bluuebunny/crossref_metadata_2025 huggingface dataset:
Requirement of the adenovirus E1A transformation domain 1 for inhibition of PC12 cell neuronal differentiation.
Authors: Heasley L E , Benedict S , Gleavy J , Johnson G L
Published: August 2013

Expression of the adenovirus early gene E1A inhibits the nerve growth factor (NGF)-induced differentiation of PC12 pheochromocytoma cells. Expression of the 12S form of E1A, which lacks the transcription activation region, also inhibited PC12 cell differentiation in a manner similar to the wild-type gene. Three cellular proteins--the retinoblastoma susceptibility gene product referred to as 105(Rb)-, 107-, and 300-kDa proteins--stably interacted with the different E1A polypeptides. Analysis of the association of these cellular proteins with mutant E1A polypeptides demonstrated that a functional domain 1, which is minimally involved in the association of the 300-kDa protein with E1A, was sufficient to inhibit neuronal differentiation. Deletion of transformation domain 2, which encodes sequences necessary for the binding of the 105(Rb)- and 107-kDa proteins, did not influence the ability of the mutant E1A polypeptide to inhibit PC12 cell differentiation. E1A was also shown to alter the expression of mRNAs for the early response genes c-fos, c-myc, egr-1, and c-jun and their regulation in response to NGF. In clones expressing either 12S or 13S E1A, NGF stimulation of c-fos and c-myc was repressed. In contrast, basal mRNA levels for c-jun and egr-1 were constitutively elevated and not significantly affected further by challenge with NGF. Simply expressing c-jun by gene transfer, however, did not mimic the action of E1A because constitutively expressing c-jun clones differentiated in response to NGF. Thus, expression of the E1A polypeptide disrupts NGF control of early transcription events that have been shown to be critical for PC12 cell neuronal differentiation.
Transforming growth factor-beta (TGF-beta) isoforms in rat liver regeneration: messenger RNA expression and activation of latent TGF-beta.
Authors: Jakowlew S B , Mead J E , Danielpour D , Wu J , Roberts A B , Fausto N
Published: August 2013

Expression of transforming growth factor-beta s (TGF-beta s) 1-3 was studied in normal liver and during liver regeneration after partial hepatectomy in the rat to determine whether each of these isoforms might be involved in hepatocyte growth in vivo. Expression of the mRNAs for all three TGF-beta isoforms increases in the regenerating liver. In addition, the levels of expression of the mRNAs for several extracellular matrix proteins, including fibronectin, vitronectin, laminin, and collagen, also increase in the regenerating liver. Immunohistochemical staining analysis shows a similar distribution of all three TGF-beta s in normal and regenerating liver; however, in both tissues, the level of expression of TGF-beta 1 is 8- to 10-fold higher than that of TGF-beta 2 as determined by sandwich enzyme-linked immunosorbent assay. Expression of all three TGF-beta mRNAs is restricted to liver nonparenchymal cells. Although hepatocytes from normal and regenerating livers do not synthesize TGF-beta, they are sensitive to inhibition of growth by all three TGF-beta isoforms. Hepatocytes from regenerating livers are capable of activating latent TGF-beta 1 complexes in vitro, whereas normal hepatocytes are not. The different TGF-beta isoforms may function in an inhibitory paracrine mechanism that is activated during liver regeneration and may also regulate the synthesis of extracellular matrix components in the regenerating liver.
Fission yeast sts1+ gene encodes a protein similar to the chicken lamin B receptor and is implicated in pleiotropic drug-sensitivity, divalent cation-sensitivity, and osmoregulation.
Authors: Shimanuki M , Goebl M , Yanagida M , Toda T
Published: August 2013

The Schizosaccharomyces pombe sts1+ gene, identified by supersensitive mutations to a protein kinase inhibitor, staurosporine, was isolated by complementation by the use of a fission yeast genomic library. Nucleotide sequencing shows that the sts1+ gene encodes a 453 amino acid putative membrane-associated protein that is significantly similar (26% identity) to the chicken lamin B receptor. It is also highly related (53% identity) to a budding yeast ORF, YGL022. These three proteins contain a similar hydrophobicity pattern consisting of eight or nine putative transmembrane domains. By gene disruption we demonstrate that the sts1+ gene is not essential for viability. These disruptants exhibit pleiotropic defects, such as cold-sensitivity for growth and at the permissive temperature, a supersensitivity to divalent cations and several unrelated drugs including staurosporine, caffeine, chloramphenicol, sorbitol, and SDS. Disruption of the sts1+ gene does not lead to a sensitivity to thiabendazole or hydroxyurea.
In vivo import of firefly luciferase into the glycosomes of Trypanosoma brucei and mutational analysis of the C-terminal targeting signal.
Authors: Sommer J M , Cheng Q L , Keller G A , Wang C C
Published: August 2013

