bluuebunny/crossref_metadata_2025 huggingface dataset:
Secretagogue-triggered Transfer of Membrane Proteins from Neuroendocrine Secretory Granules to Synaptic-like Microvesicles
Authors: Strasser Jane E. , Arribas Monica , Blagoveshchenskaya Anastasia D. , Cutler Daniel F.
Published: July 2013

The membrane proteins of all regulated secretory organelles (RSOs) recycle after exocytosis. However, the recycling of those membrane proteins that are targeted to both dense core granules (DCGs) and synaptic-like microvesicles (SLMVs) has not been addressed. Since neuroendocrine cells contain both RSOs, and the recycling routes that lead to either organelle overlap, transfer between the two pools of membrane proteins could occur during recycling. We have previously demonstrated that a chimeric protein containing the cytosolic and transmembrane domains of P-selectin coupled to horseradish peroxidase is targeted to both the DCG and the SLMV in PC12 cells. Using this chimera, we have characterized secretagogue-induced traffic in PC12 cells. After stimulation, this chimeric protein traffics from DCGs to the cell surface, internalizes into transferrin receptor (TFnR)-positive endosomes and thence to a population of secretagogue-responsive SLMVs. We therefore find a secretagogue-dependent rise in levels of HRP within SLMVs. In addition, the levels within SLMVs of the endogenous membrane protein, synaptotagmin, as well as a green fluorescent protein-tagged version of vesicle-associated membrane protein (VAMP)/synaptobrevin, also show a secretagogue-dependent increase.
The Werner Syndrome Protein Is Involved in RNA Polymerase II Transcription
Authors: Balajee Adayabalam S. , Machwe Amrita , May Alfred , Gray Matthew D. , Oshima Junko , Martin George M. , Nehlin Jan O. , Brosh Robert , Orren David K. , Bohr Vilhelm A.
Published: July 2013

Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)–dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40–60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II–dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid–protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype.
Molecular Interaction Map of the Mammalian Cell Cycle Control and DNA Repair Systems
Authors: Kohn Kurt W.
Published: July 2013

Eventually to understand the integrated function of the cell cycle regulatory network, we must organize the known interactions in the form of a diagram, map, and/or database. A diagram convention was designed capable of unambiguous representation of networks containing multiprotein complexes, protein modifications, and enzymes that are substrates of other enzymes. To facilitate linkage to a database, each molecular species is symbolically represented only once in each diagram. Molecular species can be located on the map by means of indexed grid coordinates. Each interaction is referenced to an annotation list where pertinent information and references can be found. Parts of the network are grouped into functional subsystems. The map shows how multiprotein complexes could assemble and function at gene promoter sites and at sites of DNA damage. It also portrays the richness of connections between the p53-Mdm2 subsystem and other parts of the network.
Division of Labor among the α6β4 Integrin, β1 Integrins, and an E3 Laminin Receptor to Signal Morphogenesis and β-Casein Expression in Mammary Epithelial Cells
Authors: Muschler John , Lochter André , Roskelley Calvin D. , Yurchenco Peter , Bissell Mina J.
Published: July 2013

Contact of cultured mammary epithelial cells with the basement membrane protein laminin induces multiple responses, including cell shape changes, growth arrest, and, in the presence of prolactin, transcription of the milk protein β-casein. We sought to identify the specific laminin receptor(s) mediating the multiple cell responses to laminin. Using assays with clonal mammary epithelial cells, we reveal distinct functions for the α6β4 integrin, β1 integrins, and an E3 laminin receptor. Signals from laminin for β-casein expression were inhibited in the presence of function-blocking antibodies against both the α6 and β1 integrin subunits and by the laminin E3 fragment. The α6-blocking antibody perturbed signals mediated by the α6β4 integrin, and the β1-blocking antibody perturbed signals mediated by another integrin, the α subunit(s) of which remains to be determined. Neither α6- nor β1-blocking antibodies perturbed the cell shape changes resulting from cell exposure to laminin. However, the E3 laminin fragment and heparin both inhibited cell shape changes induced by laminin, thereby implicating an E3 laminin receptor in this function. These results elucidate the multiplicity of cell-extracellular matrix interactions required to integrate cell structure and signaling and ultimately permit normal cell function.
Insulin-induced Stimulation of Na+,K+-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10
Authors: Féraille Eric , Carranza Maria Luisa , Gonin Sandrine , Béguin Pascal , Pedemonte Carlos , Rousselot Martine , Caverzasio Joseph , Geering Käthi , Martin Pierre-Yves , Favre Hervé
Published: July 2013

Phosphorylation of the α-subunit of Na+,K+-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na+,K+-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na+,K+-ATPase α-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the α-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat α-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive86Rb uptake in opossum kidney cells expressing mutant rat α1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive86Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na+,K+-ATPase α-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.
Import into and Degradation of Cytosolic Proteins by Isolated Yeast Vacuoles
Authors: Horst Martin , Knecht Erwin C. , Schu Peter V.
Published: July 2013

