bluuebunny/crossref_metadata_2025 huggingface dataset:
MKBP, a Novel Member of the Small Heat Shock Protein Family, Binds and Activates the Myotonic Dystrophy Protein Kinase
Authors: Suzuki Atsushi , Sugiyama Yuki , Hayashi Yukiko , Nyu-i Nobuo , Yoshida Michihiko , Nonaka Ikuya , Ishiura Sho-ichi , Arahata Kiichi , Ohno Shigeo
Published: July 2002

Muscle cells are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses. The small heat shock proteins (sHSPs) such as αB-crystallin and HSP27, which are highly expressed in muscle cells, have been suggested to play roles in maintaining myofibrillar integrity against such stresses. Here, we identified a novel member of the sHSP family that associates specifically with myotonic dystrophy protein kinase (DMPK). This DMPK-binding protein, MKBP, shows a unique nature compared with other known sHSPs: (a) In muscle cytosol, MKBP exists as an oligomeric complex separate from the complex formed by αB-crystallin and HSP27. (b) The expression of MKBP is not induced by heat shock, although it shows the characteristic early response of redistribution to the insoluble fraction like other sHSPs. Immunohistochemical analysis of skeletal muscle cells shows that MKBP localizes to the cross sections of individual myofibrils at the Z-membrane as well as the neuromuscular junction, where DMPK has been suggested to be concentrated. In vitro, MKBP enhances the kinase activity of DMPK and protects it from heat-induced inactivation. These results suggest that MKBP constitutes a novel stress-responsive system independent of other known sHSPs in muscle cells and that DMPK may be involved in this system by being activated by MKBP. Importantly, since the amount of MKBP protein, but not that of other sHSP family member proteins, is selectively upregulated in skeletal muscle from DM patients, an interaction between DMPK and MKBP may be involved in the pathogenesis of DM.
NRP/B, a Novel Nuclear Matrix Protein, Associates With p110RB and Is Involved in Neuronal Differentiation
Authors: Kim Tae-Aug , Lim Jinkyu , Ota Setsuo , Raja Sandhya , Rogers Rick , Rivnay Benjamin , Avraham Hava , Avraham Shalom
Published: July 2002

The nuclear matrix is defined as the insoluble framework of the nucleus and has been implicated in the regulation of gene expression, the cell cycle, and nuclear structural integrity via linkage to intermediate filaments of the cytoskeleton. We have discovered a novel nuclear matrix protein, NRP/B (nuclear restricted protein/brain), which contains two major structural elements: a BTB domain–like structure in the predicted NH2 terminus, and a “kelch motif” in the predicted COOH-terminal domain. NRP/B mRNA (5.5 kb) is predominantly expressed in human fetal and adult brain with minor expression in kidney and pancreas. During mouse embryogenesis, NRP/B mRNA expression is upregulated in the nervous system. The NRP/B protein is expressed in rat primary hippocampal neurons, but not in primary astrocytes. NRP/B expression was upregulated during the differentiation of murine Neuro 2A and human SH-SY5Y neuroblastoma cells. Overexpression of NRP/B in these cells augmented neuronal process formation. Treatment with antisense NRP/B oligodeoxynucleotides inhibited the neurite development of rat primary hippocampal neurons as well as the neuronal process formation during neuronal differentiation of PC-12 cells. Since the hypophosphorylated form of retinoblastoma protein (p110RB) is found to be associated with the nuclear matrix and overexpression of p110RB induces neuronal differentiation, we investigated whether NRP/B is associated with p110RB. Both in vivo and in vitro experiments demonstrate that NRP/B can be phosphorylated and can bind to the functionally active hypophosphorylated form of the p110RB during neuronal differentiation of SH-SY5Y neuroblastoma cells induced by retinoic acid. Our studies indicate that NRP/B is a novel nuclear matrix protein, specifically expressed in primary neurons, that interacts with p110RB and participates in the regulation of neuronal process formation.
Dual Actions of Sphingosine-1-Phosphate: Extracellular through the Gi-coupled Receptor Edg-1 and Intracellular to Regulate Proliferation and Survival
Authors: Van Brocklyn James R. , Lee Menq-Jer , Menzeleev Ramil , Olivera Ana , Edsall Lisa , Cuvillier Olivier , Thomas Dianne M. , Coopman Peter J.P. , Thangada Shobha , Liu Catherine H. , Hla Timothy , Spiegel Sarah
Published: July 2002

