bluuebunny/crossref_metadata_2025 huggingface dataset:
The Fe(II) permease Fet4p functions as a low affinity copper transporter and supports normal copper trafficking in Saccharomyces cerevisiae
Authors: HASSETT Richard , DIX David R. , EIDE David J. , KOSMAN Daniel J.
Published: August 2015

The plasma-membrane of Saccharomycescerevisiae contains high affinity permeases for Cu(I) and Fe(II). A low affinity Fe(II) permease has also been identified, designated Fet4p. A corresponding low affinity copper permease has not been characterized, although yeast cells that lack high affinity copper uptake do accumulate this metal ion. We demonstrate in the present study that Fet4p can function as a low affinity copper permease. Copper is a non-competitive inhibitor of 55Fe uptake through Fet4p (Ki = 22µM). Fet4p-dependent 67Cu uptake was kinetically characterized, with Km and Vmax values of 35µM and 8pmol of copper/min per 106 cells respectively. A fet4-containing strain exhibited no saturable, low affinity copper uptake indicating that this uptake was attributable to Fet4p. Mutant forms of Fet4p that exhibited decreased efficiency in 55/59Fe uptake were similarly compromised in 67Cu uptake, indicating that similar amino acid residues in Fet4p contribute to both uptake processes. The copper taken into the cell by Fet4p was metabolized similarly to the copper taken into the cell by the high affinity permease, Ctr1p. This was shown by the Fet4p-dependence of copper activation of Fet3p, the copper oxidase that supports high affinity iron uptake in yeast. Also, copper-transported by Fet4p down-regulated the copper sensitive transcription factor, Mac1p. Whether supplied by Ctr1p or by Fet4p, an intracellular copper concentration of approx. 10µM caused a 50% reduction in the transcriptional activity of Mac1p. The data suggest that the initial trafficking of newly arrived copper in the yeast cell is independent of the copper uptake pathway involved, and that this copper may be targeted first to a presumably small ‘holding’pool prior to its partitioning within the cell.
The biochemical characterization of aggrecan from normal and tibial-dyschondroplastic chicken growth-plate cartilage
Authors: TSELEPIS Chris , KWAN Alvin P. L. , THORNTON David , SHEEHAN John
Published: August 2015

Tibial dyschondroplasia (TD) is a disorder of endochondral ossification characterized by the presence of an avascular, non-mineralized cartilage lesion extending from the growth plate into the metaphysis. Cells within the TD growth plate fail to differentiate to full hypertrophy, and instead appear to maintain a ‘pre-hypertrophic’or ‘transitional’status. Studies of the expression and distribution of cartilage matrix macromolecules in the TD growth plate have shown a marked decrease in the levels of aggrecan in the TD matrix. In the present study we compared the biochemical characteristics of the aggrecan molecules extracted from normal epiphyseal and TD cartilage. We have shown three major differences between normal and TD cartilage aggrecan. These are: (1) increase in molecular mass; (2) increase in the number of keratan sulphate chains; and (3) difference in the pattern of sulphation in TD aggrecan. Such changes in biochemical characteristics of the aggrecan monomers in TD cartilage may be associated with the lack of mineralization of the diseased cartilage. The present study provides a basis for further investigations into the importance of proteoglycans in normal and pathological bone development.
Identification of a novel 45 kDa protein (JP-45) from rabbit sarcoplasmic-reticulum junctional-face membrane
Authors: ZORZATO Francesco , ANDERSON Ayuk A. , OHLENDIECK Kai , FROEMMING Gabriele , GUERRINI Remo , TREVES Susan
Published: August 2015

Using a biochemical/immunological approach to analyse the protein constituents of skeletal-muscle junctional-face membrane (JFM), we identified a 45kDa protein. Its N-terminal amino acid was blocked, but the amino acid sequence obtained from several peptides after proteolytic treatment did not significantly match that of any protein present in the SwissProt and NCBI (National Center for Biotechnology Information) databases. We synthesized a peptide whose sequence matched that of one of the peptides obtained after CNBr cleavage of the 45kDa protein; the peptide was conjugated to a carrier and used to raise antibodies. The antiserum was used to study in more detail the biochemical characteristics of the novel 45kDa protein. Analysis of the proteins present in different subcellular membrane fractions show that the novel 45kDa polypeptide: (i) is an integral membrane constituent present both in neonatal and adult skeletal-muscle sarcoplasmic reticulum; (ii) is selectively localized in the JFM; (iii) is not present in microsomes obtained from rabbit heart, liver or kidney. Immunoprecitation with anti-(45kDa protein) antibody indicates that the 45kDa protein is part of a complex which can be phosphorylated in vitro by the catalytic subunit of protein kinase A.
Amino-acid-dependent signal transduction
Authors: VAN SLUIJTERS Daphne A. , DUBBELHUIS Peter F. , BLOMMAART Edward F. C. , MEIJER Alfred J.
Published: August 2015

Recent research carried out in several laboratories has indicated that, in addition to their role as intermediates in many metabolic pathways, amino acids can interact with insulin-dependent signal transduction. In this short review, the current state of this rapidly expanding field is discussed.
The pH dependence of naturally occurring low-spin forms of methaemoglobin and metmyoglobin: an EPR study
Authors: SVISTUNENKO Dimitri A. , SHARPE Martyn A. , NICHOLLS Peter , BLENKINSOP Christine , DAVIES Nathan A. , DUNNE Jacqueline , WILSON Michael T. , COOPER Chris E.
Published: August 2015