In eukaryotic cells, both lysosomal and nonlysosomal pathways are involved in degradation of cytosolic proteins. The physiological condition of the cell often determines the degradation pathway of a specific protein. In this article, we show that cytosolic proteins can be taken up and degraded by isolated Saccharomyces cerevisiae vacuoles. After starvation of the cells, protein uptake increases. Uptake and degradation are temperature dependent and show biphasic kinetics. Vacuolar protein import is dependent on cytosolic heat shock proteins of the hsp70 family and on protease-sensitive component(s) on the outer surface of vacuoles. Degradation of the imported cytosolic proteins depends on a functional vacuolar ATPase. We show that the cytosolic isoform of yeast glyceraldehyde-3-phosphate dehydrogenase is degraded via this pathway. This import and degradation pathway is reminiscent of the protein transport pathway from the cytosol to lysosomes of mammalian cells.
Rad18 Is Required for DNA Repair and Checkpoint Responses in Fission Yeast
Authors: Verkade Heather M. , Bugg Sarah J. , Lindsay Howard D. , Carr Anthony M. , O’Connell Matthew J.
Published: July 2013

To survive damage to the genome, cells must respond by activating both DNA repair and checkpoint responses. Using genetic screens in the fission yeast Schizosaccharomyces pombe, we recently isolated new genes required for DNA damage checkpoint control. We show here that one of these strains defines a new allele of the previously described rad18 gene, rad18-74. rad18 is an essential gene, even in the absence of extrinsic DNA damage. It encodes a conserved protein related to the structural maintenance of chromosomes proteins. Point mutations in rad18 lead to defective DNA repair pathways responding to both UV-induced lesions and, as we show here, double-stranded breaks. Furthermore, rad18p is required to maintain cell cycle arrest in the presence of DNA damage, and failure of this leads to highly aberrant mitoses. A gene encoding a BRCT-containing protein, brc1, was isolated as an allele-specific high-copy suppressor of rad18-74. brc1is required for mitotic fidelity and for cellular viability in strains with rad18 mutations but is not essential for DNA damage responses. Mutations in rad18 and brc1are synthetically lethal with a topoisomerase II mutant (top2-191), indicating that these proteins play a role in chromatin organization. These studies show a role for chromatin organization in the maintenance or activation of responses to DNA damage.
A Nuclear Protein Involved in Apoptotic-like DNA Degradation inStylonychia: Implications for Similar Mechanisms in Differentiating and Starved Cells
Authors: Maercker Christian , Kortwig Heike , Nikiforov Mikhail A. , Allis C. David , Lipps Hans J.
Published: July 2013

Ciliates are unicellular eukaryotic organisms containing two types of nuclei: macronuclei and micronuclei. After the sexual pathway takes place, a new macronucleus is formed from a zygote nucleus, whereas the old macronucleus is degraded and resorbed. In the course of macronuclear differentiation, polytene chromosomes are synthesized that become degraded again after some hours. Most of the DNA is eliminated, and the remaining DNA is fragmented into small DNA molecules that are amplified to a high copy number in the new macronucleus. The protein Pdd1p (programmed DNA degradation protein 1) fromTetrahymena has been shown to be present in macronuclear anlagen in the DNA degradation stage and also in the old macronuclei, which are resorbed during the formation of the new macronucleus. In this study the identification and localization of a Pdd1p homologous protein in Stylonychia (Spdd1p) is described. Spdd1p is localized in the precursor nuclei in the DNA elimination stage and in the old macronuclei during their degradation, but also in macronuclei and micronuclei of starved cells. In all of these nuclei, apoptotic-like DNA breakdown was detected. These data suggest that Spdd1p is a general factor involved in programmed DNA degradation inStylonychia.
The Rib43a Protein Is Associated with Forming the Specialized Protofilament Ribbons of Flagellar Microtubules inChlamydomonas
Authors: Norrander Jan M. , deCathelineau Aimee M. , Brown Jennifer A. , Porter Mary E. , Linck Richard W.
Published: July 2013

Ciliary and flagellar microtubules contain a specialized set of three protofilaments, termed ribbons, that are composed of tubulin and several associated proteins. Previous studies of sea urchin sperm flagella identified three of the ribbon proteins astektins, which form coiled-coil filaments in doublet microtubules and which are associated with basal bodies and centrioles. To study the function of tektins and other ribbon proteins in the assembly of flagella and basal bodies, we have begun an analysis of ribbons from the unicellular biflagellate, Chlamydomonas reinhardtii, and report here the molecular characterization of the ribbon protein rib43a. Using antibodies against rib43a to screen an expression library, we recovered a full-length cDNA clone that encodes a 42,657-Da polypeptide. On Northern blots, the rib43a cDNA hybridized to a 1.7-kb transcript, which was up-regulated upon deflagellation, consistent with a role for rib43a in flagellar assembly. The cDNA was used to isolate RIB43a, an ∼4.6-kb genomic clone containing the complete rib43a coding region, and restriction fragment length polymorphism analysis placed the RIB43agene on linkage group III. Sequence analysis of theRIB43a gene indicates that the substantially coiled-coil rib43a protein shares a high degree of sequence identity with clones from Trypanosoma cruzi and Homo sapiens(genomic, normal fetal kidney, and endometrial and germ cell tumors) but little sequence similarity to other proteins including tektins. Affinity-purified antibodies against native and bacterially expressed rib43a stained both flagella and basal bodies by immunofluorescence microscopy and stained isolated flagellar ribbons by immuno-electron microscopy. The structure of rib43a and its association with the specialized protofilament ribbons and with basal bodies is relevant to the proposed role of ribbons in forming and stabilizing doublet and triplet microtubules and in organizing their three-dimensional structure.
Dissociation from BiP and Retrotranslocation of Unassembled Immunoglobulin Light Chains Are Tightly Coupled to Proteasome Activity
Authors: Chillarón Josep , Haas Ingrid G.
Published: July 2013