The compartmentalization of glycolytic enzymes into specialized organelles, the glycosomes, allows the bloodstream form of Trypanosoma brucei to rely solely on glycolysis for its energy production. The biogenesis of glycosomes in these parasites has been studied intensively as a potential target for chemotherapy. We have adapted the recently developed methods for stable transformation of T. brucei to the in vivo analysis of glycosomal protein import. Firefly luciferase, a peroxisomal protein in the lantern of the insect, was expressed in stable transformants of the procyclic form of T. brucei, where it was found to accumulate inside the glycosomes. Mutational analysis of the peroxisomal targeting signal serine-lysine-leucine (SKL) located at the C-terminus of luciferase showed that replacement of the serine residue (Serine548) with a small neutral amino acid (A, C, G, H, N, P, T) still resulted in an import efficiency of 50-100% of the wild-type luciferase. Lysine549 could be substituted with an amino acid capable of hydrogen bonding (H, M, N, Q, R, S), whereas the C-terminal leucine550 could be replaced with a subset of hydrophobic amino acids (I, M, Y). Thus, a peroxisome-like C-terminal SKL-dependent targeting mechanism may function in T. brucei to import luciferase into the glycosomes. However, a few significant differences exist between the glycosomal targeting signals identified here and the tripeptide sequences that direct proteins to mammalian or yeast peroxisomes.
Disruption of epithelial cell-cell adhesion by exogenous expression of a mutated nonfunctional N-cadherin.
Authors: Fujimori T , Takeichi M
Published: August 2013

Cadherins, a family of transmembrane cell-cell adhesion receptors, require interactions with the cytoskeleton for normal function. To assess the mechanisms of these interactions, we studied the effect of exogenous expression of a mutant N-cadherin, cN390 delta; on epithelial cell-cell adhesion. The intracellular domain of cN390 delta was intact but its extracellular domain was largely deleted so that this molecule was not functional for cell adhesion. cDNA of cN390 delta was attached to the metallothionein promoter, and introduced into the keratinocyte line PAM212 expressing endogenous E- and P-cadherin. When the expression of cN390 delta was induced by Zn2+, cadherin-dependent adhesion of the transfected cells was inhibited, resulting in the dispersion of cell colonies, although their contacts were maintained under high cell density conditions. In these cultures, cN390 delta was expressed not only on the free surfaces of the cells but also at cell-cell junctions. The endogenous cadherins were concentrated at cell-cell junctions under normal conditions. As a result of cN390 delta expression, however, the endogenous cadherins localizing at the cell-cell junctions were largely diminished, suggesting that these molecules were replaced by the mutant molecules at these sites. As a control, we transfected the same cell line with cDNA of a truncated form of N-cadherin cadherin whose intracellular C terminus had been deleted leaving the extracellular domain intact. This molecule had no effect on cell-cell adhesion, nor did it localize to cell-cell contact sites. We also found that the association of the endogenous cadherins with alpha- and beta-catenins and plakoglobin was not affected by the expression of cN390 delta, which also formed a complex with these molecules, suggesting that no competition occurred between the endogenous and exogenous cadherins for these cytoplasmic proteins. These and other additional results suggest that the nonfunctional cadherins whose intracellular domain is intact occupy the sites where the endogenous cadherins should localize, through interactions with the cytoskeleton, and inhibit the cadherin adhesion system.
Phosphorylation sites at the C-terminus of the platelet-derived growth factor receptor bind phospholipase C gamma 1.
Authors: Kashishian A , Cooper J A
Published: August 2013

We have identified two tyrosine phosphorylation sites, Tyr 1009 and Tyr 1021, in the C-terminal noncatalytic region of the human platelet-derived growth factor (PDGF) receptor beta subunit. Mutant receptors with phenylalanine substitutions at either or both of these tyrosines were expressed in dog epithelial cells. Mutation of Tyr 1021 markedly reduced the PDGF-stimulated binding of phospholipase C (PLC) gamma 1 but had no effect on binding of the GTPase activator protein of Ras or of phosphatidylinositol 3 kinase. Mutation of Tyr 1009 reduced binding of PLC gamma 1 less severely. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, also reduced the PDGF-dependent binding of a transiently expressed fusion protein containing the two Src-homology 2 domains from PLC gamma 1. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, greatly reduced PDGF-stimulated tyrosine phosphorylation of PLC gamma 1 but did not prevent the tyrosine phosphorylation of other cell proteins, including mitogen-activated protein kinase. We conclude that Tyr 1021, and possibly Tyr 1009, is a binding site for PLC gamma 1.
Suppression of yeast geranylgeranyl transferase I defect by alternative prenylation of two target GTPases, Rho1p and Cdc42p.
Authors: Ohya Y , Qadota H , Anraku Y , Pringle J R , Botstein D
Published: August 2013

Geranylgeranyl transferase I (GGTase I), which modifies proteins containing the sequence Cys-Ali-Ali-Leu (Ali: aliphatic) at their C-termini, is indispensable for growth in the budding yeast Saccharomyces cerevisiae. We report here that GGTase I is no longer essential when Rho1p and Cdc42p are simultaneously overproduced. The lethality of a GGTase I deletion is most efficiently suppressed by provision of both Rho1p and Cdc42p with altered C-terminal sequences (Cys-Ali-Ali-Met) corresponding to the C-termini of substrates of farnesyl transferase (FTase). Under these circumstances, the FTase, normally not essential for growth of yeast, becomes essential.
Basic fibroblast growth factor modulates integrin expression in microvascular endothelial cells.
Authors: Klein S , Giancotti F G , Presta M , Albelda S M , Buck C A , Rifkin D B
Published: August 2013

