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At least 955 records · Page 53Linked to original sources
PubMed2004

Wave2 activates serum response element via its VCA region and functions downstream of Rac.

WAVE2 is a member of the WASP/WAVE family of protein effectors of actin reorganization and cell movement. In this report, we demonstrate that WAVE2 overexpression induces serum response element (SRE) activation through serum response factor. A WAVE2 mutant lacking the VCA region did not induce SRE activation and actin polymerization. WAVE2-induced SRE activation was blocked by exposure of cells to Latrunculin A, or overexpression of actin mutant R62D. The DeltaVCA mutant inhibited Rac V12-induced SRE activation, suggesting that WAVE2 lies downstream of Rac. Similar deletion of the VCA domain of WASP attenuated Cdc42 V12-mediated SRE activation, suggesting that WAVE2 acts in relation to Rac as WASP acts in relation to Cdc42. WAVE2 overexpression did not activate NF-kappaB.

Actins↗
PubMed2005

Subconfluent endothelial cells form podosomes downstream of cytokine and RhoGTPase signaling.

Adhesion, migration and invasion of endothelial cells are prerequisites for the formation of blood vessels and have to be controlled on a subcellular level. We report that subconfluent human umbilical vein endothelial cells (HUVEC) are able to constitutively form podosomal adhesions that are sites of matrix metalloprotease concentration and matrix degradation. Importantly, incubation of serum-starved cells with VEGF or TNFalpha revealed the dependence of podosomes on cytokine signaling. Podosome formation was also stimulated by addition of monocytes to HUVEC. Microinjection/application of specific inhibitors or active/inactive mutants showed that regulatory pathways include Src kinase and RhoGTPase signaling, N-WASP activation and Arp2/3 complex-dependent actin nucleation. In sum, our data show that HUVEC displaying a migratory phenotype constitutively form f-actin-rich adhesions with podosomal characteristics downstream of cytokine signaling. We propose that HUVEC podosomes play an important role in endothelial cell migration and invasion.

Actin-Related Protein 2↗
PubMed2005

The Cbl RING finger C-terminal flank controls epidermal growth factor receptor fate downstream of receptor ubiquitination.

Evolutionarily conserved sequences of the E3/protein-ubiquitin ligase Cbl regulate epidermal growth factor receptor (EGF-R) signaling and degradation. These sequences encompass Cbl's tyrosine kinase-binding domain, linker region, RING finger (RF), and an uncharacterized flank C-terminal to the RF (residues 420-436). The latter domain, designated the RF tail, extends beyond Cbl's ubiquitin-conjugating enzyme (Ubc)-binding domain and has no known function. We report structure-function studies evaluating the impact of Cbl RF tail truncations on EGF-R fate in HEK 293 cells. All of the truncation mutants exhibit greatly reduced binding to activated EGF-R and lack proline-rich sequences that mediate direct Cbl association with SH3 proteins such as Grb2, yet a subset of mutants collectively enhances EGF-R ubiquitination, downregulation, and degradation. Significantly, EGF-R degradation correlates better with RF tail-dependent degradation of the Cbl substrate Sprouty2 than with EGF-R ubiquitination: expression of the RF tail truncation mutant Cbl 1-433 enhanced EGF-R ubiquitination while impeding Sprouty2 degradation, and Cbl 1-433 failed to enhance EGF-R downregulation or degradation. Our results suggest that EGF-R fate is controlled by a checkpoint downstream of receptor ubiquitination whose regulation by the Cbl RF tail may require Sprouty2 degradation.

Amino Acid Sequence↗
PubMed2006

Phosphorylation of DARPP-32 regulates breast cancer cell migration downstream of the receptor tyrosine kinase DDR1.

Cell migration plays a central role in processes such as development, wound healing and cancer metastasis. Here we describe a novel interaction between DDR1, a receptor tyrosine kinase activated by collagen, and the phosphoprotein DARPP-32 in mammary epithelial cells. DARPP-32 expression was readily detected in non-transformed mammary cell lines, but was strongly reduced or even absent in breast tumor cell lines, such as MCF7. Transfection of MCF7 cells with DARPP-32 resulted in severely impaired cell migration, while DARPP-32 transfection into the DDR1-deficient breast cancer cell line MDA-MB-231 did not alter migration. Co-expression of both DDR1 and DARPP-32 in MDA-MB-231 cells inhibited migration, thereby supporting a critical role of the DDR1/DARPP-32 complex in motility. Mutational substitution of the phosphorylation sites Thr-34 or Thr-75 on DARPP-32 revealed that phosphorylation of Thr-34 is necessary for the ability of DARPP-32 to impair breast tumor cell migration. Thus, DARPP-32 signaling downstream of DDR1 is a potential new target for effective anti-metastatic breast cancer therapy.