Unassembled immunoglobulin light chains expressed by the mouse plasmacytoma cell line NS1 (κNS1) are degraded in vivo with a half-life of 50–60 min in a way that closely resembles endoplasmic reticulum (ER)-associated degradation ( Knittler et al., 1995 ). Here we show that the peptide aldehydes MG132 and PS1 and the specific proteasome inhibitor lactacystin effectively increased the half-life of κNS1, arguing for a proteasome-mediated degradation pathway. Subcellular fractionation and protease protection assays have indicated an ER localization of κNS1upon proteasome inhibition. This was independently confirmed by the analysis of the folding state of κNS1and size fractionation experiments showing that the immunoglobulin light chain remained bound to the ER chaperone BiP when the activity of the proteasome was blocked. Moreover, kinetic studies performed in lactacystin-treated cells revealed a time-dependent increase in the physical stability of the BiP–κNS1complex, suggesting that additional proteins are present in the older complex. Together, our data support a model for ER-associated degradation in which both the release of a soluble nonglycosylated protein from BiP and its retrotranslocation out of the ER are tightly coupled with proteasome activity.
ATPase-defective Mammalian VPS4 Localizes to Aberrant Endosomes and Impairs Cholesterol Trafficking
Authors: Bishop Naomi , Woodman Philip
Published: July 2013

The yeast vacuolar sorting protein Vps4p is an ATPase required for endosomal trafficking that couples membrane association to its ATPase cycle. To investigate the function of mammalian VPS4 in endosomal trafficking, we have transiently expressed wild-type or ATPase-defective human VPS4 (hVPS4) in cultured cells. Wild-type hVPS4 was cytosolic, whereas a substantial fraction of hVPS4 that was unable to either bind or hydrolyze ATP was localized to membranes, including those of specifically induced vacuoles. Vacuoles were exclusively endocytic in origin, and subsets of enlarged vacuoles stained with markers for each stage of the endocytic pathway. Sorting of receptors from the early endosome to the recycling compartment or to thetrans-Golgi network was not significantly affected, and no mutant hVPS4 associated with these compartments. However, many hVPS4-induced vacuoles were substantially enriched in cholesterol relative to the endosomal compartments of untransfected cells, indicating that expression of mutant hVPS4 gives rise to a kinetic block in postendosomal cholesterol sorting. The phenotype described here is largely consistent with the defects in vacuolar sorting associated with class E vps mutants in yeast, and a role for mammalian VPS4 is discussed in this context.
Vps52p, Vps53p, and Vps54p Form a Novel Multisubunit Complex Required for Protein Sorting at the Yeast Late Golgi
Authors: Conibear Elizabeth , Stevens Tom H.
Published: July 2013

The late Golgi of the yeast Saccharomyces cerevisiaereceives membrane traffic from the secretory pathway as well as retrograde traffic from post-Golgi compartments, but the machinery that regulates these vesicle-docking and fusion events has not been characterized. We have identified three components of a novel protein complex that is required for protein sorting at the yeast late Golgi compartment. Mutation of VPS52, VPS53, orVPS54 results in the missorting of 70% of the vacuolar hydrolase carboxypeptidase Y as well as the mislocalization of late Golgi membrane proteins to the vacuole, whereas protein traffic through the early part of the Golgi complex is unaffected. Avps52/53/54 triple mutant strain is phenotypically indistinguishable from each of the single mutants, consistent with the model that all three are required for a common step in membrane transport. Native coimmunoprecipitation experiments indicate that Vps52p, Vps53p, and Vps54p are associated in a 1:1:1 complex that sediments as a single peak on sucrose velocity gradients. This complex, which exists both in a soluble pool and as a peripheral component of a membrane fraction, colocalizes with markers of the yeast late Golgi by immunofluorescence microscopy. Together, the phenotypic and biochemical data suggest that VPS52, VPS53, andVPS54 are required for the retrograde transport of Golgi membrane proteins from an endosomal/prevacuolar compartment. The Vps52/53/54 complex joins a growing list of distinct multisubunit complexes that regulate membrane-trafficking events.
Functions and Functional Domains of the GTPase Cdc42p
Authors: Kozminski Keith G. , Chen Ann J. , Rodal Avital A. , Drubin David G.
Published: July 2013

