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At least 1,081 records · Page 60Linked to original sources
PubMed2005

Amino-acid limitation induces transcription from the human C/EBPbeta gene via an enhancer activity located downstream of the protein coding sequence.

For animals, dietary protein is critical for the nutrition of the organism and, at the cellular level, protein nutrition translates into amino acid availability. Amino acid deprivation triggers the AAR (amino acid response) pathway, which causes enhanced transcription from specific target genes. The present results show that C/EBPbeta (CCAAT/enhancer-binding protein beta) mRNA and protein content were increased following the deprivation of HepG2 human hepatoma cells of a single amino acid. Although there was a modest increase in mRNA half-life following histidine limitation, the primary mechanism for the elevated steady-state mRNA was increased transcription. Transient transfection documented that C/EBPbeta genomic fragments containing the 8451 bp 5' upstream of the transcription start site did not contain amino-acid-responsive elements. However, deletion analysis of the genomic region located 3' downstream of the protein coding sequence revealed that a 93 bp fragment contained an amino-acid-responsive activity that functioned as an enhancer. Exogenous expression of ATF4 (activating transcription factor 4), known to activate other genes through amino acid response elements, caused increased transcription from reporter constructs containing the C/EBPbeta enhancer in cells maintained in complete amino acid medium. Chromatin immunoprecipitation demonstrated that RNA polymerase II is bound at the C/EBPbeta promoter and at the 93 bp regulatory region in vivo, whereas ATF4 binds to the enhancer region only. Immediately following amino acid removal, the kinetics of binding for ATF4, ATF3, and C/EBPbeta itself to the 93 bp regulatory region were similar to those observed for the amino-acid-responsive asparagine synthetase gene. Collectively the findings show that expression of C/EBPbeta, which contributes to the regulation of amino-acid-responsive genes, is itself controlled by amino acid availability through transcription.

Activating Transcription Factor 4↗
PubMed2007

Clustering of monosialyl-Gb5 initiates downstream signalling events leading to invasion of MCF-7 breast cancer cells.

Invasion is a complex process controlled by secretion and activation of proteases, alteration of integrin levels and GSL (glycosphingolipid) patterns. Differential organization of GSLs with specific membrane proteins and signal transducers in GEMs (GSL-enriched microdomains), initiates signalling events to modify cellular phenotype. Although the GSL monosialyl-Gb5 has been linked with invasion, its functional role in invasion is poorly described and understood. To investigate this problem, we induced the invasion of human breast cancer cells and subsequently explored the underlying mechanism. In the present study, the invasion of human MCF-7 breast cancer cells is highly dependent on clustering of monosialyl-Gb5, and the subsequent activation of monosialyl-Gb5-associated focal adhesion kinase and cSrc in GEM leading to the downstream activation of extracellular-signal-regulated kinase (ERK). As a result, we observed increased expression levels and activity of matrix metalloproteinases-2 and -9, which correlated with decreased expression of integrins alpha1 and beta1. Together these results suggest that the organization of crucial molecules in GEMs of MCF-7 cells is critical for their invasive properties.

Breast Neoplasms↗
PubMed2004

Identification of novel effectors of invasive cell growth downstream of phosphoinositide 3-kinase.

Conventional approaches to identifying cancer targets are complicated by the chromosomal instability of tumour cells, and typically result in a large number of differentially expressed candidate genes with uncertain disease relevance. Here we present a novel approach which aims to elucidate the molecular changes that are induced after loss of tumour suppressor function. Using gene silencing tools, we mimic the loss of tumour suppressor function to identify key regulators of tumour initiation and progression. Loss of function of the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) correlates with increased invasive cell growth due to the resulting chronic activation of the PI 3-kinase (phosphoinositide 3-kinase) pathway. Induced activation of PI 3-kinase either by inhibiting PTEN expression or by using p110*, a constitutively active PI 3-kinase, increased signalling and the invasive growth potential of cells. Using this unbiased approach we have identified novel downstream effectors of PI 3-kinase/PTEN signalling that mediate the behaviour of cells with a hyperactive PI 3-kinase pathway. These molecules represent candidate targets for therapeutic intervention in patients with PTEN-deficient tumours.