The paramagnetic species in human metHb and horse metmyoglobin (metMb) have been studied at low temperature using EPR spectroscopy. The high-spin (HS) haem signal in aquometMb has a greater rhombic distortion than the HS metHb signal. Nevertheless, the individual line width (g = 6) is smaller in metMb than in metHb, consistent with non-identical signals from the α and β Hb subunits. Three low-spin (LS) haem forms are present in metHb, while metMb has only two. The major LS form in both proteins is the alkaline species (with OH- at the sixth co-ordination position). The minor LS forms are assigned to different histidine hemichromes in equilibrium with the normal HS species at low temperature. LS forms disappear when the haem is bound by a ligand, such as fluoride, which ensures 100% occupancy of the HS state both at room temperature and at 25K. The small differences in effective g-factors of the histidine hemichromes are interpreted in terms of different distances between the distal histidine and haem iron. The pH dependence of the inter-conversion of the different paramagnetic species is consistent with a model whereby protonation of a residue with a pK of 5.69 (metHb) or 6.12 (metMb), affects ligand binding and transformation from the HS to the LS form. Chemical and spectroscopic considerations suggest that the residue is unlikely to be the proximal or distal histidine. We therefore propose a model where protonation of this distant amino acid causes a conformational change at the iron site. Identical effects are seen in frozen human blood, suggesting that this effect may have physiological significance.
Specific interactions among transmembrane 4 superfamily (TM4SF) proteins and phosphoinositide 4-kinase
Authors: YAUCH Robert L. , HEMLER Martin E.
Published: August 2015

In earlier work we established that phosphoinositide 4-kinase (PI 4-kinase) may associate with transmembrane 4 superfamily (TM4SF, tetraspanin) proteins, but critical specificity issues were not addressed. Here we demonstrate that at least five different TM4SF proteins (CD9, CD63, CD81, CD151 and A15/TALLA1) can associate with a similar or identical 55kDa type II PI 4-kinase. These associations were specific, since we found no evidence for other phosphoinositide kinases (e.g. phosphoinositide 3-kinase and phosphoinositide-4-phosphate 5-kinase) associating with TM4SF proteins, and many other TM4SF proteins (including CD82 and CD53) did not associate with PI 4-kinase. CD63–PI 4-kinase complexes were almost entirely intracellular, and thus are distinct from other TM4SF–PI 4-kinase complexes (e.g. involving CD9), which are largely located in the plasma membrane. These results suggest that a specific subset of TM4SF proteins may recruit PI 4-kinase to specific membrane locations, and thereby influence phosphoinositide-dependent signalling.
Different receptors use inositol trisphosphate to mobilize Ca2+ from different intracellular pools
Authors: SHORT Alison D. , WINSTON Gavin P. , TAYLOR Colin W.
Published: August 2015

In cells expressing different receptors linked to Ins(1,4,5)P3 formation, maximal stimulation of any one of them often releases all the Ins(1,4,5)P3-sensitive Ca2+ stores, suggesting that Ins(1,4,5)P3 is used similarly by many receptors. In single HEK-293 cells, ATP and carbamylcholine (CCh) stimulated Ca2+ release from intracellular stores via a pathway that was entirely dependent on Ins(1,4,5)P3. After stimulation with maximal concentrations of ATP or CCh in Ca2+-free medium, there was no response to a second stimulation with the same agonist, indicating that each agonist had emptied the Ins(1,4,5)P3-sensitive stores to which it had access. However, the Ca2+ release evoked by the second agonist was unaffected by prior stimulation with the first. We conclude that Ins(1,4,5)P3 mediates the effects of both receptors, but Ins(1,4,5)P3 is more versatile than hitherto supposed, because the spatial organization of the signalling pathways apparently allows Ins(1,4,5)P3 made in response to each agonist to interact with different Ins(1,4,5)P3 receptors.
Regulation of Nramp1 mRNA stability by oxidants and protein kinase C in RAW264.7 macrophages expressing Nramp1Gly169
Authors: LAFUSE William P. , ALVAREZ Gail R. , ZWILLING Bruce S.
Published: August 2015

The murine Nramp1 (natural-resistance-associated macrophage protein) locus confers innate resistance against intracellular macrophage pathogens. The gene encodes a transporter molecule, which is rapidly recruited to the phagosome. Nramp1 functions as an iron transporter by transporting iron into the phagosome. Within the phagosome iron mediates anti-microbial killing by hydroxyl radical formation through the iron-catalysed Fenton/Haber–Weiss reaction. In addition to its effects on the growth of intracellular pathogens, Nramp1 exerts a wide range of pleiotropic effects in activated macrophages. One of these pleiotropic effects is the increased stability of a variety of mRNA species, including Nramp1 mRNA. In the present study, the stability of Nramp1 mRNA in Mycobacterium avium infected RAW264.7 macrophages expressing either the Nramp1Gly169 resistant allele or the Nramp1Asp169 susceptible allele was examined. Nramp1 mRNA stability was greater in Nramp1Gly169 macrophages than in Nramp1Asp169 macrophages. The increase in Nramp1 mRNA stability in resistant macrophages was inhibited by antioxidants and protein kinase C (PKC) inhibitors, suggesting that Nramp1 mRNA stability is regulated by an oxidant-generated signalling pathway that requires PKC activity. This was corroborated by treating Nramp1Asp169 macrophages with menadione, which generates reactive oxygen species within cells. Menadione increased Nramp1 mRNA stability to the level observed in resistant macrophages; this increase was also inhibited by a PKC inhibitor. Further, PKC activity was found to be greater in M. avium-infected Nramp1Gly169 macrophages than in infected Nramp1Asp169 macrophages and inhibited by treatment with an antioxidant.
Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labelling: binding characteristics and amino acid sequences
Authors: ZHANG Ying-Yi , HAMMARBERG Tove , RADMARK Olof , SAMUELSSON Bengt , NG Carol F. , FUNK Colin D. , LOSCALZO Joseph
Published: August 2015