Cdc42p, a Rho family GTPase of the Ras superfamily, is a key regulator of cell polarity and morphogenesis in eukaryotes. Using 37 site-directed cdc42 mutants, we explored the functions and interactions of Cdc42p in the budding yeast Saccharomyces cerevisiae. Cytological and genetic analyses of thesecdc42 mutants revealed novel and diverse phenotypes, showing that Cdc42p possesses at least two distinct essential functions and acts as a nodal point of cell polarity regulation in vivo. In addition, mapping the functional data for each cdc42mutation onto a structural model of the protein revealed as functionally important a surface of Cdc42p that is distinct from the canonical protein-interacting domains (switch I, switch II, and the C terminus) identified previously in members of the Ras superfamily. This region overlaps with a region (α5-helix) recently predicted by structural models to be a specificity determinant for Cdc42p-protein interactions.
The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes
Authors: Antonin Wolfram , Holroyd Claudia , Tikkanen Ritva , Höning Stefan , Jahn Reinhard
Published: July 2013

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.
Phosphorylation-dependent Localization of Microtubule-associated Protein MAP2c to the Actin Cytoskeleton
Authors: Ozer Rachel S. , Halpain Shelley
Published: July 2013

Microtubule-associated protein 2 (MAP2) is a neuronal phosphoprotein that promotes net microtubule growth and actin cross-linking and bundling in vitro. Little is known about MAP2 regulation or its interaction with the cytoskeleton in vivo. Here we investigate the in vivo function of three specific sites of phosphorylation on MAP2. cAMP-dependent protein kinase activity disrupts the MAP2–microtubule interaction in living HeLa cells and promotes MAP2c localization to peripheral membrane ruffles enriched in actin. cAMP-dependent protein kinase phosphorylates serines within three KXGS motifs, one within each tubulin-binding repeat. These highly conserved motifs are also found in homologous proteins tau and MAP4. Phosphorylation at two of these sites was detected in brain tissue. Constitutive phosphorylation at these sites was mimicked by single, double, and triple mutations to glutamic acid. Biochemical and microscopy-based assays indicated that mutation of a single residue was adequate to disrupt the MAP2–microtubule interaction in HeLa cells. Double or triple point mutation promoted MAP2c localization to the actin cytoskeleton. Specific association between MAP2c and the actin cytoskeleton was demonstrated by retention of MAP2c–actin colocalization after detergent extraction. Specific phosphorylation states may enhance the interaction of MAP2 with the actin cytoskeleton, thereby providing a regulated mechanism for MAP2 function within distinct cytoskeletal domains.
Role for the Silencing Protein Dot1 in Meiotic Checkpoint Control
Authors: San-Segundo Pedro A. , Roeder G. Shirleen
Published: July 2013

During the meiotic cell cycle, a surveillance mechanism called the “pachytene checkpoint” ensures proper chromosome segregation by preventing meiotic progression when recombination and chromosome synapsis are defective. The silencing protein Dot1 (also known as Pch1) is required for checkpoint-mediated pachytene arrest of thezip1 and dmc1 mutants ofSaccharomyces cerevisiae. In the absence ofDOT1, the zip1 and dmc1mutants inappropriately progress through meiosis, generating inviable meiotic products. Other components of the pachytene checkpoint include the nucleolar protein Pch2 and the heterochromatin component Sir2. Indot1, disruption of the checkpoint correlates with the loss of concentration of Pch2 and Sir2 in the nucleolus. In addition to its checkpoint function, Dot1 blocks the repair of meiotic double-strand breaks by a Rad54-dependent pathway of recombination between sister chromatids. In vegetative cells, mutation ofDOT1 results in delocalization of Sir3 from telomeres, accounting for the impaired telomeric silencing in dot1.
Conditional Expression of a Truncated Fragment of Nonmuscle Myosin II-A Alters Cell Shape but Not Cytokinesis in HeLa Cells
Authors: Wei Qize , Adelstein Robert S.
Published: July 2013

A truncated fragment of the nonmuscle myosin II-A heavy chain (NMHC II-A) lacking amino acids 1–591, ΔN592, was used to examine the cellular functions of this protein. Green fluorescent protein (GFP) was fused to the amino terminus of full-length human NMHC II-A, NMHC II-B, and ΔN592 and the fusion proteins were stably expressed in HeLa cells by using a conditional expression system requiring absence of doxycycline. The HeLa cell line studied normally expressed only NMHC II-A and not NMHC II-B protein. Confocal microscopy indicated that the GFP fusion proteins of full-length NMHC II-A, II-B, and ΔN592 were localized to stress fibers. However, in vitro assays showed that baculovirus-expressed ΔN592 did not bind to actin, suggesting that ΔN592 was localized to actin stress fibers through incorporation into endogenous myosin filaments. There was no evidence for the formation of heterodimers between the full-length endogenous nonmuscle myosin and truncated nonmuscle MHCs. Expression of ΔN592, but not full-length NMHC II-A or NMHC II-B, induced cell rounding with rearrangement of actin filaments and disappearance of focal adhesions. These cells returned to their normal morphology when expression of ΔN592 was repressed by addition of doxycycline. We also show that GFP-tagged full-length NMHC II-A or II-B, but not ΔN592, were localized to the cytokinetic ring during mitosis, indicating that, in vertebrates, the amino-terminus part of mammalian nonmuscle myosin II may be necessary for localization to the cytokinetic ring.
Wpkci, Encoding an Altered Form ofPKCI, Is Conserved Widely on the Avian W Chromosome and Expressed in Early Female Embryos: Implication of Its Role in Female Sex Determination
Authors: Hori Tetsuya , Asakawa Shuichi , Itoh Yuichiro , Shimizu Nobuyoshi , Mizuno Shigeki
Published: July 2013