Animals↗
PubMed1999

Changes in nuclear morphology during apoptosis correlate with vimentin cleavage by different caspases located either upstream or downstream of Bcl-2 action.

BACKGROUND: Upon Fas stimulation, procaspase-8 is recruited to the death-inducing signalling complex where autoactivation of caspase-8 occurs. Active caspase-8 can directly activate downstream caspases (e.g. caspase-3, 6, and 7) for the execution of apoptosis (mitochondria-independent pathway), while caspase-8 can also lead to executioner caspase activation through mitochondrial damage (mitochondria-dependent pathway). Caspase activation results in the dismantling of intracellular structure through specific proteolysis. RESULTS: We have found that an intermediate filament protein, vimentin, is cleaved at multiple sites by caspases at an early stage of apoptosis in Jurkat cells. The sequences of the two major cleavage sites in vimentin (IDVD/V and DSVD/F) suggested that these sites are cleaved by caspase-8 and caspase-3, respectively, or by close homologues of these proteases. The IDVD/V site can be cleaved by caspase-8 in vitro, and its cleavage is less sensitive to DEVD-CHO and Bcl-2 over-expression than that of the DSVD/F site in Jurkat cells. Over-expression of a mutant vimentin which was insensitive to caspase cleavage at these sites delayed the appearance of apoptotic nuclei in Jurkat cells. CONCLUSION: The specific cleavage of vimentin can be used as an apoptotic marker of both apical- and mitochondria-dependent caspase activation. Apoptotic cleavage of vimentin most likely results in disruption of its filamentous structure, which may facilitate nuclear condensation and subsequent fragmentation through disruption of the cytoskeletal network.

Amino Acid Sequence↗
PubMed2000

Mechanism of catabolite repression in the bgl operon of Escherichia coli: involvement of the anti-terminator BglG, CRP-cAMP and EIIAGlc in mediating glucose effect downstream of transcription initiation.

BACKGROUND: Expression of the bgl operon of Escherichia coli, involved in the regulated uptake and utilization of aromatic beta-glucosides, is extremely sensitive to the presence of glucose in the growth medium. We have analysed the mechanism by which glucose exerts its inhibitory effect on bgl expression. RESULTS: Our studies show that initiation of transcription from the bgl promoter is only marginally sensitive to glucose. Instead, glucose exerts a more significant inhibition on the elongation of transcription beyond the rho-independent terminator present within the leader sequence. Transcriptional analyses using plasmids that carry mutations in bglG or within the terminator, suggest that the target for glucose-mediated repression is the anti-terminator protein, BglG. Introduction of multiple copies of bglG or the presence of mutations that inhibit its phosphorylation by Enzyme IIBgl (BglF), result in loss of glucose repression. Studies using crp, cya and crr strains show that both CRP-cAMP and the Enzyme IIAGlc (EIIAGlc) are involved in the regulation. Although transcription initiation is normal in a crp, cya double mutant, no detectable transcription is seen downstream of the terminator, which is restored by a mutation within the terminator. Transcription past the terminator is also partly restored by the addition of exogenous cAMP to glucose-grown cultures of a crp+ strain. Glucose repression is lost in the crr mutant strain. CONCLUSIONS: The results summarized above indicate that glucose repression in the bgl operon is mediated at the level of transcription anti-termination, and glucose affects the activity of BglG by altering its phosphorylation by BglF. The CRP-cAMP complex is also involved in this regulation. The results using the crr mutant suggest a negative role for EIIAGlc in the catabolite repression of the bgl genes.