Sphingosine-1-phosphate (SPP), a bioactive lipid, acts both intracellularly and extracellularly to cause pleiotropic biological responses. Recently, we identified SPP as a ligand for the G protein–coupled receptor Edg-1 (Lee, M.-J., J.R. Van Brocklyn, S. Thangada, C.H. Liu, A.R. Hand, R. Menzeleev, S. Spiegel, and T. Hla. 1998. Science. 279:1552–1555). Edg-1 binds SPP with remarkable specificity as only sphinganine-1-phosphate displaced radiolabeled SPP, while other sphingolipids did not. Binding of SPP to Edg-1 resulted in inhibition of forskolin-stimulated cAMP accumulation, in a pertussis toxin–sensitive manner. In contrast, two well-characterized biological responses of SPP, mitogenesis and prevention of apoptosis, were clearly unrelated to binding to Edg-1 and correlated with intracellular uptake. SPP also stimulated signal transduction pathways, including calcium mobilization, activation of phospholipase D, and tyrosine phosphorylation of p125FAK, independently of edg-1 expression. Moreover, DNA synthesis in Swiss 3T3 fibroblasts was significantly and specifically increased by microinjection of SPP. Finally, SPP suppresses apoptosis of HL-60 and pheochromocytoma PC12 cells, which do not have specific SPP binding or expression of Edg-1 mRNA. Conversely, sphinganine-1-phosphate, which binds to and signals via Edg-1, does not have any significant cytoprotective effect. Thus, SPP is a prototype for a novel class of lipid mediators that act both extracellularly as ligands for cell surface receptors and intracellularly as second messengers.
Tumor Suppressor PTEN Inhibits Integrin- and Growth Factor–mediated Mitogen-activated Protein (MAP) Kinase Signaling Pathways
Authors: Gu Jianguo , Tamura Masahito , Yamada Kenneth M.
Published: July 2002

The tumor suppressor PTEN dephosphorylates focal adhesion kinase (FAK) and inhibits integrin-mediated cell spreading and cell migration. We demonstrate here that expression of PTEN selectively inhibits activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. PTEN expression in glioblastoma cells lacking the protein resulted in inhibition of integrin-mediated MAP kinase activation. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)- induced MAPK activation were also blocked. To determine the specific point of inhibition in the Ras/Raf/ MEK/ERK pathway, we examined these components after stimulation by fibronectin or growth factors. Shc phosphorylation and Ras activity were inhibited by expression of PTEN, whereas EGF receptor autophosphorylation was unaffected. The ability of cells to spread at normal rates was partially rescued by coexpression of constitutively activated MEK1, a downstream component of the pathway. In addition, focal contact formation was enhanced as indicated by paxillin staining. The phosphatase domain of PTEN was essential for all of these functions, because PTEN with an inactive phosphatase domain did not suppress MAP kinase or Ras activity. In contrast to its effects on ERK, PTEN expression did not affect c-Jun NH2-terminal kinase (JNK) or PDGF-stimulated Akt. Our data suggest that a general function of PTEN is to down-regulate FAK and Shc phosphorylation, Ras activity, downstream MAP kinase activation, and associated focal contact formation and cell spreading.
Distinct Mutants of Retrograde Intraflagellar Transport (IFT) Share Similar Morphological and Molecular Defects
Authors: Piperno Gianni , Siuda Edward , Henderson Scott , Segil Margarethe , Vaananen Heikki , Sassaroli Massimo
Published: July 2002

A microtubule-based transport of protein complexes, which is bidirectional and occurs between the space surrounding the basal bodies and the distal part of Chlamydomonas flagella, is referred to as intraflagellar transport (IFT). The IFT involves molecular motors and particles that consist of 17S protein complexes. To identify the function of different components of the IFT machinery, we isolated and characterized four temperature-sensitive (ts) mutants of flagellar assembly that represent the loci FLA15, FLA16, and FLA17. These mutants were selected among other ts mutants of flagellar assembly because they displayed a characteristic bulge of the flagellar membrane as a nonconditional phenotype. Each of these mutants was significantly defective for the retrograde velocity of particles and the frequency of bidirectional transport but not for the anterograde velocity of particles, as revealed by a novel method of analysis of IFT that allows tracking of single particles in a sequence of video images. Furthermore, each mutant was defective for the same four subunits of a 17S complex that was identified earlier as the IFT complex A. The occurrence of the same set of phenotypes, as the result of a mutation in any one of three loci, suggests the hypothesis that complex A is a portion of the IFT particles specifically involved in retrograde intraflagellar movement.
Distinct Roles for the p110α and hVPS34 Phosphatidylinositol 3′-Kinases in Vesicular Trafficking, Regulation of the Actin Cytoskeleton, and Mitogenesis
Authors: Siddhanta Uma , McIlroy James , Shah Amishi , Zhang Yitao , Backer Jonathan M.
Published: July 2002