Two W chromosome–linked cDNA clones, p5fm2 and p5fm3, were obtained from a subtracted (female minus male) cDNA library prepared from a mixture of undifferentiated gonads and mesonephroi of male or female 5-d (stages 26–28) chicken embryos. These two clones were demonstrated to be derived from the mRNA encoding an altered form of PKC inhibitor/interacting protein (PKCI), and its gene was namedWpkci. The Wpkci gene reiterated ∼40 times tandemly and located at the nonheterochromatic end of the chicken W chromosome. The W linkage and the moderate reiteration ofWpkci were conserved widely in Carinatae birds. The chicken PKCI gene, chPKCI, was shown to be a single-copy gene located near the centromere on the long arm of the Z chromosome. Deduced amino acid sequences of Wpkci and chPKCI showed ∼65% identity. In the deduced sequence of Wpkci, the HIT motif, which is essential for PKCI function, was absent, but the α-helix region, which was conserved among the PKCI family, and a unique Leu- and Arg-rich region, were present. Transcripts from bothWpkci and chPKCI genes were present at significantly higher levels in 3- to 6-d (stages 20–29) embryos. These transcripts were detected in several embryonic tissues, including undifferentiated left and right gonads. When the green fluorescent protein–fused form of Wpkci was expressed in male chicken embryonic fibroblast, it was located almost exclusively in the nucleus. A model is presented suggesting that Wpkci may be involved in triggering the differentiation of ovary by interfering with PKCI function or by exhibiting its unique function in the nuclei of early female embryos.
Sequential Activation of ERK and Repression of JNK by Scatter Factor/Hepatocyte Growth Factor in Madin-Darby Canine Kidney Epithelial Cells
Authors: Paumelle Réjane , Tulasne David , Leroy Catherine , Coll Jean , Vandenbunder Bernard , Fafeur Véronique
Published: July 2013

The scattering of Madin-Darby canine kidney (MDCK) epithelial cells by scatter factor/hepatocyte growth factor (SF/HGF) is associated with transcriptional induction of the urokinase gene, which occurs essentially through activation of an EBS/AP1 response element. We have investigated the signal transduction pathways leading to this transcriptional response. We found that SF/HGF induces rapid and sustained phosphorylation of the extracellular signal-regulated kinase (ERK) MAPK while stimulating weakly and then repressing phosphorylation of the JUN N-terminal kinase (JNK) MAPK for several hours. This delayed repression of JNK was preceded by phosphorylation of the MKP2 phosphatase, and both MKP2 induction and JNK dephosphorylation were under the control of MEK, the upstream kinase of ERK. ERK and MKP2 stimulate the EBS/AP1-dependent transcriptional response to SF/HGF, but not JNK, which inhibits this response. We further demonstrated that depending on cell density, the RAS-ERK-MKP2 pathway controls this transrepressing effect of JNK. Together, these data demonstrate that in a sequential manner SF/HGF activates ERK and MKP2, which in turn dephosphorylates JNK. This sequence of events provides a model for efficient cell scattering by SF/HGF at low cell density.
Colony-stimulating Factor-1 Receptor Utilizes Multiple Signaling Pathways to Induce Cyclin D2 Expression
Authors: Dey Arunangsu , She Hongyun , Kim Leopold , Boruch Allan , Guris Deborah L. , Carlberg Kristen , Sebti Saı̈d M. , Woodley David T. , Imamoto Akira , Li Wei
Published: July 2013

Colony-stimulating factor-1 (CSF-1) induces expression of immediate early gene, such as c-myc and c-fos and delayed early genes such as D-type cyclins (D1 and D2), whose products play essential roles in the G1 to S phase transition of the cell cycle. Little is known, however, about the cytoplasmic signal transduction pathways that connect the surface CSF-1 receptor to these genes in the nucleus. We have investigated the signaling mechanism of CSF-1-induced D2 expression. Analyses of CSF-1 receptor autophosphorylation mutants show that, although certain individual mutation has a partial inhibitory effect, only multiple combined mutations completely block induction of D2 in response to CSF-1. We report that at least three parallel pathways, the Src pathway, the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, and the c-myc pathway, are involved. Induction of D2 is partially inhibited in Src−/− bone marrow-derived macrophages and by Src inhibitor PP1 and is enhanced in v-Src-overexpressing cells. Activation of myc's transactivating activity selectively induces D2 but not D1. Blockade of c-myc expression partially blocks CSF-1-induced D2 expression. Complete inhibition of the MEK/ERK pathway causes 50% decrease of D2 expression. Finally, simultaneous inhibition of Src, MEK activation, and c-myc expression additively blocks CSF-1-induced D2 expression. This study indicates that multiple signaling pathways are involved in full induction of a single gene, and this finding may also apply broadly to other growth factor-inducible genes.
Mutational Analysis Suggests That Activation of the Yeast Pheromone Response Mitogen-activated Protein Kinase Pathway Involves Conformational Changes in the Ste5 Scaffold Protein
Authors: Sette Claudio , Inouye Carla J. , Stroschein Shannon L. , Iaquinta Phillip J. , Thorner Jeremy
Published: July 2013

