bluuebunny/crossref_metadata_2025 huggingface dataset:
The T-cell antigen CD5 acts as a receptor and substrate for the protein-tyrosine kinase p56lck.
Authors: Raab M , Yamamoto M , Rudd C E
Published: October 2015

CD5 is a T-cell-specific antigen which binds to the B-cell antigen CD72 and acts as a coreceptor in the stimulation of T-cell growth. CD5 associates with the T-cell receptor zeta chain (TcR zeta)/CD3 complex and is rapidly phosphosphorylated on tyrosine residues as a result of TcR zeta/CD3 ligation. However, despite this, the mechanism by which CD5 generates intracellular signals is unclear. In this study, we demonstrate that CD5 is coupled to the protein-tyrosine kinase p56lck and can act as a substrate for p56lck. Coexpression of CD5 with p56lck in the baculovirus expression system resulted in the phosphorylation of CD5 on tyrosine residues. Further, anti-CD5 and anti-p56lck coprecipitated each other in a variety of detergents, including Nonidet P-40 and Triton X-100. Anti-CD5 also precipitated the kinase from various T cells irrespective of the expression of TcR zeta/CD3 or CD4. No binding between p59fyn(T) and CD5 was detected in T cells. The binding of p56lck to CD5 induced a 10- to 15-fold increase in p56lck catalytic activity, as measured by in vitro kinase analysis. In vivo labelling with 32P(i) also showed a four- to fivefold increase in Y-394 occupancy in p56lck when associated with CD5. The use of glutathione S-transferase-Lck fusion proteins in precipitation analysis showed that the SH2 domain of p56lck could recognize CD5 as expressed in the baculovirus expression system. CD5 interaction with p56lck represents a novel variant of a receptor-kinase complex in which receptor can also serve as substrate. The CD5-p56lck interaction is likely to play roles in T-cell signalling and T-B collaboration.
Characterization of tobacco protein kinase NPK5, a homolog of Saccharomyces cerevisiae SNF1 that constitutively activates expression of the glucose-repressible SUC2 gene for a secreted invertase of S. cerevisiae.
Authors: Muranaka T , Banno H , Machida Y
Published: October 2015

We have isolated a cDNA (cNPK5) that encodes a protein kinase of 511 amino acids from suspension cultures of tobacco cells. The predicted kinase domain of NPK5 is 65% identical in terms of amino acid sequence to that of the SNF1 serine/threonine protein kinase of Saccharomyces cerevisiae, which plays a central role in catabolite repression in yeast cells. SNF1 positively regulates transcription of various glucose-repressible genes of the yeast, such as the SUC2 gene for a secreted invertase, in response to glucose deprivation: snf1 mutants cannot utilize sucrose as a carbon source. Expression of cNPK5 in yeast cells allowed the snf1 mutant cells to utilize sucrose for growth and caused constitutive expression of the SUC2 gene in wild-type cells even in the presence of glucose, an indication that the NPK5 protein is present in a constitutively active form in S. cerevisiae. On the other hand, expression of cNPK5 failed to suppress the growth defect of the snf4 mutant cells in the presence of sucrose and to induce expression of the SUC2 gene. These results indicate that SNF4 is required for the induction of SUC2 expression by NPK5, as by SNF1, even if NPK5 is constitutively active in S. cerevisiae. The recombinant NPK5 protein is capable of autophosphorylation in vitro in a reaction that requires Mn2+ rather than Mg2+ ions but is inhibited by Ca2+ ions. Both dicotyledonous and monocotyledonous plants have several copies of the NPK5-related gene, which probably constitute a small gene family. NPK5-related genes were found to be expressed in the roots, leaves, and stems of tobacco plants. The high degree of structural conservation and the functional similarity of NPK5 to SNF1 lead us to speculate that NPK5 (or a related kinase) also plays a role in sugar metabolism in higher plants.
Cloning and characterization of an evolutionarily divergent DNA-binding subunit of mammalian TFIIIC.
Authors: Lagna G , Kovelman R , Sukegawa J , Roeder R G
Published: October 2015

Transcription factor IIIC (TFIIIC) is required for the assembly of a preinitiation complex on 5S RNA, tRNA, and adenovirus VA RNA genes and contains two separable components, TFIIIC1 and TFIIIC2. TFIIIC2 binds to the 3' end of the internal control region of the VAI RNA gene and contains five polypeptides ranging in size from 63 to 220 kDa; the largest of these directly contacts DNA. Here we describe the cloning of cDNAs encoding all (rat) or part (human) of the 220-kDa subunit (TFIIIC alpha). Surprisingly, TFIIIC alpha has no homology to any of the yeast TFIIIC subunits already cloned, suggesting a significant degree of evolutionary divergence for RNA polymerase III factors. Antibodies raised against the N terminus of recombinant human TFIIIC alpha specifically inhibit binding of natural TFIIIC to DNA. Furthermore, immunodepletion assays indicate that TFIIIC alpha is absolutely required for RNA polymerase III transcription of 5S RNA, tRNA, and VAI RNA genes but not for the 7SK RNA and U6 small nuclear RNA genes. Transcription from the tRNA and VAI RNA genes in TFIIIC-depleted nuclear extracts can be restored by addition of purified TFIIIC. In contrast, restoration of 5S RNA gene transcription requires readdition of both TFIIIC and TFIIIA, indicating a promoter-independent interaction between these factors. Immunoprecipitation experiments demonstrate a tight association of all five polypeptides previously identified in the TFIIIC2 fraction, confirming the multisubunit structure of the human factor.
The Drosophila nuclear receptors FTZ-F1 alpha and FTZ-F1 beta compete as monomers for binding to a site in the fushi tarazu gene.
Authors: Ohno C K , Ueda H , Petkovich M
Published: October 2015

