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PubMed1993

Bcl-2 associates with the ras-related protein R-ras p23.

Apoptosis is an important but poorly understood mechanism of cell regulation. Growth factor deprivation can trigger apoptosis in a variety of cells, suggesting the existence of a signal transduction pathway responding to external signals and leading to apoptosis. Overexpression of the proto-oncogene bcl-2 can override these signals and block apoptosis, indicating that the bcl-2 protein (Bcl-2) is an important component of the apoptotic response. The identification of Bcl-2-binding proteins might help explain how Bcl-2 acts to regulate apoptosis. Here we use the yeast two-hybrid system to show that the human ras-related protein R-ras p23 (ref 16-18) binds to Bcl-2. This association is also detected in immunoprecipitates from human cell extracts. The association requires full-length Bcl-2 but the C-terminal 60 amino acids of R-ras p23 are sufficient for the interaction. These results provide evidence of a putative component of a signal transduction pathway involved in the regulation of apoptosis.

HeLa Cells↗

PubMed2003

Pharmacologic inactivation of kinase suppressor of ras-1 abrogates Ras-mediated pancreatic cancer.

Inhibition of the kinase suppressor of ras-1 (KSR1) gene by continuous infusion of phosphorothioate antisense oligonucleotides (ODNs) prevented growth of K-Ras-dependent human PANC-1 pancreatic and A549 non-small-cell lung carcinoma xenografts in nude mice, effected regression of established PANC-1 tumors and inhibited A549 lung metastases, all without apparent toxicity. These studies suggest KSR1 antisense ODNs as a treatment for Ras-dependent human malignancies, in particular pancreatic cancer, which lacks effective curative therapy.

Animals↗

PubMed1998

NF1 inactivation cooperates with N-ras in in vivo lymphogenesis activating Erk by a mechanism independent of its Ras-GTPase accelerating activity.

We crossed transgenic mice overexpressing the N-ras proto-oncogene (RasTg) with mice carrying one inactivated copy of the NF1 tumor suppressor gene (NF1+/-) to assess their possible cooperation in tumorigenesis. We have found a significant increase in the incidence of lymphomas in animals with both lesions (RasTg NF1+/-), as compared with animals with single lesions. The mechanism of this cooperation appears to be independent of the NF1 GTPase activating activity since the level of Ras-GTP in primary cultures of tumor tissue do not differ among animals with double and with single lesions. Nevertheless, the finding of significantly higher levels of Erk-1 and Erk-2 activation in lymphomas in the RasTg NF1+/- than in the RasTg group suggests that this cooperative effect may be in part explained by increased signaling through the Erk pathways. Consistent with a role for Erk activation in transformation is the additional observation that Erk-1 and Erk-2 activation is significantly increased in lymphomas as compared with normal spleen. This activation is likely to occur by phosphorylation of previously synthesized and inactive Erk proteins since, despite differences in activation, Erk-1 and Erk-2 expression is similar in normal and lymphoid tissue in all groups. The observed cooperation in in vivo lymphomagenesis between N-ras overexpression and NF1 inactivation emphasizes the importance of searching for additional functions for the NF1 protein and of intensifying the screening for NF1 mutations in human lymphomas.

3T3 Cells↗

PubMed2003

Alterations of RAS signalling in Chinese multiple myeloma patients: absent BRAF and rare RAS mutations, but frequent inactivation of RASSF1A by transcriptional silencing or expression of a non-functional variant transcript.

The methylation status, mutation and expression of RASSF1A, and mutations of RAS and BRAF were studied in 52 patients with multiple myeloma (MM), one plasma cell leukaemia (PCL) patient and four MM-derived cell lines. Aberrant methylation of RASSF1A was found in nine of 32 MM patients and in one of four MM cell lines (U266), where the associated loss of transcription was reversible by demethylation treatment. RASSF1A transcription was further investigated on anti-CD138-sorted plasma cell-enriched bone marrow samples from 10 MM, one PCL and three reactive plasmacytosis patients. While the wild-type RASSF1A transcript was detected in all three reactive plasmacytosis and the PCL samples, we found no detectable wild-type transcripts in six of 10 MM samples studied. In two MM samples, only the non-functional variant transcript was detected, whereas the other four showed loss of transcription. In great contrast to western data, RAS mutations were identified in only four of 31 (13%) MM patients. While no RASSF1A or BRAF mutation (V599E) was detected in any of the primary MM studied (n = 21), the latter was found in the U266 cell line. Taken together, these data indicate that alterations of RAS signalling are critical in MM pathogenesis. In our current studies of Chinese MM patients, these alterations involved frequent RASSF1A inactivation (60%) as a result of transcriptional silencing or expression of a non-functional variant transcript.