5-Lipoxygenase (5LO) catalyses the first two steps in the biosynthesis of leukotrienes, which are inflammatory mediators derived from arachidonic acid. 5LO activity is stimulated by ATP; however, a consensus ATP-binding site or nucleotide-binding site has not been found in its protein sequence. In the present study, affinity and photoaffinity labelling of 5LO with 5′-p-fluorosulphonylbenzoyladenosine (FSBA) and 2-azido-ATP showed that 5LO bound to the ATP analogues quantitatively and specifically and that the incorporation of either analogue inhibited ATP stimulation of 5LO activity. The stoichiometry of the labelling was 1.4mol of FSBA/mol of 5LO (of which ATP competed with 1mol/mol) or 0.94mol of 2-azido-ATP/mol of 5LO (of which ATP competed with 0.77mol/mol). Labelling with FSBA prevented further labelling with 2-azido-ATP, indicating that the same binding site was occupied by both analogues. Other nucleotides (ADP, AMP, GTP, CTP and UTP) also competed with 2-azido-ATP labelling, suggesting that the site was a general nucleotide-binding site rather than a strict ATP-binding site. Ca2+, which also stimulates 5LO activity, had no effect on the labelling of the nucleotide-binding site. Digestion with trypsin and peptide sequencing showed that two fragments of 5LO were labelled by 2-azido-ATP. These fragments correspond to residues 73–83 (KYWLNDDWYLK, in single-letter amino acid code) and 193–209 (FMHMFQSSWNDFADFEK) in the 5LO sequence. Trp-75 and Trp-201 in these peptides were modified by the labelling, suggesting that they were immediately adjacent to the C-2 position of the adenine ring of ATP. Given the stoichiometry of the labelling, the two peptide sequences of 5LO were probably near each other in the enzyme's tertiary structure, composing or surrounding the ATP-binding site of 5LO.
Novel consensus sequence for the Golgi apparatus casein kinase, revealed using proline-rich protein-1 (PRP1)-derived peptide substrates
Authors: BRUNATI Anna Maria , MARIN Oriano , BISINELLA Alfredo , SALVIATI Alessandro , PINNA Lorenzo A.
Published: August 2015

Previous studies have shown that the Golgi apparatus casein kinase (G-CK) recognizes phosphoacceptor sites specified by the triplet SXE/Sp, which is found in several phosphoproteins, besides casein itself. In the present study, we report that G-CK can phosphorylate, with comparable efficiency, sequences surrounding Ser-22 of salivary proline-rich protein-1 (PRP1), which do not conform to the SXE/Sp motif. By using a series of peptide substrates derived from the PRP1 Ser-22 site, we also have shown that the optimal consensus sequence recognized by G-CK in this case was SXQXX(D/E)3, where the acidic residues at positions n+5 to n+7 and, to a lesser extent, the glutamine residue at position n+2 are the critical determinants.
Shared control of hepatic glycogen synthesis by glycogen synthase and glucokinase
Authors: GOMIS Roger R. , FERRER Juan C. , GUINOVART Joan J.
Published: August 2015

We have used recombinant adenoviruses (AdCMV-RLGS and AdCMV-GK) to overexpress the liver isoforms of glycogen synthase (GS) and glucokinase (GK) in primary cultured rat hepatocytes. Glucose activated overexpressed GS in a dose-dependent manner and caused the accumulation of larger amounts of glycogen in the AdCMV-RLGS-treated hepatocytes. The concentration of intermediate metabolites of the glycogenic pathway, such as glucose 6-phosphate (Glc-6-P) and UDP-glucose, were not significantly altered. GK overexpression also conferred on the hepatocyte an enhanced capacity to synthesize glycogen in response to glucose, as described previously [Seoane, Gómez-Foix, O'Doherty, Gómez-Ara, Newgard and Guinovart (1996) J. Biol. Chem. 271, 23756–23760], although, in this case, they accumulated Glc-6-P. When GS and GK were simultaneously overexpressed, the accumulation of glycogen was enhanced in comparison with cells overexpressing either GS or GK. Our results are consistent with the hypothesis that liver GS catalyses the rate-limiting step of hepatic glycogen synthesis. However, hepatic glycogen deposition from glucose is submitted to a system of shared control in which the ‘controller’, GS, is, in turn, controlled by GK. This control is indirectly exerted through Glc-6-P, which ‘switches on’ GS dephosphorylation and activation.
Identification of Glu-519 as the catalytic nucleophile in β-mannosidase 2A from Cellulomonas fimi
Authors: STOLL Dominik , HE Shouming , WITHERS Stephen G. , WARREN R. Antony J.
Published: August 2015

Incubation of the β-mannosidase Man2A from Cellulomonas fimi with 2-deoxy-2-fluoro-β-d-mannosyl fluoride (2FManβF) resulted in time-dependent inactivation of the enzyme (inactivation rate constant ki = 0.57min-1, dissociation constant for the inactivator Ki = 0.41mM) through the accumulation of a covalent 2-deoxy-2-fluoro-α-d-mannosyl–β-mannosidase 2A (2FMan–Man2A) enzyme intermediate, as observed by electrospray ionization mass spectrometry. The stoichiometry of inactivation was 1:1. Removal of excess inactivator and regeneration of active enzyme by transglycosylation of the covalently attached inhibitor to gentiobiose [Glcβ(1–6)Glc] demonstrated that the covalent intermediate was catalytically competent. Comparison by MS of the peptic digests of 2FMan–Man2A with peptic digests of native Man2A revealed a peptide of m/z 1520 that was unique to 2FMan–Man2A, and one of m/z 1036.5 that was unique to a Man2A peptide. Their sequences, determined by collision-induced fragmentation, were CSEFGFQGPPTW and FGFQGPPTW, corresponding to residues 517–528 and 520–528 of Man2A respectively. The difference in mass of 483.5 between the two peptides equals the sum of the masses of the tripeptide CSE plus that of 2-fluoromannose. It was concluded that in 2FMan–Man2A, the 2-fluoromannose esterified to Glu-519 blocks hydrolysis of the Glu-519–Phe-520 peptide bond, and that Glu-519 is the catalytic nucleophile in this enzyme. This residue is conserved in all members of family 2 of the glycosyl hydrolases. This represents the first ever labelling and identification of an active-site nucleophile in a β-mannosidase.
Identification of Rab6 as an N-ethylmaleimide-sensitive fusion protein-binding protein
Authors: HAN Sang Yeul , PARK Dong Yoon , PARK Sang Dai , HONG Seung Hwan
Published: August 2015