Bacterial Proteins↗
PubMed1999

Msn1p/Mss10p, Mss11p and Muc1p/Flo11p are part of a signal transduction pathway downstream of Mep2p regulating invasive growth and pseudohyphal differentiation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, a network of signal transduction pathways governs the switch from yeast-type growth to pseudohyphal and invasive growth that occurs in response to nutrient limitation. Important elements of this network have been identified, including nutrient signal receptors, GTP-binding proteins, components of the pheromone-dependent MAP kinase cascade and several transcription factors. However, the structural and functional mapping of these pathways is far from complete. Here, we present data regarding three genes, MSN1/MSS10, MSS11 and MUC1/FLO11, which form an essential part of the signal transduction network establishing invasive growth. Both MSN1 and MSS11 are involved in the co-regulation of starch degradation and invasive growth. Msn1p and Mss11p act downstream of Mep2p and Ras2p and regulate the transcription of both STA2 and MUC1. We show that MUC1 mediates the effect of Msn1p and Mss11p on invasive growth. In addition, our results suggest that the activity of Msn1p is independent of the invasive growth MAP kinase cascade, but the Mss11p is required for the activation of pseudohyphal and invasive growth by Ste12p. We also show that starch metabolism in S. cerevisiae is subject to regulation by components of the MAP kinase cascade.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

Variegation and silencing of the Heat Shock Cognate 80 gene are relieved by a bipartite downstream regulatory element.

Transgenes are important tools for plant molecular biologists. However, some aspects of their regulation are not completely understood. First, the contribution of elements separate from the promoter has been documented for several genes, but a general requirement for such elements is still unclear. Second, transgene expression can be subject to variability from chromosomal position effects and from epigenetic effects, often due to the presence of repeated sequences. While studying the regulation of the developmentally expressed Heat Shock Cognate 80 (HSC80) gene, several peculiarities were found which involve both of these questions. First, HSC80 transgenes are subject to silencing and variegation at a high frequency. Second, a bipartite element downstream of the transcriptional start is necessary to achieve efficient expression from the HSC80 promoter in stable transformants. Finally, this bipartite element is not required for expression in a transient system, where the promoter alone is sufficient. These results together support a role for chromatin structure in the regulation of HSC80.

Arabidopsis↗
PubMed2002

HFR1, a phytochrome A-signalling component, acts in a separate pathway from HY5, downstream of COP1 in Arabidopsis thaliana.

HFR1, a basic helix-loop-helix protein, is known to be required for a subset of phytochrome A (phyA)-dependent photoresponses. To investigate the role of HFR1 in light signalling, we have examined the genetic interaction between HFR1 and HY5, a positive regulator of light signalling, and COP1, a repressor of photomorphogenesis. Double mutant analysis suggests that HFR1 mediates phyA-dependent inhibition of hypocotyl elongation independently of HY5. HFR1 was shown to be necessary for a subset of cop1-triggered photomorphogenic phenotypes in the dark, including inhibition of hypocotyl elongation, gravitropic hypocotyl growth, and expression of the light-inducible genes CAB and RBCS. Phenotypic analysis of the triple mutant cop1hy5hfr1 indicated that both HFR1 and HY5 are required for cop1-mediated photomorphogenic seedling development in darkness, consistent with their additive roles in phyA-dependent signalling. Taken together, these results suggest that HFR1 might act downstream of COP1, in a separate pathway from HY5, to mediate photomorphogenesis in Arabidopsis.

Arabidopsis↗
PubMed2002

ABI5 acts downstream of ABI3 to execute an ABA-dependent growth arrest during germination.

The development of a germinating embryo into an autotrophic seedling is arrested under conditions of water deficit. This ABA-mediated developmental checkpoint requires the bZIP transcription factor ABI5. Here, we used abi3-1, which is also unable to execute this checkpoint, to investigate the relative role of ABI3 and ABI5 in this process. In wild-type Arabidopsis plants, ABI3 expression and activity parallel those described for ABI5 following stratification. During this process, transcript levels of late embryogenesis genes such as AtEm1 and AtEm6 are also re-induced, which might be responsible for the acquired osmotic tolerance in germinated embryos whose growth is arrested. ABI5 expression is greatly reduced in abi3-1 mutants, which has low AtEm1 or AtEm6 expression. Cross complementation experiments showed that 35S-ABI5 could complement abi3-1, whereas 35S-ABI3 cannot complement abi5-4. These results indicate that ABI5 acts downstream of ABI3 to reactivate late embryogenesis programmes and to arrest growth of germinating embryos. Although ABI5 is consistently located in the nucleus, chromosomal immunoprecipitation (ChIP) experiments revealed that ABA increases ABI5 occupancy on the AtEm6 promoter.