We have examined the roles of the p85/ p110α and hVPS34 phosphatidylinositol (PI) 3′-kinases in cellular signaling using inhibitory isoform-specific antibodies. We raised anti-hVPS34 and anti-p110α antibodies that specifically inhibit recombinant hVPS34 and p110α, respectively, in vitro. We used the antibodies to study cellular processes that are sensitive to low-dose wortmannin. The antibodies had distinct effects on the actin cytoskeleton; microinjection of anti-p110α antibodies blocked insulin-stimulated ruffling, whereas anti-hVPS34 antibodies had no effect. The antibodies also had different effects on vesicular trafficking. Microinjection of inhibitory anti-hVPS34 antibodies, but not anti-p110α antibodies, blocked the transit of internalized PDGF receptors to a perinuclear compartment, and disrupted the localization of the early endosomal protein EEA1. Microinjection of anti-p110α antibodies, and to a lesser extent anti-hVPS34 antibodies, reduced the rate of transferrin recycling in CHO cells. Surprisingly, both antibodies inhibited insulin-stimulated DNA synthesis by 80%. Injection of cells with antisense oligonucleotides derived from the hVPS34 sequence also blocked insulin-stimulated DNA synthesis, whereas scrambled oligonucleotides had no effect. Interestingly, the requirement for p110α and hVPS34 occurred at different times during the G1–S transition. Our data suggest that different PI 3′-kinases play distinct regulatory roles in the cell, and document an unexpected role for hVPS34 during insulin-stimulated mitogenesis.
Homotypic Fusion of Immature Secretory Granules during Maturation in a Cell-free Assay
Authors: Urbé Sylvie , Page Lesley J. , Tooze Sharon A.
Published: July 2002

The biogenesis of secretory granules embodies several morphological and biochemical changes. In particular, in neuroendocrine cells maturation of secretory granules is characterized by an increase in size which has been proposed to reflect homotypic fusion of immature secretory granules (ISGs). Here we describe an assay that provides the first biochemical evidence for such a fusion event and allows us to analyze its regulation. The assay reconstitutes homotypic fusion between one population of ISGs containing a [35S]sulfate-labeled substrate, secretogranin II (SgII), and a second population containing the prohormone convertase PC2. Both substrate and enzyme are targeted exclusively to ISGs. Fusion is measured by quantification of a cleavage product of SgII produced by PC2. With this assay we show that fusion only occurs between ISGs and not between ISGs and MSGs, is temperature dependent, and requires ATP and GTP and cytosolic proteins. NSF (N-ethylmaleimide–sensitive fusion protein) is amongst the cytosolic proteins required, whereas we could not detect a requirement for p97. The ability to reconstitute ISG fusion in a cell-free assay is an important advance towards the identification of molecules involved in the maturation of secretory granules and will increase our understanding of this process.
Nuclear Import of the Parsley bZIP Transcription Factor CPRF2 Is Regulated by Phytochrome Photoreceptors
Authors: Kircher Stefan , Wellmer Frank , Nick Peter , Rügner Alexander , Schäfer Eberhard , Harter Klaus
Published: July 2002

In plants, light perception by photoreceptors leads to differential expression of an enormous number of genes. An important step for differential gene expression is the regulation of transcription factor activities. To understand these processes in light signal transduction we analyzed the three well-known members of the common plant regulatory factor (CPRF) family from parsley (Petroselinum crispum). Here, we demonstrate that these CPRFs, which belong to the basic- region leucine-zipper (bZIP) domain-containing transcription factors, are differentially distributed within parsley cells, indicating different regulatory functions within the regulatory networks of the plant cell. In particular, we show by cell fractionation and immunolocalization approaches that CPRF2 is transported from the cytosol into the nucleus upon irradiation due to action of phytochrome photoreceptors. Two NH2-terminal domains responsible for cytoplasmic localization of CPRF2 in the dark were characterized by deletion analysis using a set of CPRF2-green fluorescent protein (GFP) gene fusion constructs transiently expressed in parsley protoplasts. We suggest that light-induced nuclear import of CPRF2 is an essential step in phytochrome signal transduction.
Ca2+-induced Ca2+ Release in Chromaffin Cells Seen from inside the ER with Targeted Aequorin
Authors: Alonso Maria Teresa , Barrero Maria José , Michelena Pedro , Carnicero Estela , Cuchillo Inmaculada , García Antonio G. , García-Sancho Javier , Montero Mayte , Alvarez Javier
Published: July 2002