Breast Neoplasms↗
PubMed2002

Epstein-Barr virus BHRF1 functions downstream of Bid cleavage and upstream of mitochondrial dysfunction to inhibit TRAIL-induced apoptosis in BJAB cells.

We have previously reported that TNF-related apoptosis inducing ligand (TRAIL) causes cleavage of Bid via activation of caspase-8 and the loss of mitochondrial membrane potential (DeltaPsim), resulting in apoptosis. Experiments with BJAB clones expressing Epstein-Barr virus (EBV) anti-apoptotic protein BHRF1 showed that BHRF1 drastically inhibited TRAIL-mediated apoptosis. Although Western blot analysis demonstrated that TRAIL-induced Bid cleavage was not inhibited by BHRF1, the decrease in DeltaPsim caused by TRAIL was effectively blocked by BHRF1. These findings suggest that in BJAB cells, BHRF1 acts downstream of Bid cleavage and upstream of mitochondrial damage, resulting in inhibition of TRAIL-induced apoptosis.

Apoptosis↗
PubMed1988

Cloning and characterization of an alpha 1-antitrypsin like gene 12 KB downstream of the genuine alpha 1-antitrypsin gene.

Cosmid clones containing alpha 1-antitrypsin (alpha 1AT) gene sequences were observed to contain alpha 1AT-like sequences approximately 12 kb downstream of the authentic alpha 1AT gene. Restriction mapping suggested the alpha 1AT-like gene lacks promoter sequences. Cosmid clones from one library contained a truncated alpha 1AT-like gene with a deletion encompassing 1745 bp, including the whole exon IV and part of exon V. Sequencing of exon II of this truncated gene revealed a nucleotide homology of 76% but included critical mutations in the start codon (ATG - greater than ATA) and the 3' exon-intron junction. These results strongly suggest that the truncated alpha 1AT-like gene is a pseudogene, which is present at a frequency of 0.30 in the Dutch population.

Animals↗
PubMed1992

Calculated enhancement of open base pair probability downstream of a (TATA)2 box.

The modified self-consistent phonon approximation is generalized to calculate the open base pair probability of a (TATA)2 insert between two semi-infinite poly(dA-dC) . poly(dG-dT) helices. An iterative method based entirely on the Green's function method is developed to compute the open interbase hydrogen bond probability of the insert and near bases versus temperature. The open interbase hydrogen bond probability is calculated for two temperatures and compared with perfect homopolymer open base pair probabilities at room temperature. The probability of opening is enhanced by a factor of two for the major groove bond of the AT pair at the transcriptional downstream end of the (TATA)2 insert. The total base pair opening probability of that pair is enhanced by 54%. The probability of the next inline GC base pair to be open is increased by a factor of ten.

Base Composition↗
PubMed1999

Presence of up-stream and downstream components of a mitogen-activated protein kinase pathway in the PSD of the rat forebrain.

We previously reported the presence of Erk2 type mitogen-activated protein kinase (MAPK) and enrichment of its substrates in the post-synaptic density (PSD) fraction, and suggested a role for MAPK in the synaptic transmission and its modulation [Suzuki, T., Okumura-Noji, K., Nishida, E., ERK2-type mitogen-activated protein kinase (MAPK) and its substrates in post-synaptic density fractions from the rat brain, Neurosci. Res., 22 (1995) 277-285.]. In this paper, synaptic localization of the upstream and downstream components of a MAPK cascade was examined. We found that RSK1, Sos1, N-Shc 66 kDa, N-Shc 52 kDa, and Grb2 were present in the PSD fraction, and cPLA(2) was present in the synaptic plasma membrane fraction. RSK2, Sos2, and N-Shc 46 kDa were not present in the PSD fraction. Post-synaptic localization of RSK1 and Sos1 was confirmed by immunohistochemical examination at the electron microscopic level: the two immunoreactivities were localized in the PSDs, both in the spines and dendrites. These results suggest that all the MAPK cascade components examined were associated with PSD or the synaptic plasma membrane, suggesting the role(s) of the MAPK cascade for synaptic transmission and its regulation at post-synaptic sites.