Ste5 is essential for pheromone response and binds components of a mitogen-activated protein kinase (MAPK) cascade: Ste11 (MEKK), Ste7 (MEK), and Fus3 (MAPK). Pheromone stimulation releases Gβγ (Ste4-Ste18), which recruits Ste5 and Ste20 (p21-activated kinase) to the plasma membrane, activating the MAPK cascade. A RING-H2 domain in Ste5 (residues 177–229) negatively regulates Ste5 function and mediates its interaction with Gβγ. Ste5(C177A C180A), carrying a mutated RING-H2 domain, cannot complement a ste5Δ mutation, yet supports mating even in ste4Δ ste5Δ cells when artificially dimerized by fusion to glutathioneS-transferase (GST). In contrast, wild-type Ste5 fused to GST permits mating of ste5Δ cells, but does not allow mating of ste4Δ ste5Δ cells. This differential behavior provided the basis of a genetic selection for STE5 gain-of-function mutations. MATaste4Δ ste5Δ cells expressing Ste5-GST were mutagenized chemically and plasmids conferring the capacity to mate were selected. Three independent single-substitution mutations were isolated. These constitutive STE5 alleles induce cell cycle arrest, transcriptional activation, and morphological changes normally triggered by pheromone, even when Gβγ is absent. The first, Ste5(C226Y), alters the seventh conserved position in the RING-H2 motif, confirming that perturbation of this domain constitutively activates Ste5 function. The second, Ste5(P44L), lies upstream of a basic segment, whereas the third, Ste5(S770K), is situated within an acidic segment in a region that contacts Ste7. None of the mutations increased the affinity of Ste5 for Ste11, Ste7, or Fus3. However, the positions of these novel-activating mutations suggested that, in normal Ste5, the N terminus may interact with the C terminus. Indeed, in vitro, GST-Ste5(1-518) was able to associate specifically with radiolabeled Ste5(520-917). Furthermore, both the P44L and S770K mutations enhanced binding of full-length Ste5 to GST-Ste5(1-518), whereas they did not affect Ste5 dimerization. Thus, binding of Gβγ to the RING-H2 domain may induce a conformational change that promotes association of the N- and C-terminal ends of Ste5, stimulating activation of the MAPK cascade by optimizing orientation of the bound kinases and/or by increasing their accessibility to Ste20-dependent phosphorylation (or both). In accord with this model, the novel Ste5 mutants copurified with Ste7 and Fus3 in their activated state and their activation required Ste20.
A Conserved Interaction between Moe1 and Mal3 Is Important for Proper Spindle Formation inSchizosaccharomyces pombe
Authors: Chen Chang-Rung , Chen Jing , Chang Eric C.
Published: July 2013

Moe1 is a conserved fission yeast protein that negatively affects microtubule stability/assembly. We conducted a two-hybrid screen to search for Moe1-binding proteins and isolated Mal3, a homologue of human EB1. We show that Moe1 and Mal3 expressed in bacteria form a complex and that Moe1 and Mal3 expressed in fission yeast cosediment with microtubules. Deletion of either moe1 ormal3 does not result in lethality; however, deletion of both moe1 and mal3 leads to cell death in the cold. The resulting cells appear to die of chromosome missegregation, which correlates with the presence of abnormal spindles. We investigated the cause for the formation of monopolar spindles and found that only one of the two spindle pole bodies (SPBs) contains γ-tubulin, although both SPBs appear to be equal in size and properly inserted in the nuclear membrane. Moreover, the moe1 mal3 double null mutant in the cold contains abnormally short and abundant interphase microtubule bundles. These data suggest that Moe1 and Mal3 play a role in maintaining proper microtubule dynamics/integrity and distribution of γ-tubulin to the SPBs during mitosis. Finally, we show that human Moe1 and EB1 can each rescue the phenotype of the moe1 mal3 double null mutant and form a complex, suggesting that these proteins are part of a well-conserved mechanism for regulating spindle functioning.
Distinct FTDP-17 Missense Mutations in Tau Produce Tau Aggregates and Other Pathological Phenotypes in Transfected CHO Cells
Authors: Vogelsberg-Ragaglia Vanessa , Bruce Jennifer , Richter-Landsberg Christiane , Zhang Bin , Hong Ming , Trojanowski John Q. , Lee Virginia M.-Y.
Published: July 2013

Multiple tau gene mutations are pathogenic for hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), with filamentous tau aggregates as the major lesions in the CNS of these patients. Recent studies have shown that bacterially expressed recombinant tau proteins with FTDP-17 missense mutations cause functional impairments, i.e., a reduced ability of mutant tau to bind to or promote the assembly of microtubules. To investigate the biological consequences of FTDP-17 tau mutants and assess their ability to form filamentous aggregates, we engineered Chinese hamster ovary cell lines to stably express tau harboring one or several different FTDP-17 mutations and showed that different tau mutants produced distinct pathological phenotypes. For example, ΔK, but not several other single tau mutants (e.g., V337 M, P301L, R406W), developed insoluble amorphous and fibrillar aggregates, whereas a triple tau mutant (VPR) containing V337M, P301L, and R406W substitutions also formed similar aggregates. Furthermore, the aggregates increased in size over time in culture. Significantly, the formation of aggregated ΔK and VPR tau protein correlated with reduced affinity of these mutants to bind microtubules. Reduced phosphorylation and altered proteolysis was also observed in R406W and ΔK tau mutants. Thus, distinct pathological phenotypes, including the formation of insoluble filamentous tau aggregates, result from the expression of different FTDP-17 tau mutants in transfected Chinese hamster ovary cells and implies that these missense mutations cause diverse neurodegenerative FTDP-17 syndromes by multiple mechanisms.
Human Orthologs of Yeast Vacuolar Protein Sorting Proteins Vps26, 29, and 35: Assembly into Multimeric Complexes
Authors: Haft Carol Renfrew , Sierra Maria de la Luz , Bafford Richard , Lesniak Maxine A. , Barr Valarie A. , Taylor Simeon I.
Published: July 2013