The striped pattern of fushi tarazu (ftz) expression found in the blastoderm of the Drosophila melanogaster embryo is generated largely through complex interactions between multiple transcription factors that bind to the zebra element of the ftz gene. A motif in the zebra element, the FTZ-F1 recognition element (F1RE), has been shown to bind a transcription factor, FTZ-F1 alpha, that is a member of the nuclear receptor family. We recently identified a second, related member of this family, FTZ-F1 beta, that also binds to this motif. To investigate the possibility that FTZ-F1 alpha and FTZ-F1 beta coregulate ftz transcription through the F1RE, we have studied the DNA binding properties of FTZ-F1 alpha and FTZ-F1 beta. We demonstrate that recombinant FTZ-F1 alpha and FTZ-F1 beta proteins produce similar in vitro DNase I footprint patterns on a 14-nucleotide region of the zebra element and bind to this site with similar affinities and sequence specificities. Using wild-type and N-terminally truncated receptors, we have determined that FTZ-F1 alpha and FTZ-F1 beta both bind as monomers to the 9-bp F1RE in the zebra element, as well as to an imperfect inverted F1RE repeat present in the Drosophila alcohol dehydrogenase gene. A polyclonal antibody raised against FTZ-F1 beta identifies a predominant F1RE-binding component in embryonic nuclear extracts. Although FTZ-F1 alpha is also present in these extracts, FTZ-F1 alpha and FTZ-F1 beta do not appear to form heterodimers with each other. Cotransfection assays in mammalian cell culture indicate that both receptors contribute to the net transcriptional activity of a reporter gene through their direct interaction with the F1RE. These data suggest that FTZ-F1 alpha and FTZ-F1 beta likely coregulate common target genes by competition for binding to a 9-bp recognition element.
Species-specific functional interactions of DNA polymerase alpha-primase with simian virus 40 (SV40) T antigen require SV40 origin DNA.
Authors: Schneider C , Weisshart K , Guarino L A , Dornreiter I , Fanning E
Published: October 2015

Physical and functional interactions of simian virus 40 (SV40) and polyomavirus large-T antigens with DNA polymerase alpha-primase were analyzed to elucidate the molecular basis for the species specificity of polymerase alpha-primase in viral DNA replication. SV40 T antigen associated more efficiently with polymerase alpha-primase in crude human extracts than in mouse extracts, while polyomavirus T antigen interacted preferentially with polymerase alpha-primase in mouse extracts. The apparent species specificity of complex formation was not observed when purified polymerase alpha-primases were substituted for the crude extracts. Several functional interactions between T antigen and purified polymerase alpha-primase, including stimulation of primer synthesis and primer elongation on M13 DNA in the presence or absence of the single-stranded DNA binding protein RP-A, also proved to be independent of the species from which polymerase alpha-primase had been purified. However, the human DNA polymerase alpha-primase was specifically required for primosome assembly and primer synthesis on SV40 origin DNA in the presence of T antigen and RP-A.
Analysis of the role of the Shc and Grb2 proteins in signal transduction by the v-ErbB protein.
Authors: Meyer S , LaBudda K , McGlade J , Hayman M J
Published: October 2015

The epidermal growth factor receptor, EGFR, has been implicated in cell transformation in both mammalian and avian species. The v-ErbB oncoprotein is an oncogenic form of the chicken EGFR. The tyrosine kinase activity of this oncoprotein is required for transformation, but no transformation-specific cellular substrates have been described to date. Recently activation of the ras signal transduction pathway by the EGFR has been shown to involve the Shc and Grb2 proteins. In this communication, we demonstrate that the Shc proteins are phosphorylated on tyrosine residues and are complexed with Grb2 and the chicken EGFR following ligand activation of this receptor. In fibroblasts and erythroid cells transformed by the avian erythroblastosis virus (AEV) strains H and ES4, the Shc proteins are found to be constitutively phosphorylated on tyrosine residues. The tyrosine-phosphorylated forms of the AEV strain H v-ErbB protein are found in a complex with Shc and Grb2, but the Shc proteins do not bind to the AEV strain ES4 v-ErbB protein. Mutant forms of the v-ErbB protein (in which several of the tyrosines that become autophosphorylated have been deleted by truncation) are unable to transform erythroid cells but can still transform fibroblasts. Analysis of cells transformed by one of these mutants revealed that the truncated v-ErbB protein could no longer bind to either Shc or Grb2, but this oncoprotein still gave rise to tyrosine-phosphorylated Shc proteins that complexed with Grb2 and led to activation of mitogen-activated protein (MAP) kinase. The results suggest that stable binding of Grb2 and Shc to the v-ErbB protein is not necessary to activate this signal transduction pathway and assuming that the mutant activate MAP kinase in erythroid cells in a manner similar to that of fibroblasts, that activation of this pathway is not sufficient to transform erythroid cells.
Glucagon gene expression is negatively regulated by hepatocyte nuclear factor 3 beta.
Authors: Philippe J , Morel C , Prezioso V R
Published: October 2015