Animals↗
PubMed2003

Antizyme is a target of sex-lethal in the Drosophila germline and appears to act downstream of hedgehog to regulate sex-lethal and cyclin B.

The sex determination master switch, Sex-lethal, has been shown to regulate the mitosis of early germ cells in Drosophila melanogaster. Sex-lethal is an RNA binding protein that regulates splicing and translation of specific targets in the soma, but the germline targets are unknown. In an experiment aimed at identifying targets of Sex-lethal in early germ cells, the RNA encoded by gutfeeling, the Drosophila homolog of Ornithine Decarboxylase Antizyme, was isolated. gutfeeling interacts genetically with Sex-lethal. It is not only a target of Sex-lethal, but also appears to regulate the nuclear entry and overall levels of Sex-lethal in early germ cells. This regulation of Sex-lethal by gutfeeling appears to occur downstream of the Hedgehog signal. We also show that Hedgehog, Gutfeeling, and Sex-lethal function to regulate Cyclin B, providing a link between Sex-lethal and mitosis.

Animals↗
PubMed2003

Molecular link in the sequential induction of the Spemann organizer: direct activation of the cerberus gene by Xlim-1, Xotx2, Mix.1, and Siamois, immediately downstream from Nodal and Wnt signaling.

To elucidate the molecular basis of organizer functions in Xenopus, we sought the target genes of the LIM homeodomain protein Xlim-1, which is one of the organizer-specific transcriptional activators. We found that an activated form of Xlim-1, Xlim-1/3m, initiates ectopic expression of the head-inducing organizer factor gene cerberus in animal caps. Thus, we analyzed the cerberus promoter using reporter assays. We show that three consecutive TAAT motifs of the homeodomain-binding sites between positions -141 and -118, collectively designated the "3xTAAT element," are crucial for the response of the cerberus promoter to Xlim-1/3m, and for its activation in the dorsal region of the embryo. Because cooperative activation of the cerberus promoter by Xnr1 and Xwnt8 also requires the 3xTAAT element, we focused on homeodomain transcriptional activators downstream from either Nodal or Wnt signaling. We found that wild-type Xlim-1 synergistically activates the cerberus promoter with Mix.1 and Siamois through the 3xTAAT element, and this synergy requires the LIM domains of Xlim-1. In contrast, Xotx2 acts synergistically with Mix.1 and Siamois through the TAATCT sequence at -95. Electrophoretic mobility shift assays revealed that Xlim-1, Siamois, and Mix.1 are likely to bind as a complex, in a LIM domain-dependent manner, to the region containing the 3xTAAT element. These data suggest that cerberus is a direct target for Xlim-1, Mix.1, Siamois, and Xotx2. Therefore, we propose a model for the molecular link in the inductive sequence from the formation of the organizer to anterior neural induction.

Animals↗
PubMed2003

Zebrafish hhex, nk2.1a, and pax2.1 regulate thyroid growth and differentiation downstream of Nodal-dependent transcription factors.

During zebrafish development, the thyroid primordium initiates expression of molecular markers such as hhex and nk2.1a in the endoderm prior to pharynx formation. As expected for an endodermally derived organ, initiation of thyroid development depends on Nodal signalling. We find that it also depends on three downstream effectors of Nodal activity, casanova (cas), bonnie and clyde (bon), and faust (fau)/gata5. Despite their early Nodal-dependent expression in the endoderm, both hhex and nk2.1a are only required relatively late during thyroid development. In hhex and nk2.1a loss-of-function phenotypes, thyroid development is initiated and arrests only after the primordium has evaginated from the pharyngeal epithelium. Thus, like pax2.1, both hhex and nk2.1a have similarly late roles in differentiation or growth of thyroid follicular cells, and here, we show that all three genes act in parallel rather than in a single pathway. Our functional analysis suggests that these genes have similar roles as in mammalian thyroid development, albeit in a different temporal mode of organogenesis.