The presence and physiological role of Ca2+-induced Ca2+ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca2+] ([Ca2+]c). Using targeted aequorin, we have directly monitored [Ca2+] changes inside the ER ([Ca2+]ER) in bovine adrenal chromaffin cells. Ca2+ entry induced by cell depolarization triggered a transient Ca2+ release from the ER that was highly dependent on [Ca2+]ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca2+ release was quantal in nature due to modulation by [Ca2+]ER. Whereas caffeine released essentially all the Ca2+ from the ER, inositol 1,4,5-trisphosphate (InsP3)- producing agonists released only 60–80%. Both InsP3 and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca2+ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca2+]c measurements showed that the wave of [Ca2+]c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca2+ pool that can release Ca2+ both via InsP3 receptors or CICR.
The Junction-associated Protein AF-6 Interacts and Clusters with Specific Eph Receptor Tyrosine Kinases at Specialized Sites of Cell–Cell Contact in the Brain
Authors: Buchert Michael , Schneider Stefan , Meskenaite Virginia , Adams Mark T. , Canaani Eli , Baechi Thomas , Moelling Karin , Hovens Christopher M.
Published: July 2002

The AF-6/afadin protein, which contains a single PDZ domain, forms a peripheral component of cell membranes at specialized sites of cell–cell junctions. To identify potential receptor-binding targets of AF-6 we screened the PDZ domain of AF-6 against a range of COOH-terminal peptides selected from receptors having potential PDZ domain-binding termini. The PDZ domain of AF-6 interacts with a subset of members of the Eph subfamily of RTKs via its COOH terminus both in vitro and in vivo. Cotransfection of a green fluorescent protein-tagged AF-6 fusion protein with full-length Eph receptors into heterologous cells induces a clustering of the Eph receptors and AF-6 at sites of cell–cell contact. Immunohistochemical analysis in the adult rat brain reveals coclustering of AF-6 with Eph receptors at postsynaptic membrane sites of excitatory synapses in the hippocampus. Furthermore, AF-6 is a substrate for a subgroup of Eph receptors and phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor. The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates. AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain.
Cell Surface Heparan Sulfate Proteoglycan Syndecan-2 Induces the Maturation of Dendritic Spines in Rat Hippocampal Neurons
Authors: Ethell Iryna M. , Yamaguchi Yu
Published: July 2002

Dendritic spines are small protrusions that receive synapses, and changes in spine morphology are thought to be the structural basis for learning and memory. We demonstrate that the cell surface heparan sulfate proteoglycan syndecan-2 plays a critical role in spine development. Syndecan-2 is concentrated at the synapses, specifically on the dendritic spines of cultured hippocampal neurons, and its accumulation occurs concomitant with the morphological maturation of spines from long thin protrusions to stubby and headed shapes. Early introduction of syndecan-2 cDNA into immature hippocampal neurons, by transient transfection, accelerates spine formation from dendritic protrusions. Deletion of the COOH-terminal EFYA motif of syndecan-2, the binding site for PDZ domain proteins, abrogates the spine-promoting activity of syndecan-2. Syndecan-2 clustering on dendritic protrusions does not require the PDZ domain-binding motif, but another portion of the cytoplasmic domain which includes a protein kinase C phosphorylation site. Our results indicate that syndecan-2 plays a direct role in the development of postsynaptic specialization through its interactions with PDZ domain proteins.
Dissection of Cell Division Processes in the One Cell Stage Caenorhabditis elegans Embryo by Mutational Analysis
Authors: Gönczy Pierre , Schnabel Heinke , Kaletta Titus , Amores Ana Duran , Hyman Tony , Schnabel Ralf
Published: July 2002

To identify novel components required for cell division processes in complex eukaryotes, we have undertaken an extensive mutational analysis in the one cell stage Caenorhabditis elegans embryo. The large size and optical properties of this cell permit observation of cell division processes with great detail in live specimens by simple differential interference contrast (DIC) microscopy. We have screened an extensive collection of maternal-effect embryonic lethal mutations on chromosome III with time-lapse DIC video microscopy. Using this assay, we have identified 48 mutations in 34 loci which are required for specific cell division processes in the one cell stage embryo. We show that mutations fall into distinct phenotypic classes which correspond, among others, to the processes of pronuclear migration, rotation of centrosomes and associated pronuclei, spindle assembly, chromosome segregation, anaphase spindle positioning, and cytokinesis. We have further analyzed pronuclear migration mutants by indirect immunofluorescence microscopy using antibodies against tubulin and ZYG-9, a centrosomal marker. This analysis revealed that two pronuclear migration loci are required for generating normal microtubule arrays and four for centrosome separation. All 34 loci have been mapped by deficiencies to distinct regions of chromosome III, thus paving the way for their rapid molecular characterization. Our work contributes to establishing the one cell stage C. elegans embryo as a powerful metazoan model system for dissecting cell division processes.
Integrin-mediated Activation of Focal Adhesion Kinase Is Required for Signaling to Jun NH2-terminal Kinase and Progression through the G1 Phase of the Cell Cycle
Authors: Oktay Maja , Wary Kishore K. , Dans Michael , Birge Raymond B. , Giancotti Filippo G.
Published: July 2002