During angiogenesis capillary endothelial cells undergo a coordinated set of modifications in their interactions with extracellular matrix components. In this study we have investigated the effect of the prototypical angiogenic factor basic fibroblast growth factor (bFGF) on the expression and function of several integrins in microvascular endothelial cells. Immunoprecipitation experiments with antibodies to individual subunits indicated that microvascular cells express at their surface several integrins. These include the alpha 1 beta 1, alpha 2 beta 1, and alpha 3 beta 1 laminin/collagen receptors; the alpha 6 beta 1 laminin receptor; the alpha 5 beta 1 and alpha v beta 1 fibronectin receptors; the alpha 6 beta 4 basement membrane receptor; and the alpha v beta 3 and alpha v beta 5 vitronectin receptors. Treatment with bFGF caused a significant increase in the surface expression of the alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha 6 beta 4, and alpha v beta 5 integrins. In contrast, the level of expression of the alpha 1 beta 1 and alpha v beta 3 integrins was decreased in bFGF-treated cells. Immunoprecipitation of metabolically labeled cells indicated that bFGF increases the biosynthesis of the alpha 3, alpha 5, alpha 6, beta 4, and beta 5 subunits and decreases the production of the alpha v and beta 3 subunits. These results suggest that bFGF modulates integrin expression by altering the biosynthesis of individual alpha or beta subunits. In accordance with the upregulation of several integrins observed in bFGF-treated cells, these cells adhered better to fibronectin, laminin, vitronectin, and type I collagen than did untreated cells. The largest differences in beta 1 integrin expression occurred approximately 72 h after exposure to bFGF, at a time when the expression of the endothelial cell-to-cell adhesion molecule endoCAM was also significantly upregulated. In contrast, a shorter exposure to bFGF (24-48 h) was required for the maximal induction of plasminogen activator production in the same cells. Taken together, these results show that bFGF causes significant changes in the level of expression and function of several integrins in microvascular endothelial cells.
The 170-kDa glucose-regulated stress protein is an endoplasmic reticulum protein that binds immunoglobulin.
Authors: Lin H Y , Masso-Welch P , Di Y P , Cai J W , Shen J W , Subjeck J R
Published: August 2013

Anoxia, glucose starvation, calcium ionophore A23187, EDTA, glucosamine, and several other conditions that adversely affect the function of the endoplasmic reticulum (ER) induce the synthesis of the glucose-regulated class of stress proteins (GRPs). The primary GRPs induced by these stresses migrate at 78 and 94 kDa (GRP78 and GRP94). In addition, another protein of approximately 150-170 kDa (GRP170) has been previously observed and is coordinately induced with GRP78 and GRP94. To characterize this novel stress protein, we have prepared an antisera against purified GRP170. Immunofluorescence, Endoglycosidase H sensitivity, and protease resistance of this protein in microsomes indicates that GRP170 is an ER lumenal glycoprotein retained in a pre-Golgi compartment. Immunoprecipitation of GRP170 with our antibody coprecipitates the GRP78 (also referred to as the B cell immunoglobulin-binding protein) and GRP94 members of this stress protein family in Chinese hamster ovary cells under stress conditions. ATP depletion, by immunoprecipitation in the presence of apyrase, does not affect the interaction between GRP78 and GRP170 but results in the coprecipitation of an unidentified 60-kDa protein. In addition, GRP170 is found to be coprecipitated with immunoglobulin (Ig) in four different B cell hybridomas expressing surface IgM, cytoplasmic Ig light chain only, cytoplasmic Ig heavy chain only, or an antigen specific secreted IgG. In addition, in IgM surface expressing WEHI-231 B cells, anti-IgM coprecipitates GRP78, GRP94, as well as GRP170; antibodies against GRP170 and GRP94 reciprocally coprecipitate GRP94/GRP170 as well as GRP78. Results suggest that this 170-kDa GRP is a retained ER lumenal glycoprotein that is constitutively present and that may play a role in immunoglobulin folding and assembly in conjunction or consecutively with GRP78 and GRP94.
Rab GDI: a solubilizing and recycling factor for rab9 protein.
Authors: Soldati T , Riederer M A , Pfeffer S R
Published: August 2013

Rab proteins are thought to function in the processes by which transport vesicles identify and/or fuse with their respective target membranes. The bulk of these proteins are membrane associated, but a measurable fraction can be found in the cytosol. The cytosolic forms of rab3A, rab11, and Sec4 occur as equimolar complexes with a class of proteins termed "GDIs," or "GDP dissociation inhibitors." We show here that the cytosolic form of rab9, a protein required for transport between late endosomes and the trans Golgi network, also occurs as a complex with a GDI-like protein, with an apparent mass of approximately 80 kD. Complex formation could be reconstituted in vitro using recombinant rab9 protein, cytosol, ATP, and geranylgeranyl diphosphate, and was shown to require an intact rab9 carboxy terminus, as well as rab9 geranylgeranylation. Monoprenylation was sufficient for complex formation because a mutant rab9 protein bearing the carboxy terminal sequence, CLLL, was prenylated in vitro by geranylgeranyl transferase I and was efficiently incorporated into 80-kD complexes. Purified, prenylated rab9 could also assemble into 80-kD complexes by addition of purified, rab3A GDI. Finally, rab3A-GDI had the capacity to solubilize rab9GDP, but not rab9GTP, from cytoplasmic membranes. These findings support the proposal that GDI proteins serve to recycle rab proteins from their target membranes after completion of a rab protein-mediated, catalytic cycle. Thus GDI proteins have the potential to regulate the availability of specific intracellular transport factors.
Gradients in the concentration and assembly of myosin II in living fibroblasts during locomotion and fiber transport.
Authors: Kolega J , Taylor D L
Published: August 2013