Adult↗

PubMed1999

Separate roles for H-Ras and Rac in signaling by transforming growth factor (TGF)-beta. H-Ras is essential for activation of MAP kinase, partially required for transcriptional activation by TGF-beta, but not required for signaling of growth suppression by TGF-beta.

The signaling components located downstream of the transforming growth factor (TGF)-beta receptor are poorly understood. We constructed adenoviral vectors expressing a dominant-negative form of either H-Ras (AdCARasY57) or Rac (AdCARacN17), and used them to examine the roles of H-Ras and Rac in TGF-beta signaling using arterial endothelial cells in primary culture, and several established cells including a mink lung epithelial cell line (Mv1Lu). The rapid activation of p42/44 MAP kinase (MAPK) by TGF-beta1 was eliminated completely, and transcriptional activation by TGF-beta1 of the plasminogen activator inhibitor-1 gene was reduced by 50% in both endothelial cells and Mv1Lu when they were infected with AdCARasY57. However, the antiproliferative effect of TGF-beta, as assessed by the induction of the mRNA for Cdk4/6-specific inhibitor p15INK4B and by DNA synthesis, was not affected in AdCARasY57-infected cells. A MAPK kinase (MEK)1/2 inhibitor, U0126 also abolished MAPK activation and partially inhibited transcriptional activation by TGF-beta, suggesting that MAPK may be partially involved in this pathway. MAPK activation, transcriptional activation and growth suppression by TGF-beta were all unaffected in cells infected with AdCARacN17, although the DNA synthesis elicited by serum mitogens was suppressed completely in the infected cells. Our data indicate that H-Ras is essential for mitogen-activated protein kinase activation, partly required for transcriptional activation by TGF-beta, but not critically involved in the signaling that exerts the antiproliferative effect of TGF-beta. The results also suggest that Rac may not serve as an essential molecule in signaling by TGF-beta in the cells tested.

Adenoviridae↗

PubMed2006

Involvement of the Ras-Ras-activated Rab5 guanine nucleotide exchange factor RIN2-Rab5 pathway in the hepatocyte growth factor-induced endocytosis of E-cadherin.

E-cadherin is a key cell-cell adhesion molecule at adherens junctions (AJs) and undergoes endocytosis when AJs are disrupted by the action of an extracellular signal, such as hepatocyte growth factor (HGF)/scatter factor. Rab5 small G protein has been implicated in the HGF-induced endocytosis of E-cadherin, but the molecular mechanism for the regulation of Rab5 activity remains unknown. We first studied this mechanism by using the cell-free assay system for the endocytosis of E-cadherin of the AJ-enriched fraction from rat livers. HGF induced activation of Ras small G protein, which then bound to RIN2, a Rab5 GDP/GTP exchange factor with the Vps9p-like guanine nucleotide exchange factor and Ras association domains, and activated it. Activated RIN2 then activated Rab5, eventually inducing the endocytosis of E-cadherin. We then studied whether RIN2 was involved in the HGF-induced endocytosis of E-cadherin in intact Madin-Darby canine kidney cells. RIN2 localized at the cell-cell adhesion sites, and its guanine nucleotide exchange factor activity was required for the HGF-induced endocytosis of E-cadherin in Madin-Darby canine kidney cells. These results indicate that RIN2 connects Ras to Rab5 in the HGF-induced endocytosis of E-cadherin.

Animals↗

PubMed2005

Deregulated matriptase causes ras-independent multistage carcinogenesis and promotes ras-mediated malignant transformation.

Overexpression of the type II transmembrane serine protease matriptase is a highly consistent feature of human epithelial tumors. Here we show that matriptase possesses a strong oncogenic potential when unopposed by its endogenous inhibitor, HAI-1. Modest orthotopic overexpression of matriptase in the skin of transgenic mice caused spontaneous squamous cell carcinoma and dramatically potentiated carcinogen-induced tumor formation. Matriptase-induced malignant conversion was preceded by progressive interfollicular hyperplasia, dysplasia, follicular transdifferentiation, fibrosis, and dermal inflammation. Furthermore, matriptase induced activation of the pro-tumorigenic PI3K-Akt signaling pathway. This activation was frequently accompanied by H-ras or K-ras mutations in carcinogen-induced tumors, whereas matriptase-induced spontaneous carcinoma formation occurred independently of ras activation. Increasing epidermal HAI-1 expression completely negated the oncogenic effects of matriptase. The data implicate dysregulated matriptase expression in malignant epithelial transformation.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1989

The ras oncogene and tumour metastasis: observations on murine cells transfected with activated human c-Ha-ras.