The extracellular matrix exerts a stringent control on the proliferation of normal cells, suggesting the existence of a mitogenic signaling pathway activated by integrins, but not significantly by growth factor receptors. Herein, we provide evidence that integrins cause a significant and protracted activation of Jun NH2-terminal kinase (JNK), while several growth factors cause more modest or no activation of this enzyme. Integrin-mediated stimulation of JNK required the association of focal adhesion kinase (FAK) with a Src kinase and p130CAS, the phosphorylation of p130CAS, and subsequently, the recruitment of Crk. Ras and PI-3K were not required. FAK–JNK signaling was necessary for proper progression through the G1 phase of the cell cycle. These findings establish a role for FAK in both the activation of JNK and the control of the cell cycle, and identify a physiological stimulus for JNK signaling that is consistent with the role of Jun in both proliferation and transformation.
Histone Macroh2a1.2 Relocates to the Inactive X Chromosome after Initiation and Propagation of X-Inactivation
Authors: Mermoud Jacqueline E. , Costanzi Carl , Pehrson John R. , Brockdorff Neil
Published: July 2002

The histone macroH2A1.2 has been implicated in X chromosome inactivation on the basis of its accumulation on the inactive X chromosome (Xi) of adult female mammals. We have established the timing of macroH2A1.2 association with the Xi relative to the onset of X-inactivation in differentiating murine embryonic stem (ES) cells using immuno-RNA fluorescence in situ hybridization (FISH). Before X-inactivation we observe a single macroH2A1.2-dense region in both undifferentiated XX and XY ES cells that does not colocalize with X inactive specific transcript (Xist) RNA, and thus appears not to associate with the X chromosome(s). This pattern persists through early stages of differentiation, up to day 7. Then the frequency of XY cells containing a macroH2A1.2-rich domain declines. In contrast, in XX cells there is a striking relocalization of macroH2A1.2 to the Xi. Relocalization occurs in a highly synchronized wave over a 2-d period, indicating a precisely regulated association. The timing of macroH2A1.2 accumulation on the Xi suggests it is not necessary for the initiation or propagation of random X-inactivation.
A Role for Myosin-I in Actin Assembly through Interactions with Vrp1p, Bee1p, and the Arp2/3 Complex
Authors: Evangelista Marie , Klebl Bert M. , Tong Amy H.Y. , Webb Bradley A. , Leeuw Thomas , Leberer Ekkehard , Whiteway Malcolm , Thomas David Y. , Boone Charles
Published: July 2002

Type I myosins are highly conserved actin-based molecular motors that localize to the actin-rich cortex and participate in motility functions such as endocytosis, polarized morphogenesis, and cell migration. The COOH-terminal tail of yeast myosin-I proteins, Myo3p and Myo5p, contains an Src homology domain 3 (SH3) followed by an acidic domain. The myosin-I SH3 domain interacted with both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteins that link actin assembly and signaling molecules. The myosin-I acidic domain interacted with Arp2/3 complex subunits, Arc40p and Arc19p, and showed both sequence similarity and genetic redundancy with the COOH-terminal acidic domain of Bee1p (Las17p), which controls Arp2/3-mediated actin nucleation. These findings suggest that myosin-I proteins may participate in a diverse set of motility functions through a role in actin assembly.
Roles of Autocrine TGF-β Receptor and Smad Signaling in Adipocyte Differentiation
Authors: Choy Lisa , Skillington Jeremy , Derynck Rik
Published: July 2002

TGF-β inhibits adipocyte differentiation, yet is expressed by adipocytes. The function of TGF-β in adipogenesis, and its mechanism of action, is unknown. To address the role of TGF-β signaling in adipocyte differentiation, we characterized the expression of the TGF-β receptors, and the Smads which transmit or inhibit TGF-β signals, during adipogenesis in 3T3-F442A cells. We found that the cell-surface availability of TGF-β receptors strongly decreased as adipogenesis proceeds. Whereas mRNA levels for Smads 2, 3, and 4 were unchanged during differentiation, mRNA levels for Smads 6 and 7, which are known to inhibit TGF-β responses, decreased severely. Dominant negative interference with TGF-β receptor signaling, by stably expressing a truncated type II TGF-β receptor, enhanced differentiation and decreased growth. Stable overexpression of Smad2 or Smad3 inhibited differentiation and dominant negative inhibition of Smad3 function, but not Smad2 function, enhanced adipogenesis. Increased Smad6 and Smad7 levels blocked differentiation and enhanced TGF-β–induced responses. The inhibitory effect of Smad7 on adipocyte differentiation and its cooperation with TGF-β was associated with the C-domain of Smad7. Our results indicate that endogenous TGF-β signaling regulates the rate of adipogenesis, and that Smad2 and Smad3 have distinct functions in this endogenous control of differentiation. Smad6 and Smad7 act as negative regulators of adipogenesis and, even though known to inhibit TGF-β responses, enhance the effects of TGF-β on these cells.
Disruption of Nuclear Lamin Organization Blocks the Elongation Phase of DNA Replication
Authors: Moir Robert D. , Spann Timothy P. , Herrmann Harald , Goldman Robert D.
Published: July 2002