Abscisic Acid↗
PubMed2003

The abi1-1 mutation blocks ABA signaling downstream of cADPR action.

Arabidopsis thaliana abscisic acid insensitive 1-1 (abi1-1) is a dominant mutant that is insensitive to the inhibition of germination and growth by the plant hormone, abscisic acid (ABA). The mutation severely decreases the catalytic activity of the ABI1 type 2C protein phosphatase (PP2C). However, the site of action of the abi1-1/ABI1 in the ABA signal transduction pathway has not yet been determined. Using single cell assays, we showed that microinjecting mutant abi1-1 protein inhibited the activation of RD29A-GUS and KIN2-GUS in response to ABA, cyclic ADP-ribose (cADPR), and Ca2+. The inhibitory effect of the mutant protein, however, was reversed by co-microinjection of an excess amount of the ABI1 protein. In transgenic Arabidopsis plants, overexpression of abi1-1 rendered the plants insensitive to ABA during germination, whereas overexpression of ABI1 did not have any apparent effect. Moreover, transgenic plants overexpressing abi1-1 were blocked in the induction of ABA-responsive genes; however, overexpression of ABI1 did not affect gene expression. Taken together, our results demonstrate that abi1-1 is likely to be a dominant negative mutation and ABI1 likely acts downstream of cADPR in the ABA-signaling pathway. Our results on ABI1 overexpression in Arabidopsis are not compatible with a negative regulatory role of this phosphatase in ABA responses.

Abscisic Acid↗
PubMed2003

Sequences downstream of the transcription initiation site are important for proper initiation and regulation of mouse ribonucleotide reductase R2 gene transcription.

Ribonucleotide reductase is essential for the synthesis of all four dNTPs required for DNA replication. The enzyme is composed of two proteins, R1 and R2, which are both needed for activity. Expression of the R1 and R2 mRNAs is restricted to the S-phase of the cell cycle, but the R1 and R2 promoters show no obvious sequence homologies that could indicate coordination of transcription. Here we study initiation of transcription at the natural mouse R2 promoter, which contains an atypical TATA-box with the sequence TTTAAA, using a combination of in vivo reporter gene assays and in vitro transcription. Our results indicate that in constructs where sequences from the R2 5'-UTR are present, the mouse R2 TATA-box is dispensable both for unregulated, basal transcription from the R2 promoter and for S-phase specific activity. Instead, initiation of R2 transcription is directed by sequences downstream from the transcription start. We report that this region contains a conserved palindrome sequence that interacts with TAFIIs. This interaction down-regulates basal transcription from the R2 promoter, both in the absence and in the presence of the TATA-box.

5' Untranslated Regions↗
PubMed1998

Downstream targets of urokinase-type plasminogen-activator-mediated signal transduction.

We have investigated cellular signalling events induced by urokinase-type plasminogen activator (uPA) independent of its proteolytic activity. Treatment of the human fibrosarcoma cell line HT 1080 with diisopropylphosphorofluoridate-inactivated uPA (Dip-F-uPA) triggers a cascade of intracellular signals which are mediated by the specific cell surface receptor for uPA (uPAR). We have found that anti-uPAR Ig precipitate the src-type protein tyrosine kinases fyn, hck and lck, which belong to a family of structurally and functionally related effectors participating in signalling from antigen and cytokine receptors. Of the three uPAR-associated kinases, only hck is activated by uPA, whereas no changes in the activities of either fyn or lck could be detected by an in vitro immune complex kinase assay. We identified p38 and extracellular-signal-regulated kinase 2 from the mitogen-activated protein kinase family as downstream components of a set of consecutive signalling molecules which teleologically alter the program of gene expression. Exposure of cells to uPA results in a significant increase in c-fos mRNA that is partially due to an elevated rate of gene transcription. Presumably, the activation of the c-fos gene leads to the subsequent formation of the transcription factor activator protein-1 (AP-1), since accumulation of c-fos mRNA is followed by induction of target genes sensitive to AP-1 such as plasminogen activator inhibitor type 2 (PAI-2). These results provide new insights into proteolysis-independent cytokine-like effects of uPA.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

Temporal expression patterns of 39 Brachyury-downstream genes associated with notochord formation in the Ciona intestinalis embryo.

