a specific cascade of proteins, as reported in a recent article in Molecular and Cellular Biology by Jacqueline Salotti, Ph.D., and colleagues in the Eukaryotic Transcriptional Regulation Section of the Mouse Cancer Genetics Program, Center for Cancer Research (CCR).

  19. A Novel Ras Effector Pathway Found to Play Significant Role in Tumor Suppression | Poster

    Cancer.gov

    By Nancy Parrish, Staff Writer; photo by Richard Frederickson, Staff Photographer Normal cells have mechanisms to prevent the development of cancer. Among these is a type of tumor suppressor mechanism known as oncogene-induced senescence, or OIS, which halts the uncontrolled growth of cells caused by mutations in oncogenes. The oncogene Ras plays a crucial role in inducing OIS through a specific cascade of proteins, as reported in a recent article in Molecular and Cellular Biology by Jacqueline Salotti, Ph.D., and colleagues in the Eukaryotic Transcriptional Regulation Section of the Mouse Cancer Genetics Program, Center for Cancer Research (CCR).

  20. Yeast screens for inhibitors of Ras-Raf interaction and characterization of MCP inhibitors of Ras-Raf interaction.

    PubMed

    Khazak, Vladimir; Kato-Stankiewicz, Juran; Tamanoi, Fuyu; Golemis, Erica A

    2006-01-01

    Because of the central role of Ras in cancer cell signaling, there has been considerable interest in developing small molecule inhibitors of the Ras signaling pathways as potential chemotherapeutic agents. This chapter describes the use of a two-hybrid approach to identify the MCP compounds, small molecules that disrupt the interaction between Ras and its effector Raf. We first outline the reagent development and selection/counter selection methods required to successfully apply a two-hybrid approach to isolation of MCP compounds. Separately, we describe the collateral benefits of this screening approach in yielding novel antifungal compounds. We then discuss secondary physiological validation approaches to confirm the MCP compounds specifically target Ras-Raf signaling. Finally, we develop a decision tree for subsequent preclinical characterization and optimization of this class of pathway-targeted reagent.

  1. Telmisartan reduces atrial arrhythmia susceptibility through the regulation of RAS-ERK and PI3K-Akt-eNOS pathways in spontaneously hypertensive rats.

    PubMed

    Wang, Wei-Wei; Zhang, Fei-Long; Chen, Jian-Hua; Chen, Xue-Hai; Fu, Fa-Yuan; Tang, Mi-Rong; Chen, Liang-Long

    2015-08-01

    Telmisartan is an angiotensin II receptor blocker that displays unique PPAR-γ modulating activity. PPAR-γ agonists have been shown to decrease susceptibility to atrial fibrillation through their antioxidant and antiapoptotic effects. The aim of this study was to determine whether telmisartan would have a greater effect on susceptibility to atrial arrhythmia in a hypertensive rat model than valsartan, which is a traditional angiotensin II receptor blocker. In this study, spontaneously hypertensive rats were treated with 10 mg·(kg body mass)(-1)·d(-1) telmisartan (TEL group), 10 mg·(kg body mass)(-1)·d(-1) valsartan (VAL group), or vehicle (saline; SHR group) for 4 weeks. Age-matched Wistar-Kyoto rats (WKY) were used as normotensive controls. After 4 weeks of treatment, we performed echocardiographic assessment, electrophysiological analysis, histological evaluation, and Western blot analysis. Telmisartan decreased systolic blood pressure to a similar extent as valsartan. Relative to the WKY controls, atrial arrhythmia susceptibility was significantly increased in the SHR group, and was significantly decreased by both telmisartan and valsartan, albeit to a greater extent with telmisartan. Arrhythmogenic atrial remodeling, including enlargement of the left atrium, myocyte hypertrophy, interstitial fibrosis, and myocyte apoptosis, was observed in the SHR group, and was accompanied by activated RAS-ERK signaling and suppressed PI3K-Akt-eNOS signaling. The results suggest that telmisartan reduced susceptibility to atrial arrhythmia to a greater extent than valsartan, ameliorated atrial remodeling, and reversed imbalances in the RAS-ERK and PI3K-Akt-eNOS pathways.

  2. Exploring the interactions of the RAS family in the human protein network and their potential implications in RAS-directed therapies

    PubMed Central

    Bueno, Anibal; Morilla, Ian; Diez, Diego; Moya-Garcia, Aurelio A.; Lozano, José; Ranea, Juan A.G.

    2016-01-01

    RAS proteins are the founding members of the RAS superfamily of GTPases. They are involved in key signaling pathways regulating essential cellular functions such as cell growth and differentiation. As a result, their deregulation by inactivating mutations often results in aberrant cell proliferation and cancer. With the exception of the relatively well-known KRAS, HRAS and NRAS proteins, little is known about how the interactions of the other RAS human paralogs affect cancer evolution and response to treatment. In this study we performed a comprehensive analysis of the relationship between the phylogeny of RAS proteins and their location in the protein interaction network. This analysis was integrated with the structural analysis of conserved positions in available 3D structures of RAS complexes. Our results show that many RAS proteins with divergent sequences are found close together in the human interactome. We found specific conserved amino acid positions in this group that map to the binding sites of RAS with many of their signaling effectors, suggesting that these pairs could share interacting partners. These results underscore the potential relevance of cross-talking in the RAS signaling network, which should be taken into account when considering the inhibitory activity of drugs targeting specific RAS oncoproteins. This study broadens our understanding of the human RAS signaling network and stresses the importance of considering its potential cross-talk in future therapies. PMID:27713118

  3. The adaptor-like protein ROG-1 is required for activation of the Ras-MAP kinase pathway and meiotic cell cycle progression in Caenorhabditis elegans.

    PubMed

    Matsubara, Yosuke; Kawasaki, Ichiro; Urushiyama, Seiichi; Yasuda, Tomoharu; Shirakata, Masaki; Iino, Yuichi; Shibuya, Hiroshi; Yamanashi, Yuji

    2007-03-01

    The Ras-MAP kinase pathway regulates varieties of fundamental cellular events. In Caenorhabditis elegans, this pathway is required for oocyte development; however, the nature of its up-stream regulators has remained elusive. Here, we identified a C. elegans gene, rog-1, which encodes the only protein having the IRS-type phosphotyrosine-binding (PTB) domain in the worms. ROG-1 has no obvious domain structure aside from the PTB domain, suggesting that it could serve as an adaptor down-stream of protein-tyrosine kinases (PTKs). RNA interference (RNAi)-mediated down-regulation of rog-1 mRNA significantly decreased brood size. rog-1(tm1031) truncation mutants showed a severe disruption in progression of developing oocytes from pachytene to diakinesis, as was seen in worms carrying a loss-of-function mutation in the let-60 Ras or mpk-1 MAP kinase gene. Furthermore, let-60 Ras-regulated activation of MPK-1 in the gonad is undetectable in rog-1(tm1031) mutants. Conversely, a gain-of-function mutation in the let-60 Ras gene rescues the brood size reduction and germ cell abnormality in rog-1(tm1031) worms. Consistently, rog-1 is preferentially expressed in the germ cells and its expression in the gonad is essential for oocyte development. Thus, ROG-1 is a key positive regulator of the Ras-MAP kinase pathway that permits germ cells to exit from pachytene.