In this study we show the interaction of N-ethylmaleimide-sensitive fusion protein (NSF) with a small GTP-binding protein, Rab6. NSF is an ATPase involved in the vesicular transport within eukaryotic cells. Using the yeast two-hybrid system, we have isolated new NSF-binding proteins from the rat lung cDNA library. One of them was Rab6, which is involved in the vesicular transport within the Golgi and trans-Golgi network as a Ras-like GTPase. We demonstrated that the N-terminal domain of NSF interacted with the C-terminal domain of Rab6, and these proteins were co-immunoprecipitated from the rat brain extract. This interaction was maintained preferentially in the presence of hydrolysable ATP. Recombinant NSF-His6 can also bind to C-terminal Rab6–glutathione S-transferase under the conditions to allow the ATP hydrolysis. Surprisingly, Rab6 stimulates the ATPase activity of NSF by approx. 2-fold as does α-soluble NSF attachment protein receptor. Anti-Rab6 polyclonal antibodies significantly inhibited the Rab6-stimulated ATPase activity of NSF. Furthermore, we found that Rab3 and Rab4 can also associate with NSF and stimulate its ATPase activity. Taken together, we propose a model in which Rab can form an ATP hydrolysis-regulated complex with NSF, and function as a signalling molecule to deliver the signal of vesicle fusion through the interaction with NSF.
Divergence in the anti-apoptotic signalling pathways used by nerve growth factor and basic fibroblast growth factor (bFGF) in PC12 cells: rescue by bFGF involves protein kinase Cδ
Authors: WERT Maribeth M. , PALFREY H. Clive
Published: August 2015

The mechanisms whereby nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) block apoptosis in serum-deprived PC12 cells were investigated. NGF, but not bFGF, strongly activated Akt/protein kinase B, a downstream effector of phosphoinositide (phosphatidylinositol) 3-kinase (PI 3-kinase). In addition, inhibition of PI 3-kinase by LY294002 partially blocked inhibition of apoptosis by NGF, but not that by bFGF, suggesting divergence in NGF and bFGF anti-apoptotic signalling pathways. Both growth factors strongly activated mitogen-activated protein (MAP) kinases, but blockade of signalling through this pathway, either by the expression of dominant-negative Ras or by treatment with the MAP kinase/ERK kinase (MEK) inhibitor U0126, partially inhibited only bFGF, but not NGF, anti-apoptotic signalling. Use of isoform-specific protein kinase C (PKC) inhibitors such as bisindoylmaleimide-I and Gö 6983 suggested that PKCδ is a likely component of bFGF trophic signalling. A role for PKCδ was confirmed in PC12 cells expressing a dominant-negative PKCδ fragment, in which reversal of apoptosis by bFGF was partially blocked. The PKCδ signal was not mediated by the MAP kinase cascade, as bFGF activation of this pathway was not affected in cells expressing the dominant-negative PKCδ fragment. Full inhibition of bFGF anti-apoptotic signalling occurred when both the PKCδ and Ras/MAP kinase pathways were inhibited. Together, these data demonstrate that inhibition of apoptosis by bFGF in PC12 cells operates differently from that mediated by NGF, requiring the addition of signals from both the Ras/MAP kinase and PKC signalling pathways.
Peroxynitrite activates the phosphoinositide 3-kinase/Akt pathway in human skin primary fibroblasts
Authors: KLOTZ Lars-Oliver , SCHIEKE Stefan M. , SIES Helmut , HOLBROOK Nikki J.
Published: August 2015

Peroxynitrite is a potent oxidizing and nitrating species formed in a diffusion-limited reaction between nitrogen monoxide and superoxide. It induces apoptosis through unknown mechanisms and is believed to interfere with receptor tyrosine kinase signalling through nitration of tyrosine residues. One pathway emanating from receptor tyrosine kinases is that leading to activation of the anti-apoptotic kinase Akt. In the present study we provide evidence that peroxynitrite, administered to cells using two different delivery systems, results in the dose- and time-dependent activation of Akt. Akt activation is rapid and followed by phosphorylation of glycogen synthase kinase-3, an established substrate of Akt. Akt activation is inhibited in the presence of the phosphoinositide 3-kinase (PI-3K) inhibitors wortmannin and LY294002, and by treatment with the platelet-derived growth factor (PDGF) receptor (PDGFR) inhibitor AG1295, indicating a requirement for PDGFR and PI-3K in mediating peroxynitrite-induced Akt activation. Accordingly, the PDGFR-A and PDGFR-B isoforms were shown to undergo rapid tyrosine phosphorylation on treatment with peroxynitrite. Prior exposure of cells to peroxynitrite interferes with PDGF-induced Akt phosphorylation. Our findings suggest that Akt activation occurs as an acute response to peroxynitrite treatment and could play an important role in influencing cell survival and/or alter the cellular response to other growth regulatory signals.
Identification and characterization of a novel Rho-specific guanine nucleotide exchange factor
Authors: BLOMQUIST Andrea , SCHWÖRER Guntram , SCHABLOWSKI Helge , PSOMA Amalia , LEHNEN Michaela , JAKOBS Karl H. , RÜMENAPP Ulrich
Published: August 2015