Pancreatic expression of the glucagon gene depends on multiple transcription factors interacting with at least three DNA control elements: G1, the upstream promoter element, and G2 and G3, two enhancer-like sequences. We report here that the major enhancer of the rat glucagon gene, G2, interacts with three protein complexes, A1, A2, and A3. A2 is detected only in islet cells, and impairment of its binding to mutant G2 causes a marked decrease in transcriptional activity. We identify A1 as hepatocyte nuclear factor 3 beta (HNF-3 beta), a member of the HNF-3 DNA-binding protein family found in abundance in the liver which has been proposed to play a role in the formation of gut-related organs. HNF-3 beta binds G2 on a site which overlaps A2 and acts as a repressor of glucagon gene expression, as demonstrated by mutational analyses of G2 and by cotransfection of HNF-3 beta cDNA along with reporter genes containing G2 into glucagon-producing cells. Our data implicate HNF-3 beta in the control of glucagon gene expression and strengthen the idea of endodermal origin of the islet cells.
Yeast nucleotide excision repair proteins Rad2 and Rad4 interact with RNA polymerase II basal transcription factor b (TFIIH).
Authors: Bardwell A J , Bardwell L , Iyer N , Svejstrup J Q , Feaver W J , Kornberg R D , Friedberg E C
Published: October 2015

The Rad2, Rad3, Rad4, and Ss12 proteins are required for nucleotide excision repair in yeast cells and are homologs of four human proteins which are involved in a group of hereditary repair-defective diseases. We have previously shown that Rad3 protein is one of the five subunits of purified RNA polymerase II basal transcription initiation factor b (TFIIH) and that Ss12 protein physically associates with factor b (W.J. Feaver, J.Q. Svejstrup, L. Bardwell, A.J. Bardwell, S. Buratowski, K.D. Gulyas, T.F. Donahue, E.C. Friedberg, and R.D. Kornberg, Cell 75:1379-1387, 1993). Here we show that the Rad2 and Rad4 proteins interact with purified factor b in vitro. Rad2 (a single-stranded DNA endonuclease) specifically interacts with the Tfb1 subunit of factor b, and we have mapped a limited region of the Rad2 polypeptide which is sufficient for this interaction. Rad2 also interacts directly with Ss12 protein (a putative DNA helicase). The binding of Rad2 and Rad4 proteins to factor b may define intermediates in the assembly of the nucleotide excision repair repairosome. Furthermore, the loading of factor b (or such intermediates) onto promoters during transcription initiation provides a mechanism for the preferential targeting of repair proteins to actively transcribing genes.
Effect of a null mutation of the insulin-like growth factor I receptor gene on growth and transformation of mouse embryo fibroblasts.
Authors: Sell C , Dumenil G , Deveaud C , Miura M , Coppola D , DeAngelis T , Rubin R , Efstratiadis A , Baserga R
Published: October 2015

Fibroblast cell lines, designated R- and W cells, were generated, respectively, from mouse embryos homozygous for a targeted disruption of the Igf1r gene, encoding the type 1 insulin-like growth factor receptor, and from their wild-type littermates. W cells grow normally in serum-free medium supplemented with various combinations of purified growth factors, while pre- and postcrisis R- cells cannot grow, as they are arrested before entering the S phase. R- cells are able to grow in 10% serum, albeit more slowly than W cells, and with all phases of the cell cycle being elongated. An activated Ha-ras expressed from a stably transfected plasmid is unable to overcome the inability of R- cells to grow in serum-free medium supplemented with purified clones. Nevertheless, even in the presence of serum, R- cells stably transfected with Ha-ras, alone or in combination with simian virus 40 large T antigen, fail to form colonies in soft agar. Reintroduction into R- cells (or their derivatives) of a plasmid expressing the human insulin-like growth factor I receptor RNA and protein restores their ability to grow with purified growth factors or in soft agar. The signaling pathways participating in cell growth and transformation are discussed on the basis of these results.
Adenovirus E3 protein causes constitutively internalized epidermal growth factor receptors to accumulate in a prelysosomal compartment, resulting in enhanced degradation.
Authors: Hoffman P , Carlin C
Published: October 2015