Animals↗
PubMed1998

U73122 and U73343 inhibit receptor-mediated phospholipase D activation downstream of phospholipase C in CHO cells.

The aminosteroid 1-(6-¿[17beta-3-methoxyestra- 1,3,5(10)-trien- 17-yl]-amino¿hexyl)- 1H-pyrrole-2,5-dione (U73122) and its inactive analogue 1-(6-¿[17beta-3-methoxyestra-1,3,5(10)-trien- 17-yl]-amino¿hexyl-2,5-pyrrolidine-dione (U73343) are widely used to study the involvement of G protein-coupled 1-phosphatidylinositol-phosphodiesterase, or phospholipase C, in receptor-mediated cell activation. The present work shows that both aminosteroids inhibit cholecystokinin-(26-33)-peptide amide (CCK-8)-induced phospholipase D activation equipotently in Chinese hamster ovary cells expressing the cholecystokinin-A receptor (CHO-CCK(A) cells). In addition, the two aminosteroids virtually completely inhibited thapsigargin- and 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced phospholipase D activation. Since the latter two drugs mimic inositol 1,4,5-trisphosphate-mediated Ca2+ mobilisation and 1,2-diacylglycerol-mediated protein kinase C activation. respectively, this suggests that both U73122 and U73343 act downstream of phospholipase C to inhibit receptor-mediated phospholipase D activation. U73122, but not U73343. effectively inhibited both TPA/Ca2+-stimulated phospholipase D activation and TPA/phosphatidylserine-stimulated protein kinase C activation in a homogenate of CHO-CCK(A) cells. The data presented suggest that U73122 may act at the level of protein kinase C to inhibit activation of phospholipase D. The exact site of action of U73343 is presently unknown.

Animals↗
PubMed2003

Expression and regulation of NDRG2 (N-myc downstream regulated gene 2) during the differentiation of dendritic cells.

We searched for genes with expressions specific to human monocyte-derived dendritic cells (DCs) using differential display reverse transcription-polymerase chain reaction, and found that N-myc downstream regulated gene 2 (NDRG2), a member of a new family of differentiation-related genes, was expressed in DCs. While DCs derived from CD34(+) progenitor cells also showed strong NDRG2 expression, the corresponding mRNA expression was absent in other cell lines including monocytes, B cells, and NK cells. The inhibition of DC differentiation by dexamethasone or vitamin D(3) treatment down-regulated the expression of the NDRG2 gene in DCs. In addition, gene expression was induced in a myelomonocytic leukemia cell line, which is capable of differentiating into DCs in cytokine-conditioned culture. The level of NDRG2 gene expression in DCs was significantly higher than that of other members of the NDRG gene family. Finally, in contrast to the stable NDRG2 expression in CD40-stimulated DCs, the induction of DC maturation by lipopolysaccharide (LPS) resulted in the down-regulation of NDRG2 gene expression. This down-regulation is likely to be due to a modification and subsequent destabilization of NDRG2 mRNA, because co-treating with actinomycin D and LPS significantly blocked this LPS effect. Taken together, our results indicate that NDRG2 is expressed during the differentiation of DCs, and that NDRG2 gene expression is differentially regulated by maturation-inducing stimuli.

Antigens, CD34↗
PubMed1997

p160ROCK, a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesions.

p160ROCK is a serine/threonine protein kinase that binds selectively to GTP-Rho and is activated by this binding. To identify its function, we transfected HeLa cells with wild type and mutants of p160ROCK and examined morphology of the transfected cells. Transfection with wild type and mutants containing the kinase domain and the coiled-coil forming region induced focal adhesions and stress fibers, while no induction was observed with a kinase-defective mutant or a mutant containing only the kinase domain. Furthermore, Rho-induced formation of focal adhesions and stress fibers was inhibited by co-expression of a mutant defective in both kinase and Rho-binding activities. Rho, however, still induced an increase in F-actin content in these cells. These results suggest that p160ROCK works downstream of Rho to induce formation of focal adhesions and that Rho-induced actin polymerization is mediated by other effector(s).

Alanine↗
PubMed1997

Ro31-8220 inhibits protein kinase C to block the phorbol ester-stimulated release of choline- and ethanolamine-metabolites from C6 glioma cells: p70 S6 kinase and MAPKAP kinase-1beta do not function downstream of PKC in activating PLD.