Rho GTPases are implicated in a multitude of cellular processes regulated by membrane receptors, such as cytoskeletal rearrangements, gene transcription and cell growth and motility. Activation of these GTPases is under the direct control of guanine nucleotide exchange factors (GEFs), the Dbl family proteins. By searching protein databases we have identified a novel Rho-GEF, termed p114-Rho-GEF, which similarly to other Rho-GEFs contains a Dbl homology domain followed by a pleckstrin homology domain. p114-Rho-GEF interacted specifically with RhoA, in its nucleotide-free and guanosine 5′-[γ-thio]triphosphate-bound states, but not with Rac1 and Cdc42, and efficiently catalysed guanine nucleotide exchange of RhoA. Consistent with these results in vitro was our finding that the overexpression of p114-Rho-GEF in J82 and HEK-293 cells induced the formation of actin stress fibres and stimulated serum-response-factor-mediated gene transcription in a Rho-dependent manner. Rho-mediated transcriptional activation induced by M3 muscarinic acetylcholine and lysophosphatidic acid receptors was enhanced by p114-Rho-GEF, suggesting that the activity of this novel Rho-GEF, which is widely expressed in human tissues, can be controlled by G-protein-coupled receptors.
CREB (cAMP response element binding protein) and C/EBPα (CCAAT/enhancer binding protein) are required for the superstimulation of phosphoenolpyruvate carboxykinase gene transcription by adenoviral E1a and cAMP
Authors: ROUTES John M. , COLTON Lillester A. , RYAN Sharon , KLEMM Dwight J.
Published: August 2015

In the present study, we observed superstimulated levels of cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter in cells infected with wild-type adenovirus expressing 12S and 13S E1a proteins, or in cells expressing 13S E1a alone. cAMP-stimulated transcription was inhibited in cells expressing only 12S E1a, but slightly elevated in cells expressing E1a proteins with mutations in conserved regions 1 or 2, leading us to conclude that the superstimulation was mediated by conserved region 3 of 13S E1a. E1a failed to enhance cAMP-stimulated transcription from promoters containing mutations that abolish binding by cAMP response element binding protein (CREB) or CCAAT/enhancer binding proteins (C/EBPs). This result was supported by experiments in which expression of dominant-negative CREB and/or C/EBP proteins repressed E1a- and cAMP-stimulated transcription from the PEPCK gene promoter. In reconstitution experiments using a Gal4-responsive promoter, E1a enhanced cAMP-stimulated transcription when chimaeric Gal4–CREB and Gal4–C/EBPα were co-expressed. Phosphorylation of CREB on serine-133 was stimulated in cells treated with dibutyryl cAMP, whereas phosphorylation of C/EBPα was increased by E1a expression. Our data support a model in which cAMP agonists increase CREB activity and stimulate PEPCK gene transcription, a process that is enhanced by E1a through the phosphorylation of C/EBPα.
Cloning and expression of a cDNA encoding human inositol 1,4,5-trisphosphate 3-kinase C
Authors: DEWASTE Valérie , POUILLON Valérie , MOREAU Colette , SHEARS Stephen , TAKAZAWA Kazunaga , ERNEUX Christophe
Published: August 2015

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase catalyses the phosphorylation of Ins(1,4,5)P3 to Ins(1,3,4,5)P4. cDNAs encoding two isoenzymes of Ins(1,4,5)P3 3-kinase (3-kinases A and B) have been described previously. In the present study, we report the cloning of a full-length 2052bp cDNA encoding a third human isoenzyme of the Ins(1,4,5)P3 3-kinase family, referred to as isoform C. This novel enzyme has a calculated molecular mass of 75.207kDa and a Km for Ins(1,4,5)P3 of 6µM. Northern-blot analysis showed the presence of a transcript of approx. 3.9kb in various human tissues. Inositol trisphosphate 3-kinase C demonstrates enzymic activity when expressed in DH5αF′ bacteria or COS-7 cells. Calcium alone decreases the Ins(1,4,5)P3 3-kinase activity of the 3-kinase C isoenzyme in transfected COS-7 cells. This inhibitory effect is reversed in the presence of calmodulin. The recombinant bacterial 3-kinase C can be adsorbed on calmodulin–Sepharose in the presence of calcium. The present data show that Ins(1,4,5)P3 3-kinase C: (i) shares a conserved catalytic domain of about 275 amino acids with the two other mammalian isoforms, (ii) could be purified on a calmodulin–Sepharose column and (iii) could be distinguished from the A and B isoenzymes by the effects of calcium and of calmodulin.
Molecular cloning and functional characterization of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2) equilibrative nucleoside transporters from mouse brain
Authors: KISS Andor , FARAH Kalthoum , KIM Joon , GARRIOCK Robert J. , DRYSDALE Thomas A. , HAMMOND James R.
Published: August 2015