The role of nuclear lamins in DNA replication is unclear. To address this, nuclei were assembled in Xenopus extracts containing AraC, a reversible inhibitor that blocks near the onset of the elongation phase of replication. Dominant-negative lamin mutants lacking their NH2-terminal domains were added to assembled nuclei to disrupt lamin organization. This prevented the resumption of DNA replication after the release of the AraC block. This inhibition of replication was not due to gross disruption of nuclear envelope structure and function. The organization of initiation factors was not altered by lamin disruption, and nuclei resumed replication when transferred to extracts treated with CIP, an inhibitor of the cyclin-dependent kinase (cdk) 2–dependent step of initiation. This suggests that alteration of lamin organization does not affect the initiation phase of DNA replication. Instead, we find that disruption of lamin organization inhibited chain elongation in a dose-dependent fashion. Furthermore, the established organization of two elongation factors, proliferating cell nuclear antigen, and replication factor complex, was disrupted by ΔNLA. These findings demonstrate that lamin organization must be maintained in nuclei for the elongation phase of DNA replication to proceed.
Recruitment of Protein Phosphatase 1 to the Nuclear Envelope by a-Kinase Anchoring Protein Akap149 Is a Prerequisite for Nuclear Lamina Assembly
Authors: Steen Rikke L. , Martins Sandra B. , Taskén Kjetil , Collas Philippe
Published: July 2002

Subcellular targeting of cAMP-dependent protein kinase (protein kinase A [PKA]) and of type 1 protein phosphatase (PP1) is believed to enhance the specificity of these enzymes. We report that in addition to anchoring PKA, A-kinase anchoring protein AKAP149 recruits PP1 at the nuclear envelope (NE) upon somatic nuclear reformation in vitro, and that PP1 targeting to the NE is a prerequisite for assembly of B-type lamins. AKAP149 is an integral membrane protein of the endoplasmic reticulum/NE network. The PP1-binding domain of AKAP149 was identified as K153GVLF157. PP1 binds immobilized AKAP149 in vitro and coprecipitates with AKAP149 from purified NE extracts. Affinity isolation of PP1 from solubilized NEs copurifies AKAP149. Upon reassembly of somatic nuclei in interphase extract, PP1 is targeted to the NE. Targeting is inhibited by a peptide containing the PP1-binding domain of AKAP149, abolished in nuclei assembled with membranes immunodepleted of AKAP149, and restored after reincorporation of AKAP149 into nuclear membranes. B-type lamins do not assemble into a lamina when NE targeting of PP1 is abolished, and is rescued upon recruitment of PP1 to the NE. We propose that kinase and phosphatase anchoring at the NE by AKAP149 plays in a role in modulating nuclear reassembly at the end of mitosis.
Essential Role of Gab1 for Signaling by the C-Met Receptor in Vivo
Authors: Sachs Martin , Brohmann Henning , Zechner Dietmar , Müller Thomas , Hülsken Jörg , Walther Ingrid , Schaeper Ute , Birchmeier Carmen , Birchmeier Walter
Published: July 2002