Assembly and motor activity of myosin II affect shape, contractility, and locomotion of nonmuscle cells. We used fluorescent analogues and imaging techniques to elucidate the state of assembly and three-dimensional distribution of myosin II in living Swiss 3T3 fibroblasts. An analogue of myosin II that was covalently cross-linked in the 10S conformation and unable to assemble served as an indicator of the cytoplasmic volume accessible to 10S myosin II. Ratio-imaging of an analogue that can undergo 10S-->6S conversion versus the volume indicator revealed localized concentration of assembly-competent myosin II. In stationary serum-deprived cells and in cells locomoting at the edge of a wound, it was most concentrated in the peripheral cytoplasm, where fibers containing myosin II assemble, and least concentrated in the perinuclear cytoplasm, where they disassemble. Furthermore, fluorescence photobleaching recovery showed myosin II to be less mobile in the periphery than in perinuclear cytoplasm. These results indicate a gradient in the assembly of myosin II. Three-dimensional microscopy of living cells revealed that fibers containing myosin II were localized in the cortical cytoplasm, whereas myosin II was diffusely distributed in the deeper cytoplasm, suggesting that myosin II is assembled preferentially near the cell surface. Localized protein phosphorylation may play a role, because a kinase inhibitor, staurosporine, abolished the gradient of myosin II assembly.
PBP74, a new member of the mammalian 70-kDa heat shock protein family, is a mitochondrial protein.
Authors: Dahlseid J N , Lill R , Green J M , Xu X , Qiu Y , Pierce S K
Published: August 2013

The cloning of a cDNA encoding a new member of the highly conserved mammalian 70-kDa heat shock protein (hsp 70) family termed PBP74 was recently reported. Critical to an understanding of the function of this new hsp 70 is delineating its subcellular localization. Here we use a variety of immunological and biochemical approaches both in vitro and in vivo to demonstrate that PBP74 is imported into and resides in mitochondria. By confocal immunofluorescence microscopy PBP74 is detected in mitochondria, colocalizing with the mitochondrial 60-kDa heat shock protein. To address the inherent problem of serological cross-reactivity among the hsp70 family members, an influenza virus hemagglutinin epitope tag was introduced into the PBP74 cDNA. The epitope-tagged PBP74 protein transiently expressed in L cells localized to mitochondria. Moreover, deletion of the N-terminal 46-amino acid presequence results in a cytosolic localization of the epitope-tagged protein. Cell fractionation studies demonstrated PBP74 in purified mitochondria in a protease-protected location. After coupled transcription-translation the precursor of PBP74 is imported into isolated yeast mitochondria, where it becomes processed to the mature protein. According to a subfractionation of the mitochondria, the imported protein was found to be localized in the matrix space. Import in vitro is time- and temperature-dependent, requires matrix ATP, and is abolished upon depletion of the membrane potential across the mitochondrial inner membrane. Similarly, in mammalian cells PBP74 is synthesized as a pre-protein that requires membrane potential-dependent import into mitochondria for its maturation. Taken together, our data demonstrate that PBP74 is a mammalian mitochondrial hsp70.
In vitro assembly of coiled bodies in Xenopus egg extract.
Authors: Bauer D W , Murphy C , Wu Z , Wu C H , Gall J G
Published: August 2013

When demembranated sperm nuclei are placed in a Xenopus egg extract, they become surrounded by a nuclear envelope and then swell to form morphologically typical pronuclei. Granules ranging from < 1.0 to approximately 3.0 microns in diameter appear within such nuclei. Bell et al. identified four nucleolar proteins in these "prenucleolar bodies" by immunofluorescent staining (fibrillarin, nucleolin, B23/NO38, 180-kDa nucleolar protein). By in situ hybridization we show that these bodies also contain U3 and U8 small nuclear RNAs (snRNAs), known to be involved in pre-rRNA processing. Moreover, they contain all the snRNAs involved in pre-mRNA splicing (U1, U2, U4, U5, and U6), as well as U7, which is required for histone pre-mRNA 3' end formation. In addition to the nucleolar antigens previously identified, we demonstrated staining with antibodies against the Sm epitope, trimethylguanosine, and coilin. Because the composition of these prenucleolar bodies is closer to that of coiled bodies than to nucleoli, we propose that they be referred to as coiled bodies. The existence of large coiled bodies in transcriptionally inactive pronuclei suggests that they may play a role in the import, assembly, and storage of RNA processing components but are not themselves sites of processing. In transcriptionally active nuclei coiled bodies could serve as sites for initial preassembly and distribution of snRNP complexes for the three major RNA processing pathways: pre-mRNA splicing, pre-rRNA processing, and histone pre-mRNA 3' end formation.
Cell cycle regulation of a Xenopus Wee1-like kinase.
Authors: Mueller P R , Coleman T R , Dunphy W G
Published: August 2013

Using a polymerase chain reaction-based strategy, we have isolated a gene encoding a Wee1-like kinase from Xenopus eggs. The recombinant Xenopus Wee1 protein efficiently phosphorylates Cdc2 exclusively on Tyr-15 in a cyclin-dependent manner. The addition of exogenous Wee1 protein to Xenopus cell cycle extracts results in a dose-dependent delay of mitotic initiation that is accompanied by enhanced tyrosine phosphorylation of Cdc2. The activity of the Wee1 protein is highly regulated during the cell cycle: the interphase, underphosphorylated form of Wee1 (68 kDa) phosphorylates Cdc2 very efficiently, whereas the mitotic, hyperphosphorylated version (75 kDa) is weakly active as a Cdc2-specific tyrosine kinase. The down-modulation of Wee1 at mitosis is directly attributable to phosphorylation, since dephosphorylation with protein phosphatase 2A restores its kinase activity. During interphase, the activity of this Wee1 homolog does not vary in response to the presence of unreplicated DNA. The mitosis-specific phosphorylation of Wee1 is due to at least two distinct kinases: the Cdc2 protein and another activity (kinase X) that may correspond to an MPM-2 epitope kinase. These studies indicate that the down-regulation of Wee1-like kinase activity at mitosis is a multistep process that occurs after other biochemical reactions have signaled the successful completion of S phase.
Computer analysis of the binding reactions leading to a transmembrane receptor-linked multiprotein complex involved in bacterial chemotaxis.
Authors: Bray D , Bourret R B
Published: August 2013