Sorting nexin (SNX) 1 and SNX2 are mammalian orthologs of Vps5p, a yeast protein that is a subunit of a large multimeric complex, termed the retromer complex, involved in retrograde transport of proteins from endosomes to the trans-Golgi network. We report the cloning and characterization of human orthologs of three additional components of the complex: Vps26p, Vps29p, and Vps35p. The close structural similarity between the yeast and human proteins suggests a similarity in function. We used both yeast two-hybrid assays and expression in mammalian cells to define the binding interactions among these proteins. The data suggest a model in which hVps35 serves as the core of a multimeric complex by binding directly to hVps26, hVps29, and SNX1. Deletional analyses of hVps35 demonstrate that amino acid residues 1–53 and 307–796 of hVps35 bind to the coiled coil-containing domain of SNX1. In contrast, hVps26 binds to amino acid residues 1–172 of hVps35, whereas hVps29 binds to amino acid residues 307–796 of hVps35. Furthermore, hVps35, hVps29, and hVps26 have been found in membrane-associated and cytosolic compartments. Gel filtration chromatography of COS7 cell cytosol showed that both recombinant and endogenous hVps35, hVps29, and hVps26 coelute as a large complex (∼220–440 kDa). In the absence of hVps35, neither hVps26 nor hVps29 is found in the large complex. These data provide the first insights into the binding interactions among subunits of a putative mammalian retromer complex.
Expression and Partial Characterization of Kinesin-related Proteins in Differentiating and Adult Skeletal Muscle
Authors: Ginkel Laura M. , Wordeman Linda
Published: July 2013

Using pan-kinesin antibodies to screen a differentiating C2C12 cell library, we identified the kinesin proteins KIF3A, KIF3B, and conventional kinesin heavy chain to be present in differentiating skeletal muscle. We compared the expression and subcellular localization characteristics of these kinesins in myogenic cells to others previously identified in muscle, neuronal, and mitotic systems (KIF1C, KIF3C, and mitotic-centromere-associated kinesin). Because members of the KIF3 subfamily of kinesin-related proteins showed altered subcellular fractionation characteristics in differentiating cells, we focused our study of kinesins in muscle on the function of kinesin-II. Kinesin-II is a motor complex comprised of dimerized KIF3A and KIF3B proteins and a tail-associated protein, KAP. The Xenopus homologue of KIF3B, Xklp3, is predominantly localized to the region of the Golgi apparatus, and overexpression of motorless-Xklp3 in Xenopus A6 cells causes mislocalization of Golgi components ( Le Bot et al., 1998 ). In C2C12 myoblasts and myotubes, KIF3B is diffuse and punctate, and not primarily associated with the Golgi. Overexpression of motorless-KIF3B does not perturb localization of Golgi components in myogenic cells, and myofibrillogenesis is normal. In adult skeletal muscle, KIF3B colocalizes with the excitation–contraction-coupling membranes. We propose that these membranes, consisting of the transverse-tubules and sarcoplasmic reticulum, are dynamic structures in which kinesin-II may function to actively assemble and maintain in myogenic cells.
Endosome to Golgi Transport of Ricin Is Regulated by Cholesterol
Authors: Grimmer Stine , Iversen Tore-Geir , van Deurs Bo , Sandvig Kirsten
Published: July 2013

We have here studied the role of cholesterol in transport of ricin from endosomes to the Golgi apparatus. Ricin is endocytosed even when cells are depleted for cholesterol by using methyl-β-cyclodextrin (mβCD). However, as here shown, the intracellular transport of ricin from endosomes to the Golgi apparatus, measured by quantifying sulfation of a modified ricin molecule, is strongly inhibited when the cholesterol content of the cell is reduced. On the other hand, increasing the level of cholesterol by treating cells with mβCD saturated with cholesterol (mβCD/chol) reduced the intracellular transport of ricin to the Golgi apparatus even more strongly. The intracellular transport routes affected include both Rab9-independent and Rab9-dependent pathways to the Golgi apparatus, since both sulfation of ricin after induced expression of mutant Rab9 (mRab9) to inhibit late endosome to Golgi transport and sulfation of a modified mannose 6-phosphate receptor (M6PR) were inhibited after removal or addition of cholesterol. Furthermore, the structure of the Golgi apparatus was affected by increased levels of cholesterol, as visualized by pronounced vesiculation and formation of smaller stacks. Thus, our results indicate that transport of ricin from endosomes to the Golgi apparatus is influenced by the cholesterol content of the cell.
Head and/or CaaX Domain Deletions of Lamin Proteins Disrupt Preformed Lamin A and C But Not Lamin B Structure in Mammalian Cells
Authors: Izumi Masako , Vaughan O. Anthony , Hutchison Christopher J. , Gilbert David M.
Published: July 2013