Mammalian cells express at least two subtypes of equilibrative nucleoside transporters, i.e. ENT1 and ENT2, which can be distinguished functionally by their sensitivity and resistance respectively to inhibition by nitrobenzylthioinosine. The ENT1 transporters exhibit distinctive species differences in their sensitivities to inhibition by dipyridamole, dilazep and draflazine (human>mouse>rat). A comparison of the ENT1 structures in the three species would facilitate the identification of the regions involved in the actions of these cardioprotective agents. We now report the molecular cloning and functional expression of the murine (m)ENT1 and mENT2 transporters. mENT1 and mENT2 encode proteins containing 458 and 456 residues respectively, with a predicted 11-transmembrane-domain topology. mENT1has 88% and 78% amino acid identity with rat ENT1 and human ENT1 respectively; mENT2 is more highly conserved, with 94% and 88% identity with rat ENT2 and human ENT2 respectively. We have also isolated two additional distinct cDNAs that encode proteins similar to mENT1; these probably represent distinct mENT1 isoforms or alternative splicing products. One cDNA encodes a protein with two additional amino acids (designated mENT1b) that adds a potential protein kinase CK2 phosphorylation site in the central intracellular loop of the transporter, and is similar, in this regard, to the human and rat ENT1 orthologues. The other cDNA has a 5′-untranslated region sequence that is distinct from that of full-length mENT1. Microinjection of mENT1, mENT1b or mENT2 cRNA into Xenopus oocytes resulted in enhanced uptake of [3H]uridine by the oocytes relative to that seen in water-injected controls. mENT1-mediated, but not mENT2-mediated, [3H]uridine uptake was inhibited by nitrobenzylthioinosine and dilazep. Dipyridamole inhibited both mENT1 and mENT2, but was significantly more effective against mENT1. Adenosine inhibited both systems with a similar potency, as did a range of other purine and pyrimidine nucleosides. These results are compatible with the known characteristics of the native mENT1 and mENT2 transporters.
β1-Integrin and PTEN control the phosphorylation of protein kinase C
Authors: PAREKH Davey B. , KATSO Roy M.T. , LESLIE Nick R. , DOWNES C. Peter , PROCYK Katarzyna J. , WATERFIELD Michael D. , PARKER Peter J.
Published: August 2015

Phosphorylation of protein kinase C (PKC) provides an amplitude control that operates in conjunction with allosteric effectors. Under many conditions, PKC isotypes appear to be highly phosphorylated; however, the cellular inputs that maintain these phosphorylations are not characterized. In the present work, it is shown that there is a differential phosphorylation of PKCδ in adherent versus suspension cultures of transfected HEK-293 cells. It is established that integrin activation is sufficient to trigger PKCδ phosphorylation and that this signals through phosphoinositide 3-kinase (PI3-kinase) to stimulate the phosphorylation of two sites, T505 and S662. The loss of signal input to PKCδ in suspension culture is dependent on the tumour suppressor gene PTEN, which encodes a bi-functional phosphotyrosine/phosphoinositide 3-phosphate phosphatase. In the PTEN-/- UM-UC-3 bladder carcinoma cell line grown in suspension, transfected PKCδ no longer accumulates in a dephospho-form on serum removal. By contrast, in a UM-UC-3-derivative cell line stably expressing PTEN, PKCδ does become dephosphorylated under these conditions. Employing the PTEN Gly129 → Glu mutant, which is selectively defective in lipid phosphatase activity, it was established that it is the lipid phosphatase activity that controls PKCδ phosphorylation. The evidence indicates that PKCδ phosphorylation and its latent activity are maintained in serum-deprived adherent cultures through integrin–matrix interactions. This control acts through a pathway involving a lipid product of PI3-kinase in a manner that can be suppressed by PTEN.
Modification of cysteine residues in the ChlI and ChlH subunits of magnesium chelatase results in enzyme inactivation
Authors: JENSEN Poul E. , REID James D. , HUNTER C. Neil
Published: August 2015

The enzyme magnesium protoporphyrin chelatase catalyses the insertion of magnesium into protoporphyrin, the first committed step in chlorophyll biosynthesis. Magnesium chelatase from the cyanobacterium Synechocystis PCC6803 has been reconstituted in a highly active state as a result of purifying the constituent proteins from strains of Escherichia coli that overproduce the ChlH, ChlI and ChlD subunits. These individual subunits were analysed for their sensitivity to N-ethylmaleimide (NEM), in order to assess the roles that cysteine residues play in the partial reactions that comprise the catalytic cycle of Mg2+ chelatase, such as the ATPase activity of ChlI, and the formation of ChlI–ChlD–MgATP and ChlH–protoporphyrin complexes. It was shown that NEM binds to ChlI and inhibits the ATPase activity of this subunit, and that prior incubation with MgATP affords protection against inhibition. Quantitative analysis of the effects of NEM binding on ChlI-catalysed ATPase activity showed that three out of four thiols per ChlI molecule are available to react with NEM, but only one cysteine residue per ChlI subunit is essential for ATPase activity. In contrast, the cysteines in ChlD are not essential for Mg2+ chelatase activity, and the formation of the ChlI–ChlD–ATP complex can proceed with NEM-treated ChlI. Neither the ATPase activity of ChlI nor NEM-modifiable cysteines are therefore required to form the ChlI–ChlD–MgATP complex. However, this complex cannot catalyse magnesium chelation in the presence of the ChlH subunit, protoporphyrin and Mg2+ ions. The simplest explanation for this is that in an intact Mg2+ chelatase complex the ATPase activity of ChlI drives the chelation process. NEM binds to ChlH and inhibits the chelation reaction, and this effect can be partially alleviated by pre-incubating ChlH with magnesium and ATP. We conclude that cysteine residues play an important role in the chelation reaction, in respect of the ChlI–MgATP association, ATP hydrolysis and in the interaction of ChlH with MgATP and protoporphyrin IX.
O-glycan variability of egg-jelly mucins from Xenopus laevis: characterization of four phenotypes that differ by the terminal glycosylation of their mucins
Authors: GUERARDEL Yann , KOL Ossarath , MAES Emmanuel , LEFEBVRE Tony , BOILLY Bénoni , DAVRIL Monique , STRECKER Gérard
Published: August 2015