The chemotactic response of bacteria is mediated by complexes containing two molecules each of a transmembrane receptor and the intracellular signaling proteins CheA and CheW. Mutants in which one or the other of the proteins of this complex are absent, inactive, or expressed at elevated amounts show altered chemotactic behavior and the phenotypes are difficult to interpret for some overexpression mutants. We have examined the possibility that these unexpected phenotypes might arise from the binding steps that lead to active complex formation. A limited genetic algorithm was used to search for sets of binding reactions and associated binding constants expected to give mutant phenotypes in accord with experimental data. Different sets of binding equilibria and different assumptions about the activity of particular receptor complexes were tried. Computer analysis demonstrated that it is possible to obtain sets of binding equilibria consistent with the observed phenotypes and provided a simple explanation for these phenotypes in terms of the distribution of active and inactive complexes formed under various conditions. Optimization methods of this kind offer a unique way to analyze reactions taking place inside living cells based on behavioral data.
end5, end6, and end7: mutations that cause actin delocalization and block the internalization step of endocytosis in Saccharomyces cerevisiae.
Authors: Munn A L , Stevenson B J , Geli M I , Riezman H
Published: August 2013

Four mutants defective in endocytosis were isolated by screening a collection of temperature-sensitive yeast mutants. Three mutations define new END genes: end5-1, end6-1, and end7-1. The fourth mutation is in END4, a gene identified previously. The end5-1, end6-1, and end7-1 mutations do not affect vacuolar protein localization, indicating that the defect in each mutant is specific for internalization at the plasma membrane. Interestingly, localization of actin patches on the plasma membrane is affected in each of the mutants. end5-1, end6-1, and end7-1 are allelic to VRP1, RVS161, and ACT1, respectively. VRP1 and RVS161 are required for correct actin localization and ACT1 encodes actin. To our surprise, the end6-1 mutation fails to complement the act1-1 mutation. Disruption of the RVS167 gene, which is homologous to END6/RVS161 and which is also required for correct actin localization, also blocks endocytosis. The end7-1 mutant allele has a glycine 48 to aspartic acid substitution in the DNase I-binding loop of actin. We propose that Vrp1p, Rvs161p, and Rvs167p are components of a cytoskeletal structure that contains actin and fimbrin and that is required for formation of endocytic vesicles at the plasma membrane.
A role for Hsp90 in retinoid receptor signal transduction.
Authors: Holley S J , Yamamoto K R
Published: August 2013

The ubiquitous heat shock protein Hsp90 appears to participate directly in the function of a broad range of cellular signal transduction components, including steroid hormone receptors; however, an evolutionarily related subclass of intracellular receptors, exemplified by the retinoid receptors RAR and RXR, had been inferred from biochemical studies to function independently of Hsp90. To examine this issue genetically, we measured mammalian and avian retinoid receptor activity in a Saccharomyces cerevisiae strain in which the expression of the yeast Hsp90 homologue could be conditionally repressed approximately 20-fold relative to wild type. We tested transcriptional activation by RAR or RXR-RAR, from two types of retinoic acid response elements, triggered by three different agonist ligands. In every condition, we found that activation was severely compromised under conditions of low Hsp90 expression. We showed that the defect was in signal transduction rather than transcription activation per se, and that high affinity hormone binding was abolished in extracts of cells producing low levels of Hsp90. We suggest that Hsp90 may function in at least one step of signal transduction by all members of the intracellular receptor superfamily.
Nuclear and nucleolar targeting of human ribosomal protein S6.
Authors: Schmidt C , Lipsius E , Kruppa J
Published: August 2013

Chimeric proteins were constructed to define the nuclear localization signals (NLSs) of human ribosomal protein S6. The complete cDNA sequence, different cDNA fragments and oligonucleotides of the human ribosomal proteins S6, respectively, were joined to the 5' end of the entire LacZ gene of Escherichia coli by using recombinant techniques. The hybrid genes were transfected into L cells, transiently expressed, and the intracellular location of the fusion proteins was determined by their beta-galactosidase activity. Three NLSs were identified in the C-terminal half of the S6 protein. Deletion mutagenesis demonstrated that a single NLS is sufficient for targeting the corresponding S6-beta-galactosidase chimera into the nucleus. Removal of all three putative NLSs completely blocked the nuclear import of the resulting S6-beta-galactosidase fusion protein, which instead became evenly distributed in the cytoplasm. Chimeras containing deletion mutants of S6 with at least one single NLS or unmodified S6 accumulated in the nucleolus. Analysis of several constructs reveals the existence of a specific domain that is essential but not sufficient for nucleolar accumulation of S6.
Domains of tau protein, differential phosphorylation, and dynamic instability of microtubules.
Authors: Trinczek B , Biernat J , Baumann K , Mandelkow E M , Mandelkow E
Published: August 2013