The nuclear lamina is an important determinant of nuclear architecture. Mutations in A-type but not B-type lamins cause a range of human genetic disorders, including muscular dystrophy. Dominant mutations in nuclear lamin proteins have been shown to disrupt a preformed lamina structure in Xenopus egg extracts. Here, a series of deletion mutations in lamins A and B1 were evaluated for their ability to disrupt lamina structure in Chinese hamster ovary cells. Deletions of either the lamin A “head” domain or the C-terminal CaaX domain formed intranuclear aggregates and resulted in the disruption of endogenous lamins A/C but not lamins B1/B2. By contrast, “head-less” lamin B1 localized to the nuclear rim with no detectable effect on endogenous lamins, whereas lamin B1 CaaX domain deletions formed intranuclear aggregates, disrupting endogenous lamins A/C but not lamins B1/B2. Filter binding assays revealed that a head/CaaX domain lamin B1 mutant interacted much more strongly with lamins A/C than with lamins B1/B2. Regulated induction of this mutant in stable cell lines resulted in the rapid elimination of all detectable lamin A protein, whereas lamin C was trapped in a soluble form within the intranuclear aggregates. In contrast to results in Xenopus egg extracts, dominant negative lamin B1 (but not lamin A) mutants trapped replication proteins involved in both the initiation and elongation phases of replication but did not effect cellular growth rates or the assembly of active replication centers. We conclude that elimination of the CaaX domain in lamin B1 and elimination of either the CaaX or head domain in lamin A constitute dominant mutations that can disrupt A-type but not B-type lamins, highlighting important differences in the way that A- and B-type lamins are integrated into the lamina.
Dual Requirement for Rho and Protein Kinase C in Direct Activation of Phospholipase D1 Through G Protein-coupled Receptor Signaling
Authors: Du Guangwei , Altshuller Yelena M. , Kim Yong , Han Jung Min , Ryu Sung Ho , Morris Andrew J. , Frohman Michael A.
Published: July 2013

G protein-coupled and tyrosine kinase receptor activation of phospholipase D1 (PLD1) play key roles in agonist-stimulated cellular responses such as regulated exocytosis, actin stress fiber formation, and alterations in cell morphology and motility. Protein Kinase C, ADP-ribosylation factor (ARF), and Rho family members activate PLD1 in vitro; however, the actions of the stimulators on PLD1 in vivo have been proposed to take place through indirect pathways. We have used the yeast split-hybrid system to generate PLD1 alleles that fail to bind to or to be activated by RhoA but that retain wild-type responses to ARF and PKC. These alleles then were employed in combination with alleles unresponsive to PKC or to both stimulators to examine the activation of PLD1 by G protein-coupled receptors. Our results demonstrate that direct stimulation of PLD1 in vivo by RhoA (and by PKC) is critical for significant PLD1 activation but that PLD1 subcellular localization and regulated phosphorylation occur independently of these stimulatory pathways.
Rab7: A Key to Lysosome Biogenesis
Authors: Bucci Cecilia , Thomsen Peter , Nicoziani Paolo , McCarthy Janice , van Deurs Bo
Published: July 2013

The molecular machinery behind lysosome biogenesis and the maintenance of the perinuclear aggregate of late endocytic structures is not well understood. A likely candidate for being part of this machinery is the small GTPase Rab7, but it is unclear whether this protein is associated with lysosomes or plays any role in the regulation of the perinuclear lysosome compartment. Previously, Rab7 has mainly been implicated in transport from early to late endosomes. We have now used a new approach to analyze the role of Rab7: transient expression of Enhanced Green Fluorescent Protein (EGFP)–tagged Rab7 wt and mutant proteins in HeLa cells. EGFP-Rab7 wt was associated with late endocytic structures, mainly lysosomes, which aggregated and fused in the perinuclear region. The size of the individual lysosomes as well as the degree of perinuclear aggregation increased with the expression levels of EGFP-Rab7 wt and, more dramatically, the active EGFP-Rab7Q67L mutant. In contrast, upon expression of the dominant-negative mutants EGFP-Rab7T22N and EGFP-Rab7N125I, which localized mainly to the cytosol, the perinuclear lysosome aggregate disappeared and lysosomes, identified by colocalization of cathepsin D and lysosome-associated membrane protein–1, became dispersed throughout the cytoplasm, they were inaccessible to endocytosed molecules such as low-density lipoprotein, and their acidity was strongly reduced, as determined by decreased accumulation of the acidotropic probe LysoTracker Red. In contrast, early endosomes associated with Rab5 and the transferrin receptor, late endosomes enriched in the cation-independent mannose 6-phosphate receptor, and thetrans-Golgi network, identified by its enrichment in TGN-38, were unchanged. These data demonstrate for the first time that Rab7, controlling aggregation and fusion of late endocytic structures/lysosomes, is essential for maintenance of the perinuclear lysosome compartment.