Transfection of cells with cloned genes or total genomic DNA offers a means for studying aspects of neoplastic behaviour. We have used this method to examine whether incorporation of the cloned 6.6-kilobase (kb) fragment of DNA containing the mutant c-Ha-ras human oncogene can confer metastatic capability on murine NIH 3T3 cells. Cells co-transfected with the mutated ras gene and the neomycin resistance marker pSV2neo were selected by culture in neomycin. On subcutaneous inoculation into MF 1 nude mice, these cells proved to be tumourigenic with short latent periods (approximately 14 days)--nude mice were used to circumvent immunological rejection of the mouse cells expressing the product of the human oncogene. Transfectants were capable of lung colonisation after intravenous injection, but there was no evidence of spontaneous metastasis at autopsy, or on histological examination of the lungs and other organs, 90 days after inoculation. Incorporation of the transfected oncogene was confirmed by Southern blotting and its expression by dot-blot hybridisation and immunoprecipitation. The results in this experimental system indicate that transfection of a mutated human ras oncogene into non-neoplastic 3T3 cells can confer part of the metastatic phenotype, namely lung colonisation, but is not by itself sufficient to induce spontaneous metastatic behaviour.

Animals↗

PubMed2006

Hitting Ras where it counts: Ras antagonism in the basolateral amygdala inhibits long-term fear memory.

Several studies have implicated the Ras/mitogen-activated protein kinase (MAPK) pathway in Pavlovian fear conditioning. RasGRF1 knockout mice show significant deficits in acquisition of long-term fear memories and long-term potentaition (LTP) in the basolateral amygdala (BLA). MAPK kinase inhibition also impairs fear conditioning and amygdaloid LTP. However, there is no direct evidence to date for the involvement of Ras itself in fear conditioning. To address this issue, we examined the effects of intra-amygdala infusions of the selective Ras antagonist farnesylthiosalicylic acid (FTS) on the acquisition and expression of conditional freezing in rats. Micro-infusions of FTS into the BLA prior to contextual fear conditioning significantly impaired acquisition of long-term contextual fear memory in a dose-dependent manner. Post-training FTS infusions had no effect on acquisition of long-term fear memory. The effects of FTS on fear conditioning were specific for the BLA. Finally, intra-amygdala infusions of FTS inhibited MAPK activation in BLA. Collectively, these results provide further evidence for the involvement of amygdaloid Ras in the acquisition of long-term conditional fear memory.

Acoustic Stimulation↗

PubMed1993

Antibody mimicking the action of RAS proteins on yeast adenylyl cyclase: implication for RAS-effector interaction.

Polyclonal antisera were raised against various subregions of Saccharomyces cerevisiae adenylyl cyclase in order to examine the molecular mechanism of interaction between adenylyl cyclase and RAS proteins. One of the antisera was found to activate adenylyl cyclase to an extent comparable to that activated by saturating amounts of yeast RAS2 protein produced in Escherichia coli. The stimulatory effect of this antiserum was shown to be additive with RAS2 protein when both antisera and RAS2 protein were present at low concentrations. At saturating amounts of RAS2 protein, the antisera did not exhibit additional stimulatory effects, suggesting that the actions of RAS2 protein and the antisera are complementary with each other. The antigenic determinant for the antibody involved in the activation was mapped to a 14-amino-acid segment, 1452-NSVDNGADVANLSY-1465, located between the leucine-rich repeats and the catalytic domain of adenylyl cyclase. Certain missense mutations affecting this 14-amino acid segment significantly reduced the response of adenylyl cyclase to both activating antibody and RAS proteins. These results suggest that this segment of adenylyl cyclase is intimately involved in the mechanism by which RAS proteins activate this downstream effector.

Adenylyl Cyclases↗

PubMed2002

Two ras pathways in fission yeast are differentially regulated by two ras guanine nucleotide exchange factors.