The dynamic instability of microtubules is thought to be regulated by MAPs and phosphorylation. Here we describe the effect of the neuronal microtubule-associated protein tau by observing the dynamics of single microtubules by video microscopy. We used recombinant tau isoforms and tau mutants, and we phosphorylated tau by the neuronal kinases MARK (affecting the KXGS motifs within tau's repeat domain) and cdk5 (phosphorylating Ser-Pro motifs in the regions flanking the repeats). The variants of tau can be broadly classified into three categories, depending on their potency to affect microtubule dynamics. "Strong" tau variants have four repeats and both flanking regions. "Medium" variants have one to three repeats and both flanking regions. "Weak" variants lack one or both of the flanking regions, or have no repeats; with such constructs, microtubule dynamics is not significantly different from that of pure tubulin. N- or C-terminal tails of tau have no influence on dynamic instability. The two ends of microtubules (plus and minus) showed different activities but analogous behavior. These results are consistent with the "jaws" model of tau where the flanking regions are considered as targeting domains whereas the addition of repeats makes them catalytically active in terms of microtubule stabilization. The dominant changes in the parameters of dynamic instability induced by tau are those in the dissociation rate and in the catastrophe rate (up to 30-fold). Other rates change only moderately or not at all (association rate increased up to twofold, rates of rescue or rapid shrinkage decreased up to approximately twofold). The order of repeats has little influence on microtubule dynamics (i.e., repeats can be re-arranged or interchanged), arguing in favor of the "distributed weak binding" model proposed by Butner and Kirschner (1991); however, we confirmed the presence of a "hotspot" of binding potential involving Lys274 and Lys281 observed by Goode and Feinstein, 1994. Phosphorylation of Ser-Pro motifs by cdk5 (mainly Ser 202, 235, and 404) in the flanking regions had a moderate effect on microtubule dynamics while phosphorylation at the "Alzheimer"-site Ser262 MARK eliminated tau's interactions with microtubules. In both cases the predominant effects of phosphorylation are on the rates of tubulin dissociation and catastrophe whereas the effects on the rates of association or rescue are comparatively small.
Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes.
Authors: Pestonjamasp K , Amieva M R , Strassel C P , Nauseef W M , Furthmayr H , Luna E J
Published: August 2013

Actin-binding proteins in bovine neutrophil plasma membranes were identified using blot overlays with 125I-labeled F-actin. Along with surface-biotinylated proteins, membranes were enriched in major actin-binding polypeptides of 78, 81, and 205 kDa. Binding was specific for F-actin because G-actin did not bind. Further, unlabeled F-actin blocked the binding of 125I-labeled F-actin whereas other acidic biopolymers were relatively ineffective. Binding also was specifically inhibited by myosin subfragment 1, but not by CapZ or plasma gelsolin, suggesting that the membrane proteins, like myosin, bind along the sides of the actin filaments. The 78- and 81-kDa polypeptides were identified as moesin and ezrin, respectively, by co-migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with antibodies specific for moesin and ezrin. Although not present in detectable amounts in bovine neutrophils, radixin (a third and closely related member of this gene family) also bound 125I-labeled F-actin on blot overlays. Experiments with full-length and truncated bacterial fusion proteins localized the actin-binding site in moesin to the extreme carboxy terminus, a highly conserved sequence. Immunofluorescence micrographs of permeabilized cells and cell "footprints" showed moesin co-localization with actin at the cytoplasmic surface of the plasma membrane, consistent with a role as a membrane-actin-linking protein.
Formation of nuclear bodies in hepatocytes of estrogen-treated roosters.
Authors: Ochs R L , Stein T W , Andrade L E , Gallo D , Chan E K , Tan E M , Brasch K
Published: August 2013

As a model for cellular growth and stimulation without accompanying proliferation, we have examined the induction and formation of nuclear bodies (NBs) in hepatocytes of estrogen-treated roosters. Four-week-old roosters were injected with a single intramuscular dose of estradiol and then killed at time points of 8 h, 48 h, and 4 wk post-injection. For immunofluorescence analyses, livers were excised and isolated hepatocyte nuclei were fixed and then labeled with antibody to the coiled body-specific protein, p80-coilin. In control animals (no estradiol) or in animals 8 h post-injection, each hepatocyte nucleus contained an average of 1.0 coiled body (CB), which appeared randomly distributed in the nucleoplasm. At 48 h post-injection, there were an average of 2.7 CBs/nucleus and many of these appeared to be in contact with the nucleolus. Pairs of CBs were also observed. By 4 wk post-injection an average of 1.5 CBs/nucleus were detected, with no apparent relationship to the nucleolus observed. By serial-section electron microscopy of intact livers, two different types of round NBs were observed, sometimes in close proximity to each other and to the expanded interchromatin granule region in maximally stimulated cells. One type of NB was a classical CB that averaged 0.35 microns in diameter and the other NB type was ring shaped, averaged 0.25 microns in diameter, was composed of a fibrous shell surrounding a hollow interior, and appeared as a simple NB when sectioned tangentially through its outer shell. Immunoelectron microscopy revealed that CBs were the only class of NBs that contained p80-coilin. From these data, we conclude that CBs proliferate in response to estrogen stimulation, possibly arising from the nucleolar surface and then increasing in number by replicative division.
The majority of yeast UPF1 co-localizes with polyribosomes in the cytoplasm.
Authors: Atkin A L , Altamura N , Leeds P , Culbertson M R
Published: August 2013