Expression of the Brachyury (Ci-Bra) gene of the ascidian Ciona intestinalis is initiated at the 64-cell stage. Gene expression is restricted to notochord precursor cells, and Ci-Bra plays a key role in notochord differentiation. In a previous study, nearly 50 cDNA clones for potential Ci-Bra-downstream genes that are expressed in notochord cells were isolated. The present determination, by whole-mount in situ hybridization, of the temporal expression patterns of 19 notochord-specific and 20 notochord-predominant genes demonstrated that the timings of initiation of the expression of various genes was not identical. The expression of several genes was initiated as early as the gastrula stage. However, the expression of most of the notochord-specific genes commenced at the neural plate stage. Partial nucleotide sequence data of these clones suggest that genes expressed earlier encode potential transcriptional factors and/or nuclear proteins, while those expressed later encode proteins implicated in cell adhesion, signal transduction, regulation of the cytoskeleton, and components of the extracellular matrix. These gene activities may be associated with changes in cell shape and adhesion during the intercalation and extension of the notochord cells.

Animals↗
PubMed2002

Activation of Rac, Cdc42 and other downstream signalling molecules by Bartonella bacilliformis during entry into human endothelial cells.

Bartonella bacilliformis is an intracellular bacterial pathogen of human endothelial cells. In vitro incubation of B. bacilliformis with human endothelial cells leads to the formation of filamentous actin extensions (filopodia) within 30 min, followed by formation of membrane rufflings or lamellipodia within 1 h of incubation. By immunofluorescence, F-actin phalloidin staining and anti-Rac antibodies were shown to co-localize in the membrane rufflings, indicating the recruitment of activated Rac at lamellipodia. Preincubation of endothelial cells with the Clostridial toxin, TcdB-10463, which inactivates the Rho-family GTPases, Rho, Rac and Cdc42, inhibited the entry of B. bacilliformis by 50-90%. Preincubation of endothelial cells with the Clostridial toxin, TcsL-1522, which specifically inactivates Rac and, to a lesser extent, Cdc42, but not Rho, inhibited entry by 30-40%. A 3.4-5.0-fold increase in activated (GTP-bound) -intracellular Rac and Cdc42 was observed in affinity precipitation assays. Increased kinase activity of p21-activated kinase (PAK), a specific downstream effector of activated Rac/Cdc42 was also observed during the time course of infection. Activation of SAPK/JNK-1 and 2, and p38 MAPKs in signalling pathways, was also detected during infection with Bartonella, as was increased binding activity of AP-1 transcription factor.

Actins↗
PubMed1995

Time-resolved signaling pathways of nerve growth factor diverge downstream of the p140trk receptor activation between chick sympathetic and dorsal root ganglion sensory neurons.

We have recently shown that the small GTP binding protein p21ras is essential for nerve growth factor (NGF)-mediated survival of peripheral embryonic chick dorsal root ganglia (DRG) sensory but not sympathetic neurons. To investigate at which level of the signaling cascade the pathways diverge, we have studied the time-resolved pattern of NGF-stimulated tyrosine phosphorylation of proteins within 4 h after addition of the neurotrophin. In both chick sympathetic neurons [embryonic day (E) 12] and DRG sensory neurons (E9) NGF induces within 1 min the autophosphorylation of the receptor tyrosine kinase p140trk. However, the pattern of substrate protein tyrosine phosphorylation downstream of p140trk is distinctly different in both neuronal subtypes. In sympathetic neurons, we observed within 1 min the tyrosine phosphorylation of a new substrate protein, p105, reaching maximal levels at 3 min. Tyrosine phosphorylation of p105 remains elevated for up to 4 h. Subsequent to p105, NGF induces the tyrosine phosphorylation of p42, a protein belonging to the family of mitogen-activated protein (MAP) kinases. This stimulation is transient, reaching maximal levels at 10 min and returning to very low levels already after 2 h. In DRG sensory neurons, tyrosine phosphorylation of p105 is weak and very short lived, disappearing already after treatment with NGF for 10 min. In contrast, activation of MAP kinase p42 in DRG sensory neurons is more stable than in sympathetic neurons. All NGF-stimulated tyrosine phosphorylation events were inhibited by preincubation of neurons with the tropomyosin-related kinase (trk) inhibitor K252a.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Neurotrophic factors prevent ceramide-induced apoptosis downstream of c-Jun N-terminal kinase activation in PC12 cells.