How a given Ras prreotein coordinates multiple signaling inputs and outputs is a fundamental issue of signaling specificity. Schizosaccharomyces pombe contains one Ras, Ras1, that has two distinct outputs. Ras1 activates Scd1, a presumptive guanine nucleotide exchange factor (GEF) for Cdc42, to control morphogenesis and chromosome segregation, and Byr2, a component of a mitogen-activated protein kinase cascade, to control mating. So far there is only one established Ras1 GEF, Ste6. Paradoxically, ste6 null (ste6 Delta) mutants are sterile but normal in cell morphology. This suggests that Ste6 specifically activates the Ras1-Byr2 pathway and that there is another GEF capable of activating the Scd1 pathway. We thereby characterized a potential GEF, Efc25. Genetic data place Efc25 upstream of the Ras1-Scd1, but not the Ras1-Byr2, pathway. Like ras1 Delta and scd1 Delta, efc25 Delta is synthetically lethal with a deletion in tea1, a critical element for cell polarity control. Using truncated proteins, we showed that the C-terminal GEF domain of Efc25 is essential for function and regulated by the N terminus. We conclude that Efc25 acts as a Ras1 GEF specific for the Scd1 pathway. While ste6 expression is induced during mating, efc25 expression is constitutive. Moreover, Efc25 overexpression renders cells hyperelongated and sterile; the latter can be rescued by activated Ras1. This suggests that Efc25 can recruit Ras1 to selectively activate Scd1 at the expense of Byr2. Reciprocally, Ste6 overexpression can block Scd1 activation. We propose that external signals can partly segregate two Ras1 pathways by modulating GEF expression and that GEFs can influence how Ras is coupled to specific effectors.

ATP-Binding Cassette Transporters↗

PubMed2005

Regulation of dendritic morphogenesis by Ras-PI3K-Akt-mTOR and Ras-MAPK signaling pathways.

Dendritic arborization and spine formation are critical for the functioning of neurons. Although many proteins have been identified recently as regulators of dendritic morphogenesis, the intracellular signaling pathways that control these processes are not well understood. Here we report that the Ras-phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway plays pivotal roles in the regulation of many aspects of dendrite formation. Whereas the PI3K-Akt-mTOR pathway alone controlled soma and dendrite size, a coordinated activation together with the Ras-mitogen-activated protein kinase signaling pathway was required for increasing dendritic complexity. Chronic inhibition of PI3K or mTOR reduced soma and dendrite size and dendritic complexity, as well as density of dendritic filopodia and spines, whereas a short-term inhibition promoted the formation of mushroom-shaped spines on cells expressing constitutively active mutants of Ras, PI3K, or Akt, or treated with the upstream activator BDNF. Together, our data underscore the central role of a spatiotemporally regulated key cell survival and growth pathway on trophic regulation of the coordinated development of dendrite size and shape.

Animals↗

PubMed1992

Mutational activation of the N-ras oncogene assessed in primary clonogenic culture of acute myeloid leukemia (AML): implications for the role of N-ras mutation in AML pathogenesis.

The number of steps involved in the pathogenesis of acute myeloid leukemia (AML) is unclear. The initiating event would be expected to exist in all leukemic cells, but subsequent events may be subclonal. If several genetic events occur, they may cooperate within the same cell or be alternatively acquired by different subclones. These possibilities cannot be adequately analyzed in DNA prepared directly from patient specimens. In this study, N-ras mutations demonstrable in DNA prepared from peripheral blood of 10 patients with AML were examined in primary in vitro colonies (AML-colony-forming units [CFU]) grown from these patients. Both colonies containing the mutant gene and colonies containing normal allele only were obtained from each patient. The proportion of colonies containing no mutant allele varied among patients (5% to 57%). A subset of mutation containing colonies appeared to have lost the normal allele in nine of 10 AML cases analyzed. In the four cases with two N-ras mutations, the two mutations were found to exist in different subclones. In these cases, macroscopic colonies (AML-MCFU) were also obtained using an assay system designed to select for earlier clonogenic cells than in the AML-CFU assay. The N12cys mutation in AML10 was found in the CFU, but not in the MCFU, and the N12asp mutation in AML43 was found in the MCFU, but not in the CFU. These results suggest that N-ras mutation is a postinitiation event in AML that contributes to the outgrowth of more malignant subclones. Where two mutations are found in a case of AML, they appear to have been acquired by separate subclones, which may show different degrees of differentiation.

Base Sequence↗

PubMed1996

Structure of the Ras-binding domain of c-Raf-1 as determined by NMR spectroscopy and identification of the region that interacts with Ras.