We have previously identified and characterized an integral membrane protein coded for by the early transcription region 3 (E3) of human group C adenoviruses that down-regulates the epidermal growth factor receptor (EGFR). The goal of this study was to characterize the early receptor trafficking events leading to enhanced EGFR degradation in adenovirus-infected cells. Specifically, we wished to determine whether adenovirus increases the rate of EGFR internalization or alters the subcellular compartmentalization of internalized EGFRs. Once the optimal time for measuring early trafficking events was determined, surface EGFRs were labeled with a cleavable biotin reagent to measure internalization rates and with a receptor-specific monoclonal antibody (MAb) conjugated to colloidal gold for intracellular localization studies. We first showed that the rate of EGFR internalization in adenovirus-infected cells is indistinguishable from the constitutive internalization rate for unoccupied EGFRs. The possibility that the E3 protein can affect trafficking of EGFRs internalized at a low constitutive rate was further supported by studies showing that adenovirus-mediated down-regulation occurs independently of EGFR oligomerization and intrinsic EGFR tyrosine kinase activity, which are required for efficient ligand-induced internalization. Other tyrosine kinases inhibited by genistein are also not required for adenovirus-induced down-regulation. When the intracellular localization of EGFRs during adenovirus-mediated down-regulation was examined by electron microscopy, there was a threefold increase in the number of EGFRs localized to multivesicular bodies. The multivesicular body has been proposed to be important for regulating intracellular membrane protein sorting, since trafficking patterns for receptors that recycle and receptors that are degraded diverge in this organelle. These data therefore suggest that adenovirus may enhance EGFR degradation by causing constitutively internalized EGFRs to accumulate in a prelysosomal compartment. This is the first example of a mechanism that efficiently down-regulates EGFR without significantly increasing the rate of internalization or that does not require EGFR tyrosine kinase activity. Since viral proteins often mimic or modify a host counterpart, this suggests that there are normal physiological conditions when receptor destruction without tyrosine signalling is beneficial.
Vertebrate mRNAs with a 5'-terminal pyrimidine tract are candidates for translational repression in quiescent cells: characterization of the translational cis-regulatory element.
Authors: Avni D , Shama S , Loreni F , Meyuhas O
Published: October 2015

The translation of mammalian ribosomal protein (rp) mRNAs is selectively repressed in nongrowing cells. This response is mediated through a regulatory element residing in the 5' untranslated region of these mRNAs and includes a 5' terminal oligopyrimidine tract (5' TOP). To further characterize the translational cis-regulatory element, we monitored the translational behavior of various endogenous and heterologous mRNAs or hybrid transcripts derived from transfected chimeric genes. The translational efficiency of these mRNAs was assessed in cells that either were growing normally or were growth arrested under various physiological conditions. Our experiments have yielded the following results: (i) the translation of mammalian rp mRNAs is properly regulated in amphibian cells, and likewise, amphibian rp mRNA is regulated in mammalian cells, indicating that all of the elements required for translation control of rp mRNAs are conserved among vertebrate classes; (ii) selective translational control is not confined to rp mRNAs, as mRNAs encoding the naturally occurring ubiquitin-rp fusion protein and elongation factor 1 alpha, which contain a 5' TOP, also conform this mode of regulation; (iii) rat rpP2 mRNA contains only five pyrimidines in its 5' TOP, yet this mRNA is translationally controlled in the same fashion as other rp mRNAs with a 5' TOP of eight or more pyrimidines; (iv) full manifestation of this mode of regulation seems to require both the 5' TOP and sequences immediately downstream; and (v) an intact translational regulatory element from rpL32 mRNA fails to exert its regulatory properties even when preceded by a single A residue.
The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation.
Authors: Nelson J A , Savereide P B , Lefebvre P A
Published: October 2015

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.
Stat4, a novel gamma interferon activation site-binding protein expressed in early myeloid differentiation.
Authors: Yamamoto K , Quelle F W , Thierfelder W E , Kreider B L , Gilbert D J , Jenkins N A , Copeland N G , Silvennoinen O , Ihle J N
Published: October 2015

Interferon regulation of gene expression is dependent on the tyrosine phosphorylation and activation of the DNA-binding activity of two related proteins of 91 kDa (STAT1) and/or 113 kDa (STAT2). Recent studies have suggested that these proteins are substrates of Janus kinases and that proteins related in STAT1 are involved in a number of signalling pathways, including those activated in myeloid cells by erythropoietin and interleukin-3 (IL-3). To clone STAT-related proteins from myeloid cells, degenerate oligonucleotides were used in PCRs to identify novel family members expressed in myeloid cells. This approach allowed the identification and cloning of the Stat4 gene, which is 52% identical to STAT1. Unlike STAT1, Stat4 expression is restricted but includes myeloid cells and spermatogonia. In the erythroid lineage, Stat4 expression is differentially regulated during differentiation. Functionally, Stat4 has the properties of other STAT family genes. In particular, cotransfection of expression constructs for Stat4 and Jak1 and Jak2 results in the tyrosine phosphorylation of Stat4 and the acquisition of the ability to bind to the gamma interferon (IFN-gamma)-activated sequence of the interferon regulatory factor 1 (IRF-1) gene. Stat4 is located on mouse chromosome 1 and is tightly linked to the Stat1 gene, suggesting that the genes arose by gene duplication. Unlike Stat1, neither IFN-alpha nor IFN-gamma activates Stat4. Nor is Stat4 activated in myeloid cells by a number of cytokines, including erythropoietin, IL-3, granulocyte colony-stimulating factor, stem cell factor, colon-stimulating factor 1, hepatocyte growth factor, IL-2, IL-4, and IL-6.
A pleiotropic element in the medium-chain acyl coenzyme A dehydrogenase gene promoter mediates transcriptional regulation by multiple nuclear receptor transcription factors and defines novel receptor-DNA binding motifs.
Authors: Carter M E , Gulick T , Moore D D , Kelly D P
Published: October 2015