Eggs from Xenopus laevis are surrounded by several layers of jelly that are needed for proper fertilization. Jelly coat is composed of high-molecular-mass glycoconjugates to which are bound many globular proteins. O-glycans released from the jelly coat of X. laevis have been partially described in previous studies. In this study, we compared the glycosylation pattern of the egg jelly coat isolated from six specimens of X. laevis. The O-glycans were released from jelly coats by alkali/borohydride treatment. Structural characterization was performed through a combination of one- and two-dimensional 1H-NMR and methylation analysis. This allowed the description of a new family of sulphated O-glycans present in jelly coats of all X. laevis. However, the jelly O-glycans showed a low extent of polymorphism between specimens. This intra-specific variability was restricted to the terminal substitution of O-linked oligosaccharides. The differential expression of two glycosyltransferase [an α-(1 → 4) galactosyltransferase and an α-(1 → 3) fucosyltransferase] activities resulted in the characterization of four phenotypes of X. laevis. Furthermore, electrophoretic analysis suggested that the high-molecular-mass fraction of jelly coat was mostly composed of mucin-type glycoproteins. Blot analysis with lectins confirmed that the glycan variability was borne by these mucin-type components. However, fertilization assays suggested that the glycan polymorphism had no repercussion on egg fertilizability.
Adrenocorticotrophic hormone stimulates phosphotyrosine phosphatase SHP2 in bovine adrenocortical cells: phosphorylation and activation by cAMP-dependent protein kinase
Authors: ROCCHI Stéphane , GAILLARD Isabelle , VAN OBBERGHEN Emmanuel , CHAMBAZ Edmond M. , VILGRAIN Isabelle
Published: August 2015

During activation of adrenocortical cells by adrenocorticotrophic hormone (ACTH), tyrosine dephosphorylation of paxillin is stimulated and this correlates with protrusion of filopodial structures and a decreased number of focal adhesions. These effects are inhibited by Na3VO4, a phosphotyrosine phosphatase inhibitor [Vilgrain, Chinn, Gaillard, Chambaz and Feige (1998) Biochem. J. 332, 533–540]. However, the tyrosine phosphatases involved in these processes remain to be identified. In this study, we provide evidence that the Src homology domain (SH)2-containing phosphotyrosine phosphatase (SHP)2, but not SHP1, is expressed in adrenocortical cells and is phosphorylated upon ACTH challenge. ACTH (10-8M) treatment of 32P-labelled adrenocortical cells resulted in an increase in phosphorylated SHP2. By probing SHP2-containing immunoprecipitates with an antibody to phosphoserine we found that SHP2 was phosphorylated on serine in ACTH-treated cells in a dose- and time-dependent manner. Furthermore, using an in vitro kinase assay, we showed that SHP2 was a target for cAMP-dependent protein kinase (PKA). Serine was identified as the only target amino acid phosphorylated in SHP2. Phosphorylation of SHP2 by PKA resulted in a dramatic stimulation of phosphatase activity measured either with insulin receptor substrate-1 or with the synthetic peptide [32P]poly(Glu/Tyr) as substrate. In an in-gel assay of SHP2-containing immunoprecipitates, phosphorylated in vitro by PKA or isolated from adrenocortical cells treated with 10nM ACTH, a pronounced activation of SHP2 activity was shown. These observations clearly support the idea that a PKA-mediated signal transduction pathway contributes to SHP2 regulation in adrenocortical cells and point to SHP2 as a possible mediator of the effects of ACTH.
Phospholipase D in rat myometrium: occurrence of a membrane-bound ARF6 (ADP-ribosylation factor 6)-regulated activity controlled by βγ subunits of heterotrimeric G-proteins
Authors: LE STUNFF Hervé , DOKHAC Lien , BOURGOIN Sylvain , BADER Marie-France , HARBON Simone
Published: August 2015

Both protein kinase C and protein tyrosine kinases have been shown to be involved in phospholipase D (PLD) activation in intact rat myometrium [Le Stunff, Dokhac and Harbon (2000) J. Pharmacol. Exp. Ther. 292, 629–637]. In this study we assessed the involvement of monomeric G-proteins in PLD activation in a cell-free system derived from myometrial tissue. Both the PLD1 and PLD2 isoforms were detected. Two forms of PLD activity, essentially membrane-bound, were found in myometrial preparations. One form was stimulated by oleate and insensitive to guanosine 5ƀ-[γ-thio] triphosphate (GTP[S]). The second required ammonium sulphate to be detected and was stimulated by GTP[S]. ADP-ribosylation factors (ARF1 and ARF6) and RhoA were immunodetected in myometrial preparations. ARF1 and RhoA were present in the membrane and cytosolic fractions whereas ARF6 was detected exclusively in the membrane fraction. A synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 [myrARF6(2–13)] totally abolished PLD activation in the presence of ammonium sulphate and GTP[S], whereas myrARF1(2–17) and the inhibitory GDP/GTP-exchange factor, Rho GDI, did not. These data are consistent with a membrane-bound ARF6-regulated PLD activity. Finally, the stimulation of PLD by ARF6 was inhibited by AlF-4 and this inhibition was counteracted by the fusion protein glutathione S-transferase-β-adrenergic receptor kinase 1 (495–689) and by the QEHA peptide (from adenylate cyclase ACII), which act as G-protein βγ-subunit scavengers. It is concluded that G-protein subunits βγ are involved in a pathway modulating PLD activation by ARF6, illustrating cross-talk between heterotrimeric and monomeric G-proteins.
The conserved redox-sensitive cysteine residue of the DNA-binding region in the c-Rel protein is involved in the regulation of the phosphorylation of the protein
Authors: GLINEUR Corine , DAVIOUD-CHARVET Elisabeth , VANDENBUNDER Bernard
Published: August 2015