The structure of the Ras-binding domain of human c-Raf-1 (residues 55 to 132) as determined in solution by NMR spectroscopy is presented. It consists of a five-stranded beta-sheet, a twelve residue alpha-helix, and an additional one-turn helix. The fold belongs to a known family whose members include ubiquitin and protein G. The surface of Raf55-132 that interacts with Ras has been identified by resonance perturbation mapping. The binding site is a spatially contiguous patch comprised of the two-N-terminal beta-strands, the loop between them, and the C-terminal end of the alpha-helix. A model of the Raf-Ras complex is presented, which was derived by analogy to the complex between protein G and a Fab fragment of IgG. In the model, edge beta-strands of each protein align in an antiparallel orientation, forming a unified beta-sheet, and side chains from both proteins are able to participate in ionic and hydrophobic interactions at the interface.

Binding Sites↗

PubMed1996

Requirement of phospholipase C gamma, the tyrosine phosphatase Syp and the adaptor proteins Shc and Nck for PDGF-induced DNA synthesis: evidence for the existence of Ras-dependent and Ras-independent pathways.

We have investigated the roles of the phosphotyrosine phosphatase Syp (also called SH-PTP2), phospholipase C (PLC) gamma1, rasGTPase Activating Protein (rasGAP) and the adapter molecules Nck and Shc in the mitogenic response induced by PDGF in fibroblasts. Two separate approaches were used to inhibit the biological activity of these signalling proteins in vivo. Either glutathione S-transferase (GST) fusion proteins containing the SH2 domains of these proteins, or antibodies specific for these polypeptides, were microinjected into cells. GST-SH2 fusion proteins are expected to act as dominant inhibitors by competing for physiological SH2-mediated interactions, while microinjected antibodies can directly block protein functions. Inhibition of PLCgamma, Syp, Shc and Nck signals blocked PDGF-stimulated cells in G1 showing a requirement for these proteins for S-phase entry. Inhibition of rasGAP, in contrast, had no effect on S-phase entry. We next examined which of these signals were required for PDGF-induced cFos expression, a Ras-dependent event important for signalling. By using the same approaches with cells expressing beta-galactosidase under the control of a c-fos promoter, we showed that PLCgamma, Syp and Shc were necessary for ligand-induced cFos expression whereas Nck and phosphatidylinositol 3-kinase alpha were not. From these results we concluded that PDGF generates Ras-dependent and Ras-independent pathways important for DNA synthesis.

3T3 Cells↗

PubMed1985

Activation of K-ras and oncogenes other than ras family in rat fibrosarcomas induced by 1,8-dinitropyrene.

Oncogenes of fibrosarcomas of rats, induced by subcutaneous injection of 1,8-dinitropyrene (1,8-DNP), were examined by NIH 3T3 cell transfection assay and Southern blot analysis. Transformants containing rat specific repetitive sequences were obtained with DNAs of 4 fibrosarcomas, 1,8-DNP1, 1,8-DNP2, 1,8-DNP3 and 1,8-DNP7. A transformant, 1,8-DNP2-2, induced by DNA of a fibrosarcoma, 1,8-DNP2, and 7 secondary transformants derived from it contained rat K-ras sequences. Another transformant, 1,8-DNP2-1, induced by the same sarcoma did not have a ras family oncogene. This indicates that the sarcoma, 1,8-DNP2, has at least 2 transforming genes. The transforming genes of 6 other transformants derived from 3 sarcomas did not contain ras family or neu transforming genes.

Animals↗

PubMed1987

Active H-ras and N-ras in rat fibrosarcomas induced by 1,6-dinitropyrene.

The activated oncogenes in rat fibrosarcomas induced by subcutaneous injection of 1,6-dinitropyrene (1,6-DNP), an environmental mutagen-carcinogen, were examined by NIH3T3 cell transfection assay and Southern blot analysis. DNAs from two of eleven fibrosarcomas, 1,6-DNP6 and 1,6-DNP9T, induced transformants which contained rat specific repetitive sequences. Three primary transformants, obtained with DNA from 1,6-DNP6, and two secondary transformants of one of these primary transformants tested, contained rat H-ras. DNA from 1,6-DNP9T; a fourth transplant of 1,6-DNP9, also induced transformants. All the primary transformants with DNA from 1,6-DNP9T, and three secondary transformants of one of them, contained rat N-ras. The DNA from the original tumor, 1,6-DNP9, however, did not give any transformants by several transfection assays. Activation of N-ras oncogene during serial transplantations is discussed.

Animals↗

PubMed1982

Comparative biochemical properties of p21 ras molecules coded for by viral and cellular ras genes.

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

Animals↗