We previously identified a complex regulatory element in the medium-chain acyl coenzyme A dehydrogenase gene promoter that confers transcriptional regulation by the retinoid receptors RAR and RXR and the orphan nuclear receptor HNF-4. In this study we demonstrate a trans-repressing regulatory function for the orphan receptor COUP-TF at this same nuclear receptor response element (NRRE-1). The transcriptional regulatory properties and receptor binding sequences of each nuclear receptor response element within NRRE-1 are also characterized. NRRE-1 consists of four potential nuclear hormone receptor hexamer binding sites, arranged as [<--1-(n)s-2-->-3-->(n)4<--4], three of which are used in alternative pairwise binding by COUP-TF and HNF-4 homodimers and by RAR-RXR heterodimers, as demonstrated by mobility shift assays and methylation interference analysis. Binding and transactivation studies with mutant NRRE-1 elements confirmed the existence of distinct retinoid, COUP-TF, and HNF-4 response elements that define novel receptor binding motifs: COUP-TF homodimers bound sites 1 and 3 (two hexamer repeat sequences arranged as an everted imperfect repeat separated by 14 bp or ER14), RAR-RXR heterodimers bound sites 1 and 2 (ER8), and HNF-4 homodimers bound sites 2 and 3 (imperfect DR0). Mixing cotransfection experiments demonstrated that the nuclear receptor dimers compete at NRRE-1 to modulate constitutive and ligand-mediated transcriptional activity. These data suggest a mechanism for the transcriptional modulation of genes encoding enzymes involved in cellular metabolism.
Enhancement or inhibition of insulin signaling by insulin receptor substrate 1 is cell context dependent.
Authors: Yamauchi K , Pessin J E
Published: October 2015

Insulin treatment of Chinese hamster ovary (CHO) cells expressing high levels of the insulin receptor (CHO/IR cells) activates both c-fos serum response element and activator protein 1 (AP-1) reporter genes approximately 10-fold. In contrast, parental CHO cells display only two- to threefold insulin stimulation of reporter gene activity. Transient transfection of parental CHO cells with an insulin receptor substrate 1 (IRS1) expression plasmid enhanced insulin downstream signaling in a biphasic manner, whereas IRS1 transfection of CHO/IR cells inhibited insulin signaling in a dose-dependent fashion. Further, expression of Grb2 in parental CHO cells had no effect on insulin signaling, whereas Grb2 increased insulin activation of reporter gene expression in CHO/IR cells. These data suggest that the expression levels of various effector molecules can either enhance or inhibit insulin downstream signaling events. To assess the relative effects of various insulin receptor, IRS1, and Grb2 levels on insulin signaling, parental CHO cells were transiently transfected with various combinations of expression plasmids encoding these proteins. Although expression of IRS1 resulted in a biphasic increase of insulin signaling in parental CHO cells, coexpression of IRS1 with the insulin receptor resulted in inhibition of signaling. This inhibition of insulin signaling directly correlated with an increased association of Grb2 with IRS1 and a concomitant sequestration of Grb2 away from Shc. Consistent with the Shc-Grb2 pathway as the major route for insulin-stimulated c-Fos and AP-1 transcriptional activation, the IRS1-mediated inhibition was reversed by transfection with an expression plasmid for Grb2. These data demonstrate that the extent of insulin-stimulated downstream signaling was dependent not only on the levels of individual signaling molecules but also on the formation of multiprotein complexes with specific stoichiometries.
Hox proteins have different affinities for a consensus DNA site that correlate with the positions of their genes on the hox cluster.
Authors: Pellerin I , Schnabel C , Catron K M , Abate C
Published: October 2015

The hox genes, members of a family of essential developmental regulators, have the intriguing property that their expression in the developing murine embryo is colinear with their chromosomal organization. Members of the hox gene family share a conserved DNA binding domain, termed the homeodomain, which mediates interactions of Hox proteins with DNA regulatory elements in the transcriptional control regions of target genes. In this study, we characterized the DNA binding properties of five representative members of the Hox family: HoxA5, HoxB4, HoxA7, HoxC8, and HoxB1. To facilitate a comparative analysis of their DNA binding properties, we produced the homeodomain regions of these Hox proteins in Escherichia coli and obtained highly purified polypeptides. We showed that these Hox proteins interact in vitro with a common consensus DNA site that contains the motif (C/G)TAATTG. We further showed that the Hox proteins recognize the consensus DNA site in vivo, as determined by their ability to activate transcription through this site in transient transfection assays. Although they interact optimally with the consensus DNA site, the Hox proteins exhibit subtle, but distinct, preferences for DNA sites that contain variations of the nucleotides within the consensus motif. In addition to their modest differences in DNA binding specificities, the Hox proteins also vary in their relative affinities for DNA. Intriguingly, their relative affinities correlate with the positions of their respective genes on the hox cluster. These findings suggest that subtle differences in DNA binding specificity combined with differences in DNA binding affinity constitute features of the "Hox code" that contribute to the selective functions of Hox proteins during murine embryogenesis.
Myocyte nuclear factor, a novel winged-helix transcription factor under both developmental and neural regulation in striated myocytes.
Authors: Bassel-Duby R , Hernandez M D , Yang Q , Rochelle J M , Seldin M F , Williams R S
Published: October 2015