The docking protein Gab1 binds phosphorylated c-Met receptor tyrosine kinase directly and mediates signals of c-Met in cell culture. Gab1 is phosphorylated by c-Met and by other receptor and nonreceptor tyrosine kinases. Here, we report the functional analysis of Gab1 by targeted mutagenesis in the mouse, and compare the phenotypes of the Gab1 and c-Met mutations. Gab1 is essential for several steps in development: migration of myogenic precursor cells into the limb anlage is impaired in Gab1−/− embryos. As a consequence, extensor muscle groups of the forelimbs are virtually absent, and the flexor muscles reach less far. Fewer hindlimb muscles exist, which are smaller and disorganized. Muscles in the diaphragm, which also originate from migratory precursors, are missing. Moreover, Gab1−/− embryos die in a broad time window between E13.5 and E18.5, and display reduced liver size and placental defects. The labyrinth layer, but not the spongiotrophoblast layer, of the placenta is severely reduced, resulting in impaired communication between maternal and fetal circulation. Thus, extensive similarities between the phenotypes of c-Met and HGF/SF mutant mice exist, and the muscle migration phenotype is even more pronounced in Gab1−/−:c-Met+/− embryos. This is genetic evidence that Gab1 is essential for c-Met signaling in vivo. Analogy exists to signal transmission by insulin receptors, which require IRS1 and IRS2 as specific docking proteins.
Cortactin Localization to Sites of Actin Assembly in Lamellipodia Requires Interactions with F-Actin and the Arp2/3 Complex
Authors: Weed Scott A. , Karginov Andrei V. , Schafer Dorothy A. , Weaver Alissa M. , Kinley Andrew W. , Cooper John A. , Parsons J. Thomas
Published: July 2002

Cortactin is an actin-binding protein that is enriched within the lamellipodia of motile cells and in neuronal growth cones. Here, we report that cortactin is localized with the actin-related protein (Arp) 2/3 complex at sites of actin polymerization within the lamellipodia. Two distinct sequence motifs of cortactin contribute to its interaction with the cortical actin network: the fourth of six tandem repeats and the amino-terminal acidic region (NTA). Cortactin variants lacking either the fourth tandem repeat or the NTA failed to localize at the cell periphery. Tandem repeat four was necessary for cortactin to stably bind F-actin in vitro. The NTA region interacts directly with the Arp2/3 complex based on affinity chromatography, immunoprecipitation assays, and binding assays using purified components. Cortactin variants containing the NTA region were inefficient at promoting Arp2/3 actin nucleation activity. These data provide strong evidence that cortactin is specifically localized to sites of dynamic cortical actin assembly via simultaneous interaction with F-actin and the Arp2/3 complex. Cortactin interacts via its Src homology 3 (SH3) domain with ZO-1 and the SHANK family of postsynaptic density 95/dlg/ZO-1 homology (PDZ) domain–containing proteins, suggesting that cortactin contributes to the spatial organization of sites of actin polymerization coupled to selected cell surface transmembrane receptor complexes.
Tangled1
Authors: Smith Laurie G. , Gerttula Suzanne M. , Han Shengcheng , Levy Joshua
Published: July 2002

Spatial control of cytokinesis in plant cells depends on guidance of the cytokinetic apparatus, the phragmoplast, to a cortical “division site” established before mitosis. Previously, we showed that the Tangled1 (Tan1) gene of maize is required for this process during maize leaf development (Cleary, A.L., and L.G. Smith. 1998. Plant Cell. 10:1875–1888.). Here, we show that the Tan1 gene is expressed in dividing cells and encodes a highly basic protein that can directly bind to microtubules (MTs). Moreover, proteins recognized by anti-TAN1 antibodies are preferentially associated with the MT-containing cytoskeletal structures that are misoriented in dividing cells of tan1 mutants. These results suggest that TAN1 protein participates in the orientation of cytoskeletal structures in dividing cells through an association with MTs.
Essential Roles of Drosophila Inner Centromere Protein (Incenp) and Aurora B in Histone H3 Phosphorylation, Metaphase Chromosome Alignment, Kinetochore Disjunction, and Chromosome Segregation
Authors: Adams Richard R. , Maiato Helder , Earnshaw William C. , Carmena Mar
Published: July 2002

We have performed a biochemical and double-stranded RNA-mediated interference (RNAi) analysis of the role of two chromosomal passenger proteins, inner centromere protein (INCENP) and aurora B kinase, in cultured cells of Drosophila melanogaster. INCENP and aurora B function is tightly interlinked. The two proteins bind to each other in vitro, and DmINCENP is required for DmAurora B to localize properly in mitosis and function as a histone H3 kinase. DmAurora B is required for DmINCENP accumulation at centromeres and transfer to the spindle at anaphase. RNAi for either protein dramatically inhibited the ability of cells to achieve a normal metaphase chromosome alignment. Cells were not blocked in mitosis, however, and entered an aberrant anaphase characterized by defects in sister kinetochore disjunction and the presence of large amounts of amorphous lagging chromatin. Anaphase A chromosome movement appeared to be normal, however cytokinesis often failed. DmINCENP and DmAurora B are not required for the correct localization of the kinesin-like protein Pavarotti (ZEN-4/CHO1/MKLP1) to the midbody at telophase. These experiments reveal that INCENP is required for aurora B kinase function and confirm that the chromosomal passengers have essential roles in mitosis.
The discrepancy between presenilin subcellular localization and γ-secretase processing of amyloid precursor protein
Authors: Cupers Philippe , Bentahir Mustapha , Craessaerts Katleen , Orlans Isabelle , Vanderstichele Hugo , Saftig Paul , De Strooper Bart , Annaert Wim
Published: July 2002