In Saccharomyces cerevisiae the UPF1 protein is required for nonsense-mediated mRNA decay, the accelerated turnover of mRNAs containing a nonsense mutation. Several lines of evidence suggest that translation plays an important role in the mechanism of nonsense mRNA decay, including a previous report that nonsense mRNAs assemble in polyribosomes. In this study we show that UPF1 and ribosomal protein L1 co-localize in the cytoplasm and that UPF1 co-sediments with polyribosomes. To detect UPF1, three copies of the influenza hemagglutinin epitope were placed at the C-terminus. The tagged protein, UPF1-3EP, retains 86% (+/- 5%) of function. Using immunological detection, we found that UPF1-3EP is primarily cytoplasmic and was not detected either in the nucleus or in the mitochondrion. UPF1-3EP and L1 co-distributed with polyribosomes fractionated in a 7-47% sucrose gradient. The sucrose sedimentation profiles for UPF1-3EP and L1 exhibited similar changes using three different sets of conditions that altered the polyribosome profile. When polyribosomes were disaggregated, UPF1-3EP and L1 accumulated in fractions coincident with 80S ribosomal particles. These results suggest that UPF1-3EP associates with polyribosomes. L3 and S3 mRNAs, which code for ribosomal proteins of the 60S and 40S ribosomal subunits, respectively, were on average about 100-fold more abundant than UPF1 mRNA. Assuming that translation rates for L3, S3, and UPF1 mRNA are similar, this result suggests that there are far fewer UPF1 molecules than ribosomes per cell. Constraints imposed by the low UPF1 abundance on the functional relationships between UPF1, polyribosomes, and nonsense mRNA turnover are discussed.
InsP3-induced Ca2+ excitability of the endoplasmic reticulum.
Authors: Keizer J , Li Y X , Stojilković S , Rinzel J
Published: August 2013

Oscillations in intracellular Ca2+ can be induced by a variety of cellular signalling processes (Woods et al., 1986; Berridge 1988; Jacob et al., 1988) and appear to play a role in secretion (Stojilković et al., 1994), fertilization (Miyazaki et al., 1993), and smooth muscle contraction (Iino and Tsukioka, 1994). Recently, great progress has been made in understanding the mechanisms involved in a particular class of Ca2+ oscillation, associated with the second messenger inositol 1,4,5-trisphosphate (InsP3) (Berridge, 1993). Working in concert with intracellular Ca2+, InsP3 controls Ca2+ release via the InsP3 receptor in the endoplasmic reticulum (ER) (Berridge and Irvine, 1989). The IP3 receptor is regulated by its coagonists InsP3 and Ca2+, which both activate and inhibit Ca2+ release (Finch et al., 1991; Bezprozvanny et al., 1991; De Young and Keizer, 1992). These processes, together with the periodic activation of Ca2+ uptake into the ER, have been identified as key features in the mechanism of InsP3-induced Ca2+ oscillations in pituitary gonadotrophs (Li et al., 1994), Xenopus laevis oocytes (Lechleiter and Clapham, 1992; Atri et al., 1993), and other cell types (Keizer and De Young, 1993). Earlier discussions and models of InsP3-induced Ca2+ oscillations focused on the nature and number of internal releasable pools of Ca2+ (Goldbeter et al., 1990; Swillens and Mercan, 1990; Somogyi and Stucki, 1991), the importance of oscillations in InsP3 (Meyer and Stryer, 1988), and other issues not based on detailed experimental findings in specific cells types.
Transient, lectin-like association of calreticulin with folding intermediates of cellular and viral glycoproteins.
Authors: Peterson J R , Ora A , Van P N , Helenius A
Published: August 2013

The soluble, calcium-binding protein calreticulin shares high sequence homology with calnexin, a transmembrane chaperone of glycoprotein folding. Our experiments demonstrated that calreticulin, like calnexin, associated transiently with numerous newly synthesized proteins in the endoplasmic reticulum. The population of proteins that bound to calreticulin was partially overlapping with those that bound to calnexin. Hemagglutinin (HA) of influenza virus was shown to associate with both calreticulin and calnexin. Using HA as a model substrate, it was found that both calreticulin- and calnexin-bound HA corresponded primarily to incompletely disulfide-bonded folding intermediates and conformationally trapped forms. Binding of all substrates was oligosaccharide-dependent and required the trimming of glucose residues from asparagine-linked core glycans by glucosidases I and II. In vitro, alpha-mannosidase digestion of calreticulin-bound HA indicated that calreticulin was specific for monoglucosylated glycans. Thus, calreticulin appeared to be a lectin with similar oligosaccharide specificity as its membrane-bound homologue, calnexin. Both are therefore likely to play an important role in glycoprotein maturation and quality control in the endoplasmic reticulum.
Serine/threonine phosphatase 1 modulates the subnuclear distribution of pre-mRNA splicing factors.
Authors: Misteli T , Spector D L
Published: August 2013