A sequence motif (CCAC box) within an upstream enhancer region of the human myoglobin gene is essential for transcriptional activity in both cardiac and skeletal muscle. A cDNA clone, myocyte nuclear factor (MNF), was isolated from a murine expression library on the basis of sequence-specific binding to the myoglobin CCAC box motif and was found to encode a novel member of the winged-helix or HNF-3/fork head family of transcription factors. Probes based on this sequence identify two mRNA species that are upregulated during myocyte differentiation, and antibodies raised against recombinant MNF identify proteins of approximately 90, 68, and 65 kDa whose expression is regulated following differentiation of myogenic cells in culture. In addition, the 90-kDa form of MNF is phosphorylated and is upregulated in intact muscles subjected to chronic motor nerve stimulation, a potent stimulus to myoglobin gene regulation. Amino acid residues 280 to 389 of MNF demonstrate 35 to 89% sequence identity to the winged-helix domain from other known members of this family, but MNF is otherwise divergent. A proline-rich amino-terminal region (residues 1 to 206) of MNF functions as a transcriptional activation domain. These studies provide the first evidence that members of the winged-helix family of transcription factors have a role in myogenic differentiation and in remodeling processes of adult muscles that occur in response to physiological stimuli.
Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides.
Authors: Gulbins E , Coggeshall K M , Baier G , Telford D , Langlet C , Baier-Bitterlich G , Bonnefoy-Berard N , Burn P , Wittinghofer A , Altman A
Published: October 2015

We recently identified Vav as a Ras-activating guanine nucleotide exchange factor (GEF) stimulated by a T-cell antigen receptor-coupled protein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-independent alternative pathway of Vav activation. Phorbol ester, 1,2-diacylglycerol, or ceramide treatment of intact T cells, Vav immunoprecipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity of gel-purified Vav was similarly stimulated by phorbol myristate acetate (PMA). Stimulation was resistant to PTK and protein kinase C inhibitors but was blocked by calphostin, a PMA and diacylglycerol antagonist. In vitro-translated Vav lacking its cysteine-rich domain, or mutated at a single cysteine residue within this domain (C528A), was not stimulated by PMA but was fully activated by p56lck. This correlated with increased binding of radiolabeled phorbol ester to COS-1 cells expressing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PMA-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stimulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate production of lipid second messengers to Ras in hematopoietic cells.
A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin.
Authors: Lu C F , Kurjan J , Lipke P N
Published: October 2015

Saccharomyces cerevisiae alpha-agglutinin is a cell wall-anchored adhesion glycoprotein. The previously identified 140-kDa form, which contains a glycosyl-phosphatidylinositol (GPI) anchor (D. Wojciechowicz, C.-F. Lu, J. Kurjan, and P. N. Lipke, Mol. Cell. Biol. 13:2554-2563, 1993), and additional forms of 80, 150, 250 to 300, and > 300 kDa had the properties of intermediates in a transport and cell wall anchorage pathway. N glycosylation and additional modifications resulted in successive increases in size during transport. The 150- and 250- to 300-kDa forms were membrane associated and are likely to be intermediates between the 140-kDa form and a cell surface GPI-anchored form of > 300 kDa. A soluble form of > 300 kDa that lacked the GPI anchor had properties of a periplasmic intermediate between the plasma membrane form and the > 300-kDa cell wall-anchored form. These results constitute experimental support for the hypothesis that GPI anchors act to localize alpha-agglutinin to the plasma membrane and that cell wall anchorage involves release from the GPI anchor to produce a periplasmic intermediate followed by linkage to the cell wall.
Murine pluripotent hematopoietic progenitors constitutively expressing a normal erythropoietin receptor proliferate in response to erythropoietin without preferential erythroid cell differentiation.
Authors: Dubart A , Feger F , Lacout C , Goncalves F , Vainchenker W , Dumenil D
Published: October 2015

Erythropoietin (EPO) is a prime regulator of the growth and differentiation of erythroid blood cells. The EPO receptor (EPO-R) is expressed in late erythroid progenitors (mature BFU-E and CFU-E), and EPO induces proliferation and differentiation of these cells. By introducing, with a retroviral vector, a normal EPO-R cDNA into murine adult bone marrow cells, we showed that EPO is also able to induce proliferation in pluripotent progenitor cells. After 7 days of coculture with virus-producing cells, bone marrow cells were plated in methylcellulose culture in the presence of EPO, interleukin-3, or Steel factor alone or in combination. In the presence of EPO alone, EPO-R virus-infected bone marrow cells gave rise to mixed colonies comprising erythrocytes, granulocytes, macrophages and megakaryocytes. The addition of interleukin-3 or Steel factor to methylcellulose cultures containing EPO did not significantly modify the number of mixed colonies. The cells which generate these mixed colonies have a high proliferative potential as shown by the size and the ability of the mixed colonies to give rise to secondary colonies. Thus, it appears that EPO has the same effect on EPO-R-expressing multipotent cell proliferation as would a combination of several growth factors. Finally, our results demonstrate that inducing pluripotent progenitor cells to proliferate via the EPO signaling pathway has no major influence on their commitment.
Tyrosine 569 in the c-Fms juxtamembrane domain is essential for kinase activity and macrophage colony-stimulating factor-dependent internalization.
Authors: Myles G M , Brandt C S , Carlberg K , Rohrschneider L R
Published: October 2015