The DNA-binding activity of the transcription nuclear factor κB (NF-κB) is regulated by a redox-control mechanism involving the reduction of a disulphide bond from a specific cysteine residue conserved in all members of the NF-κB family. Thioredoxin is involved in this redox control. DNA binding and transactivating capacity of NF-κB are up-regulated by inducible phosphorylation. Here we demonstrate that the conserved redox cysteine in the c-Rel protein is involved in the phosphorylation regulation of the protein. When this cysteine residue is mutated to an aspartic acid residue, the mutant protein loses its capacity to be phosphorylated and its DNA-binding activity. In addition, our results suggest that, when the conserved redox cysteine is chemically modified by N-ethylmaleimide and 2-chloro-1,3-dinitrobenzene, the protein c-Rel cannot be phosphorylated. In contrast, the protein in which the cysteine residue was replaced by a serine residue, creating a potential phosphorylation site, is highly phosphorylated and binds κB sequences. The protein could loose the redox regulation of the phosphorylation when the residue replacing the cysteine can be itself phosphorylated. We also show that specific inhibitors of thioredoxin reductases impair the phosphorylation of the c-Rel protein, suggesting that the redox regulation of the protein controls its phosphorylation.
Glucose and the ATP paradox in yeast
Authors: SOMSEN Oscar J. G. , HOEBEN Martijn A. , ESGALHADO Eugenia , SNOEP Jacky L. , VISSER Diana , HEIJDEN Rene T. J. M. , HEIJNEN Joseph J. , WESTERHOFF Hans V.
Published: August 2015

A sustained decrease in the intracellular ATP concentration has been observed when extra glucose was added to yeast cells growing aerobically under glucose limitation. Because glucose degradation is the main source of ATP-derived free energy, this is a counter-intuitive phenomenon, which cannot be attributed to transient ATP consumption in the initial steps of glycolysis. We present a core model for aerobic growth in which glucose supplies carbon, as well as free energy, for biosynthesis. With Metabolic Control Analysis and numerical simulations, we demonstrate that the decrease in the ATP concentration can be reproduced if the biosynthetic route is more strongly activated by carbon substrates than is the catabolic (ATP-producing) route.
The latent transforming growth factor β binding protein (LTBP) family
Authors: ÖKLÜ Rahmi , HESKETH Robin
Published: August 2015

The transforming growth factor β (TGFβ) cytokines are a multi-functional family that exert a wide variety of effects on both normal and transformed mammalian cells. The secretion and activation of TGFβs is regulated by their association with latency-associated proteins and latent TGFβ binding proteins (LTBPs). Over the past few years, three members of the LTBP family have been identified, in addition to the protoype LTBP1 first sequenced in 1990. Three of the LTBP family are expressed in a variety of isoforms as a consequence of alternative splicing. This review summarizes the differences between the isoforms in terms of the effects on domain structure and hence possible function. The close identity between LTBPs and members of the fibrillin family, mutations in which have been linked directly to Marfan's syndrome, suggests that anomalous expression of LTBPs may be associated with disease. Recent data indicating that differential expression of LTBP1 isoforms occurs during the development of coronary heart disease is considered, together with evidence that modulation of LTBP function, and hence of TGFβ activity, is associated with a variety of cancers.
A role for the actin cytoskeleton in the hormonal and growth-factor-mediated activation of protein kinase B
Authors: PEYROLLIER Karine , HAJDUCH Eric , GRAY Alexander , LITHERLAND Gary J. , PRESCOTT Alan R. , LESLIE Nick R. , HUNDAL Harinder S.
Published: August 2015

We show here that cytochalasin D-induced depolymerization of actin filaments markedly reduces the stimulus-dependent activation of protein kinase B (PKB) in four different cell types (HEK-293 cells, L6 myotubes, 3T3-L1 adipocytes and U87MG cells). HEK-293 cells expressing the pleckstrin homology (PH) domains of PKB and general receptor for phosphoinositides-1 (GRP1) fused to green fluorescent protein (GFP) were used to monitor production of 3-phosphoinositides in the plasma membrane. Disassembly of the actin cytoskeleton significantly reduced the insulin-mediated translocation of both PKB-PHŐGFP and GRP1-PHŐGFP to the plasma membrane, consistent with diminished synthesis of 3-phosphoinositides. Actin depolymerization did not affect the hormonal activation of phosphoinositide 3-kinase (PI 3-kinase), and since cytochalasin D treatment also led to reduced platelet-derived growth factor (PDGF)-induced phosphorylation of PKB in U87MG cells, a PTEN (phosphatase and tensin homologue deleted on chromosome 10) null cell line, lipid phosphatase activity was unlikely to account for any reduction in cellular 3-phosphoinositides. Withdrawal of cytochalasin D from the extracellular medium induced actin filament repolymerization, and reinstated both the recruitment of PHŐGFP fusion proteins to the plasma membrane and PKB activation in response to insulin and PDGF. Our findings indicate that an intact actin network is a crucial requirement for PI 3-kinase-mediated production of 3-phosphoinositides and, therefore, for the activation of PKB.
Organization, chromosomal localization and promoter analysis of the gene encoding human acidic fibroblast growth factor intracellular binding protein
Authors: KOLPAKOVA Elona , FRENGEN Eirik , STOKKE Trond , OLSNES Sjur
Published: August 2015

Acidic fibroblast growth factor (aFGF) intracellular binding protein (FIBP) is a protein found mainly in the nucleus that might be involved in the intracellular function of aFGF. Here we present a comparative analysis of the deduced amino acid sequences of human, murine and Drosophila FIBP analogues and demonstrate that FIBP is an evolutionarily conserved protein. The human gene spans more than 5kb, comprising ten exons and nine introns, and maps to chromosome 11q13.1. Two slightly different splice variants found in different tissues were isolated and characterized. Sequence analysis of the region surrounding the translation start revealed a CpG island, a classical feature of widely expressed genes. Functional studies of the promoter region with a luciferase reporter system suggested a strong transcriptional activity residing within 600bp of the 5´ flanking region.