HeLa cell nuclei were permeabilized and reconstituted with nuclear extract to identify soluble nuclear factors which play a role in the organization of pre-mRNA splicing factors in the mammalian cell nucleus. Permeabilized nuclei reconstituted with nuclear extract were active in transcription and DNA replication and nuclear speckles containing pre-mRNA splicing factors were maintained over several hours independent of soluble nuclear components. The characteristic rounding up of nuclear speckles in response to inhibition of RNA polymerase II seen in vivo was reproduced in permeabilized cells and was strictly dependent on a catalytic activity present in the nuclear extract. By inhibitor titration experiments and sensitivity to inhibitor 2, this activity was identified as a member of the serine/threonine protein phosphatase 1 family (PP1). Interference with PP1 activity affected the distribution of pre-mRNA splicing factors in transcriptionally active, permeabilized cells, and excess PP1 activity caused increased dephosphorylation of SR proteins in nuclear speckles. These data show that the dynamic reorganization of the mammalian cell nucleus can be studied in permeabilized cells and that PP1 is involved in the rounding up of speckles as well as the overall organization of pre-mRNA splicing factors in the mammalian cell nucleus.
Induction of p18INK4c and its predominant association with CDK4 and CDK6 during myogenic differentiation.
Authors: Franklin D S , Xiong Y
Published: August 2013

Terminal cell differentiation involves permanent withdrawal from the cell division cycle. The inhibitors of cyclin-dependent kinases (CDKs) are potential molecules functioning to couple cell cycle arrest and cell differentiation. In murine C2C12 myoblast cells, G1 CDK enzymes (CDK2, CDK4, and CDK6) associate with four CDK inhibitors: p18INK4c, p19INK4d, p21, and p27Kip1. During induced myogenesis, p21 and its associated CDK proteins underwent an initial increase followed by a decrease as cells became terminally differentiated. The level of p27 protein gradually increased, but the amount of total associated CDK proteins remained unchanged. p19 protein decreased gradually during differentiation, as did its associated CDK4 protein. In contrast, p18 protein increased 50-fold, from negligible levels in proliferating myoblasts to clearly detectable levels within 8-12 h of myogenic induction. This initial rise was followed by a precipitous increase between 12 and 24 h postinduction, with p18 protein finally accumulating to its highest level in terminally differentiated cells. Induction of p18 correlated with increased and sequential complex formation--first increasing association with CDK6 and then with CDK4 over the course of myogenic differentiation. All of the CDK6 and half of the CDK4 were complexed with p18 in terminally differentiated C2C12 cells as well as in adult mouse muscle tissue. Finally, kinase activity of CDK2 and CDK4 decreases as C2C12 cells differentiate, whereas the CDK6 kinase activity is low in both proliferating myoblasts and differentiated myotubes. Our results indicate that p18 may play a critical role in causing and/or maintaining permanent cell cycle arrest associated with mature muscle formation.
ADM-1, a protein with metalloprotease- and disintegrin-like domains, is expressed in syncytial organs, sperm, and sheath cells of sensory organs in Caenorhabditis elegans.
Authors: Podbilewicz B
Published: August 2013

A search was carried out for homologues of possible fusogenic proteins to study their function in a genetically tractable animal. The isolation, molecular, and cellular characterization of the Caenorhabditis elegans adm-1 gene (a disintegrin and metalloprotease domain) are described. A glycoprotein analogous to viral fusion proteins has been identified on the surface of guinea pig sperm (PH-30/fertilin) and is implicated in sperm-egg fusion. adm-1 is the first reported invertebrate gene related to PH-30 and a family of proteins containing snake venom disintegrin- and metalloprotease-like domains. ADM-1 shows a domain organization identical to PH-30. It contains prepro, metalloprotease, disintegrin, cysteine rich with putative fusion peptide, epidermal growth factor-like repeat, transmembrane, and cytoplasmic domains. Antibodies which recognize ADM-1 protein in immunoblots were generated. Using immunofluorescence and in situ hybridization, the products of adm-1 have been detected in specific cells during different stages of development. The localization of ADM-1 to the plasma membrane of embryonic cells and to the sheath cells of sensory organs suggests a function in cell adhesion. ADM-1 expression in the hypodermis, pharynx, vulva, and mature sperm is consistent with a putative role in somatic and gamete cell fusions.
Identification and developmental regulation of a neuron-specific subunit of cytoplasmic dynein.
Authors: Pfister K K , Salata M W , Dillman J F , Torre E , Lye R J
Published: August 2013

Cytoplasmic dynein is the microtubule minus-end-directed motor for the retrograde axonal transport of membranous organelles. Because of its similarity to the intermediate chains of flagellar dynein, the 74-kDa intermediate chain (IC74) subunit of dynein is thought to be involved in binding dynein to its membranous organelle cargo. Previously, we identified six isoforms of the IC74 cytoplasmic dynein subunit in the brain. We further demonstrated that cultured glia and neurons expressed different dynein IC74 isoforms and phospho-isoforms. Two isoforms were observed when dynein from glia was analyzed. When dynein from cultured neurons was analyzed, six IC74 isoforms were observed, although the relative amounts of the dynein isoforms from cultured neurons differed from those found in dynein from brain. To better understand the role of the neuronal IC74 isoforms and identify neuron-specific IC74 dynein subunits, the expression of the IC74 protein isoforms and mRNAs of various tissues were compared. As a result of this comparison, the identity of each of the isoform spots observed on two-dimensional gels was correlated with the products of each of the IC74 mRNAs. We also found that between the fifteenth day of gestation (E15) and the fifth day after birth (P5), the relative expression of the IC74 protein isoforms changes, demonstrating that the expression of IC74 isoforms is developmentally regulated in brain. During this time period, there is relatively little change in the abundance of the various IC74 mRNAs. The E15 to P5 time period is one of rapid process extension and initial pattern formation in the rat brain. This result indicates that the changes in neuronal IC74 isoforms coincide with neuronal differentiation, in particular the extension of processes. This suggests a role for the neuronal IC74 isoforms in the establishment or regulation of retrograde axonal transport.