The receptor (Fms) for macrophage colony-stimulating factor (M-CSF) is a member of the tyrosine kinase class of growth factor receptors. It maintains survival, stimulates growth, and drives differentiation of the macrophage lineage of hematopoietic cells. Fms accumulates on the cell surface and becomes activated for signal transduction after M-CSF binding and is then internalized via endocytosis for eventual degradation in lysosomes. We have investigated the mechanism of endocytosis as part of the overall signaling process of this receptor and have identified an amino acid segment near the cytoplasmic juxtamembrane region surrounding tyrosine 569 that is important for internalization. Mutation of tyrosine 569 to alanine (Y569A) eliminates ligand-induced rapid endocytosis of receptor molecules. The mutant Fms Y569A also lacks tyrosine kinase activity; however, tyrosine kinase activity is not essential for endocytosis because the kinase inactive receptor Fms K614A does undergo ligand-induced endocytosis, albeit at a reduced rate. Mutation of tyrosine 569 to phenylalanine had no effect on the M-CSF-induced endocytosis of Fms, and a four-amino-acid sequence containing Y-569 could support endocytosis when transferred into the cytoplasmic juxtamembrane region of a glycophorin A construct. These results indicate that tyrosine 569 within the juxtamembrane region of Fms is part of a signal recognition sequence for endocytosis that does not require tyrosine phosphorylation at this site and that this domain also influences the kinase activity of the receptor. These results are consistent with a ligand-dependent step in recognition of the potential cryptic internalization signal.
Simian virus 40 origin- and T-antigen-dependent DNA replication with Drosophila factors in vitro.
Authors: Kamakaka R T , Kaufman P D , Stillman B , Mitsis P G , Kadonaga J T
Published: October 2015

DNA replication of double-stranded simian virus 40 (SV40) origin-containing plasmids, which has been previously thought to be a species-specific process that occurs only with factors derived from primate cells, is catalyzed with an extract derived from embryos of the fruit fly Drosophila melanogaster. This reaction is dependent upon both large T antigen, the SV40-encoded replication initiator protein and DNA helicase, and a functional T-antigen binding site at the origin of DNA replication. The efficiency of replication with extracts derived from Drosophila embryos is approximately 10% of that observed with extracts prepared from human 293 cells. This activity is not a unique property of embryonic extracts, as cytoplasmic extracts from Drosophila tissue culture cells also support T-antigen-mediated replication of SV40 DNA. By using highly purified proteins, DNA synthesis is initiated by Drosophila polymerase alpha-primase in a T-antigen-dependent manner in the presence of Drosophila replication protein A (RP-A; also known as single-stranded DNA-binding protein), but neither human RP-A nor Escherichia coli single-stranded DNA-binding protein could substitute for Drosophila RP-A. In reciprocal experiments, however, Drosophila RP-A was able to substitute for human RP-A in reactions carried out with human polymerase alpha-primase. These results collectively indicate that many of the specific functional interactions among T antigen, polymerase alpha-primase, and RP-A are conserved from primates to Drosophila species. Moreover, the observation that SV40 DNA replication can be performed with Drosophila factors provides a useful assay for the study of bidirectional DNA replication in Drosophila species in the context of a complete replication reaction.
A critical role for chromatin in mounting a synergistic transcriptional response to GAL4-VP16.
Authors: Chang C , Gralla J D
Published: October 2015

The role of chromatin in mounting a synergistic transcriptional response to GAL4-VP16 was investigated. Strong synergy was observed when chromatin templates were used in vitro. The synergy was severely reduced when naked DNA templates were transcribed. In vivo synergy was strong when nonreplicating templates were used. However, the use of replicating templates, which involved transient disruptions of chromatin, led to strong reductions in synergy. In both of these low-synergy responses, transcription levels were high. We infer that strong synergy has a requirement for chromatin that may be understood in terms of the competition between multiple activator molecules and histone cores for promoter DNA.
Peroxisome assembly factor 1: nonsense mutation in a peroxisome-deficient Chinese hamster ovary cell mutant and deletion analysis.
Authors: Tsukamoto T , Shimozawa N , Fujiki Y
Published: October 2015

A cDNA encoding 35-kDa peroxisome assembly factor 1 (PAF-1), a peroxisomal integral membrane protein, was cloned from Chinese hamster ovary (CHO) cells and sequenced. The CHO PAF-1 comprised 304 amino acids, one residue shorter than rat or human PAF-1, and showed high homology to rat and human PAF-1: 90 and 86% at the nucleotide sequence level and 92 and 90% in amino acid sequence, respectively. PAF-1 from these three species contains a conserved cysteine-rich sequence at the C-terminal region which is exactly the same as that of a novel cysteine-rich RING finger motif family. PAF-1 cDNA from a peroxisome-deficient CHO cell mutant, Z65 (T. Tsukamoto, S. Yokota, and Y. Fujiki, J. Cell Biol. 110:651-660, 1990), contained a nonsense mutation at the codon for Trp-114, resulting in premature termination. Truncation in PAF-1 of either 19 amino acids from the N terminus or 92 residues from the C terminus maintained the peroxisome assembly-restoring activity when tested in both the Z65 mutant and the fibroblasts from a Zellweger patient. In contrast, deletion of 27 or 102 residues from the N or C terminus eliminated the activity. PAF-1 is encoded by free polysomal RNA, consistent with a general rule for biogenesis of peroxisomal proteins, including membrane polypeptides, implying the posttranslational transport and integration of PAF-1 into peroxisomal membrane.
Stress induction of the mammalian GRP78/BiP protein gene: in vivo genomic footprinting and identification of p70CORE from human nuclear extract as a DNA-binding component specific to the stress regulatory element.
Authors: Li W W , Sistonen L , Morimoto R I , Lee A S
Published: October 2015