The use of bisindolylmaleimide derivatives of staurosporine as selective inhibitors of protein kinase C (PKC) is in doubt following the report by Alessi [FEBS Lett. 402 (1997) 121-123] that Ro31-8220 and GF109203X are potent in vitro inhibitors of p70 S6 kinase and mitogen-activated protein kinase-activated protein kinase-1beta, as well as of PKC. Here we show that the phorbol ester-stimulated release of choline- and ethanolamine-metabolites from C6 glioma cells due to phospholipid hydrolysis by phospholipase D (PLD) is not inhibited by rapamycin or PD98059, specific inhibitors respectively of p70 S6 kinase and MAPKK (MEK) and thus of MAPKAP kinase-1beta but is still completely blocked by Ro31-8220. We conclude therefore that p70S6k and MAPKAP kinase-1beta as well as MAPK are not involved in signalling pathways downstream of PKC that regulate phorbol ester-stimulated phospholipid turnover and that the inhibitory action of Ro31-8220 occurs by blocking PKC which regulates at least one pathway to PLD activation. The PI-3 kinase inhibitor, wortmannin, inhibits the phorbol ester-stimulated release of ethanolamine- but not choline-metabolites from C6 cells suggesting that different PLD isoforms regulate the turnover of PtdEth and PtdCho in C6 cells. Both PLD isoforms are activated via PKC but the PtdEth-PLD is also regulated via a wortmannin-sensitive pathway.

Androstadienes↗
PubMed1998

A phorbol ester-binding protein is required downstream of Rab5 in endosome fusion.

Previous observations indicate that a zinc and phorbol ester binding factor is necessary for endosome fusion. To further characterize the role of this factor in the process, we used an in vitro endosome fusion assay supplemented with recombinant Rab5 proteins. Both zinc depletion and addition of calphostin C, an inhibitor of protein kinase C, inhibited endosome fusion in the presence of active Rab5. Addition of the phorbol ester PMA (phorbol 12-myristate 13-acetate) reversed the inhibition of endosome fusion caused by a Rab5 negative mutant. Moreover, PMA stimulated fusion in the presence of Rab5 immunodepleted cytosol. These results suggest that the phorbol ester binding protein is acting downstream of Rab5 in endosome fusion.

Caenorhabditis elegans Proteins↗
PubMed2000

Application of monoliths for downstream processing of clotting factor IX.

In this paper, the application of monolithic columns for downstream processing of different clotting factor IX concentrates is shown. Determination of basic chromatographic conditions as well as investigations on the regeneration of disk- and tube-shaped monolithic columns using human serum albumin as a model protein, were performed. Separation of factor IX and vitronectin, a possible impurity in commercial factor IX concentrates was accomplished using disk-shaped monolithic columns. These same applications were also carried out with identical results on up-scaled tube-shaped monolithic columns. Since these media allow very fast separations, this method can be successfully applied not only to an in-process control of the purification of factor IX but also to other biopolymers from human plasma. Besides, the same application on the up-scaled tube-shaped monolithic column was successfully carried out.

Biopolymers↗
PubMed2003

Design and scaleup of downstream processing of monoclonal antibodies for cancer therapy: from research to clinical proof of principle.

Murine monoclonal antibodies (mAb) from cell culture supernatants have been purified in order to acquire clinical grade for in vivo cancer treatment. The starting material was purified by high performance liquid chromatography (HPLC) systems ranging from the analytical scale process to a scaleup to 1 g per batch. Three columns (Protein A affinity chromatography with single-step elution, hydroxyapatite (HA) chromatography followed by linear gradient elution and endotoxin removing-gel chromatography), exploiting different properties of the mAb were applied. The final batches of antibody were subjected to a large panel of tests for the purpose of evaluating the efficacy of the downstream processing. The resulting data have allowed us to determine the maximum number of times the column can be used and to precisely and thoroughly characterize antibody integrity, specificity, and potency according to in-house reference standards. The optimized bioprocessing is rapid, efficient, and reproducible. Not less importantly, all the techniques applied are characterized by costs which are affordable to medium-sized laboratories. They represent the basis for implementing immunotherapeutic protocols transferable to clinical medicine.

Animals↗