Neurotrophic factors prevent apoptosis of PC12 cells in serum-free medium. The present study determines whether neurotrophic factors can prevent ceramide-induced apoptosis in PC12 cells and investigates the role that c-Jun N-terminal kinase (JNK) activation may play in this system. Ceramide-induced apoptosis was inhibited by nerve growth factor, basic fibroblast growth factor, pituitary adenylyl cyclase-activating peptide, 4-(8-chlorophenylthio)cyclic AMP, and the caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp fluoromethyl ketone (zVAD-FMK). It was surprising that inhibition of extracellular signal-regulated kinase and/or phosphatidylinositol 3-kinase did not markedly block the protective effects exerted by neurotrophic factors against ceramide-induced apoptosis, suggesting that neurotrophic factors can promote survival independently of these signaling pathways. Treatment of PC12 cells with ceramide resulted in a time-dependent increase in JNK activity. However, neither neurotrophic factors nor zVAD-FMK attenuated ceramide-stimulated JNK activation. Further experiments indicated that ceramide-induced apoptosis in PC12 cells requires new protein synthesis, and that nerve growth factor and zVAD-FMK can prevent apoptosis after JNK activity has been detected. These results indicate that ceramide-induced JNK activation is an early event and may be required for the expression of essential components of the apoptotic machinery. It is anticipated that neurotrophic factors inhibit ceramide-induced apoptosis by affecting signaling events downstream of JNK activation.

Amino Acid Chloromethyl Ketones↗
PubMed2001

Expression of the apolipoprotein E gene in the skin is controlled by a unique downstream enhancer.

A distal enhancer that specifies apolipoprotein E gene expression in the skin was identified and characterized by in situ hybridization in transgenic mice generated with constructs of the human apolipoprotein E/C-I/C-IV/C-II gene cluster. Transgene constructs containing the enhancer expressed high levels of apolipoprotein E mRNA in the germinative cell layer of the sebaceous gland and in epithelial cells of the hair follicle root sheath. Apolipoprotein E mRNA was also detected in basal epithelial cells of the epidermis. Expression of the human apolipoprotein E transgene at these sites was specified by a unique 1.0 kb enhancer domain located 1.7 kb downstream of the apolipoprotein E gene. No transgene expression was detected in skin epithelial cells in transgenic mice when this enhancer was deleted from the apolipoprotein E gene cluster. The enhancer was used to construct a transgene expression vector that faithfully directed a heterologous cDNA to the normal sites of apolipoprotein E gene expression in epithelial cells of the skin.

Animals↗
PubMed1999

IRS-1 expression and activation are not sufficient to activate downstream pathways and enable IGF-I growth response in estrogen receptor negative breast cancer cells.

IGF-responsive breast cancer cells activate insulin receptor substrate (IRS)-1 after IGF-I treatment. To determine if IRS-1 expression was sufficient to enable IGF-responsiveness, two IGF-I unresponsive breast cancer cell lines (MDA-MB-435A and MDA-MB-468) were transfected with IRS-1. While IGF-I caused tyrosine phosphorylation of IRS-1 in both transfected cell lines, increased MAP kinase activity was not seen. IGF-I treatment of 435A IRS-1 transfected cells resulted in minimal increased PI3 kinase activity associated with IRS-1, while IRS-2/PI3 kinase was greatly reduced. In MDA-MB-468 IRS-1 transfected cells, IGF-I caused increased IRS-1 associated PI3 kinase activity compared to parental cells, but at levels far below those observed in IGF-responsive MCF-7 cells. The transfected cells were also not responsive to IGF-I in monolayer growth. Thus, IRS-1 expression and activation alone are insufficient to mediate a proliferative response to IGF-I in breast cancer cells, and it is likely that maximal activation of downstream signaling pathways must also occur.

Animals↗