GRP78, also known as BiP, is one of the better-characterized molecular chaperones. It has been implicated in protein folding and also calcium sequestration in the endoplasmic reticulum. When the cells are subjected to endoplasmic reticulum stress, in particular the depletion of stored calcium and/or the accumulation of abnormal proteins, the rate of transcription of grp78 is enhanced. Previous studies have shown that the core region of the rat grp78 promoter (-170 to -135), which is 95% conserved with the human grp78 core (-133 to -98), is one of the key regulatory elements. Using ligation-mediated PCR, we have found that there are specific changes in factor occupancy after stress induction and the major changes occur within a cluster of bases located in the 3' half of the grp core, whereas other regulatory elements are constitutively occupied. This inducible binding to the 3' half of the human grp78 core region is observed under diverse stress signals, suggesting a common mechanism for the grp stress response. Nonetheless, the lack of constitutive in vivo protection at this region is not due to the absence of a binding factor in nuclear extracts. Using in vitro gel mobility shift assays, we detected a constitutive binding activity which exhibits specificity and affinity to the stress-inducible region. Through sodium dodecyl sulfate-polyacrylamide gel electrophoresis size fractionation and renaturation analysis, the activity is found in polypeptides with molecular sizes of 65 to 75 kDa. After a three-step purification scheme including core affinity column chromatography, we purified p70CORE, which is about 70 kDa in its monomeric form. The purified p70CORE is sufficient to form a complex specific to the stress-inducible region.
GATA-binding proteins regulate the human gonadotropin alpha-subunit gene in the placenta and pituitary gland.
Authors: Steger D J , Hecht J H , Mellon P L
Published: October 2015

The human glycoprotein hormone alpha-subunit gene is expressed in two quite dissimilar tissues, the placenta and anterior pituitary. Tissue-specific expression is determined by combinations of elements, some of which are common and others of which are specific to each tissue. In the placenta, a composite enhancer confers specific expression. It contains four protein-binding sites: two cyclic AMP (cAMP) response elements that bind CREB, a trophoblast-specific element that binds TSEB, and a sequence motif, AGATAA, that matches the consensus binding site for a family of transcription factors termed the GATA-binding proteins. In pituitary gonadotropes, the cAMP response elements remain important for expression, TSEB is absent, and elements further upstream participate in tissue-specific expression. Here we establish a regulatory role for the GATA element in both the placenta and pituitary by demonstrating that a mutation of this element decreases alpha-subunit gene expression 15-fold in JEG-3 human placental cells and 2.5-fold in alpha T3-1 mouse pituitary gonadotropes. In JEG-3 cells, human GATA-2 (hGATA-2) and hGATA-3 are highly expressed and both proteins bind to the alpha-subunit gene GATA element. In alpha T3-1 cells, the GATA motif is bound by mouse GATA-2 (mGATA-2) and an mGATA-4-related protein. Cotransfection of hGATA-2 or hGATA-3 into alpha T3-1 cells activates the alpha-subunit gene threefold. These studies establish a role for the GATA-binding proteins in placental and pituitary alpha-subunit gene expression, significantly expanding the known target genes of GATA-2, GATA-3, and perhaps GATA-4.
Cutaneous and pleural involvement in a patient with multiple myeloma
Authors: Saidane Olfa , Slouma Maroua , Haouet Slim , Abdelmoula Leila
Published: October 2015

Multiple myeloma (MM) is a malignant proliferation of a single clone of plasma cells and an excess of monoclonal immunoglobulin production. It is rarely associated with cutaneous and pleural involvement. We report a new case of a 62-year-old woman with a history of a symptomatic MM. Three months after chemotherapy initiation, she presented with subcutaneous nodules. Ultrasound-guided needle biopsy confirmed the diagnosis of cutaneous plasmacytomas. She underwent local radiation therapy leading to complete regression of subcutaneous nodules. One month later, she developed dyspnoea. Thoracic CT scan showed pleural thickening associated with pleural effusion. Pleural biopsy confirmed the diagnosis of pleural plasmacytoma. Chemotherapy including vincristine, doxorubicin and dexamethasone was administered. Cutaneous involvement and pleural effusion accompanying MM are uncommon. They are associated with poor prognosis.
Multiple embolic cerebral infarcts as the first manifestation of metastatic ovarian cancer
Authors: Bond Laura M , Skrobo Darko
Published: October 2015

A 36-year-old woman presented to the emergency department with a 3-day history of an occipital headache associated with transient visual impairment and short-term memory loss. MRI of the brain showed innumerable focal embolic infarcts of differing ages, for which a cause could not be determined. The patient was discharged and readmitted 7 weeks later with acute aphasia and a right-sided hemiplegia. CT of the abdomen revealed a right-sided ovarian mass and prominent retroperitoneal nodes, which cytology confirmed to be metastatic ovarian cancer.