We investigated the relationship between PS1 and γ-secretase processing of amyloid precursor protein (APP) in primary cultures of neurons. Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent γ-secretase cleavage, although little PS1 is present in those subcellular compartments. In contrast, almost no γ-secretase processing was observed when holo-APP or APP-C99, a direct substrate for γ-secretase, were specifically retained in the endoplasmic reticulum (ER) by a double lysine retention motif. Nevertheless, APP-C99-dilysine (KK) colocalized with PS1 in the ER. In contrast, APP-C99 did not colocalize with PS1, but was efficiently processed by PS1-dependent γ-secretase. APP-C99 resides in a compartment that is negative for ER, intermediate compartment, and Golgi marker proteins. We conclude that γ-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected. This suggests that at least one other factor than PS1, located downstream of the ER, is required for the γ-cleavage of APP-C99. In agreement, we found that intracellular γ-secretase processing of APP-C99-KK both at the γ40 and the γ42 site could be restored partially after brefeldin A treatment. Our data confirm the “spatial paradox” and raise several questions regarding the PS1 is γ-secretase hypothesis.
APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death
Authors: Fortin Andre , Cregan Sean P. , MacLaurin Jason G. , Kushwaha Neena , Hickman Emma S. , Thompson Charlie S. , Hakim Antoine , Albert Paul R. , Cecconi Francesco , Helin Kristian , Park David S. , Slack Ruth S.
Published: July 2002

p53 is a transcriptional activator which has been implicated as a key regulator of neuronal cell death after acute injury. We have shown previously that p53-mediated neuronal cell death involves a Bax-dependent activation of caspase 3; however, the transcriptional targets involved in the regulation of this process have not been identified. In the present study, we demonstrate that p53 directly upregulates Apaf1 transcription as a critical step in the induction of neuronal cell death. Using DNA microarray analysis of total RNA isolated from neurons undergoing p53-induced apoptosis a 5–6-fold upregulation of Apaf1 mRNA was detected. Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons. In both in vitro and in vivo neuronal cell death processes of p53-induced cell death, Apaf1 protein levels were increased. We addressed whether p53 directly regulates Apaf1 transcription via the two p53 consensus binding sites in the Apaf1 promoter. Electrophoretic mobility shift assays demonstrated p53–DNA binding activity at both p53 consensus binding sequences in extracts obtained from neurons undergoing p53-induced cell death, but not in healthy control cultures or when p53 or the p53 binding sites were inactivated by mutation. In transient transfections in a neuronal cell line with p53 and Apaf1 promoter–luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites. The importance of Apaf1 as a p53 target gene in neuronal cell death was evaluated by examining p53-induced apoptotic pathways in primary cultures of Apaf1-deficient neurons. Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates. Together, these results demonstrate that Apaf1 is a key transcriptional target for p53 that plays a pivotal role in the regulation of apoptosis after neuronal injury.
Sorting of Golgi resident proteins into different subpopulations of COPI vesicles
Authors: Lanoix Joel , Ouwendijk Joke , Stark Annika , Szafer Edith , Cassel Dan , Dejgaard Kurt , Weiss Matthias , Nilsson Tommy
Published: July 2002

We present evidence for two subpopulations of coatomer protein I vesicles, both containing high amounts of Golgi resident proteins but only minor amounts of anterograde cargo. Early Golgi proteins p24α2, β1, δ1, and γ3 are shown to be sorted together into vesicles that are distinct from those containing mannosidase II, a glycosidase of the medial Golgi stack, and GS28, a SNARE protein of the Golgi stack. Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride. Using synthetic peptides, we find that the cytoplasmic domain of p24β1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes. We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.
T Cell Stimulation In Vivo by Lipopolysaccharide (LPS)
Authors: Tough David F. , Sun Siquan , Sprent Jonathan
Published: July 2002

Lipopolysaccharide (LPS) from gram-negative bacteria causes polyclonal activation of B cells and stimulation of macrophages and other APC. We show here that, under in vivo conditions, LPS also induces strong stimulation of T cells. As manifested by CD69 upregulation, LPS injection stimulates both CD4 and CD8+ T cells, and, at high doses, stimulates naive (CD44lo) cells as well as memory (CD44hi) cells. However, in terms of cell division, the response of T cells after LPS injection is limited to the CD44hi subset of CD8+ cells. In contrast with B cells, proliferative responses of CD44hi CD8+ cells require only very low doses of LPS (10 ng). Based on studies with LPS-nonresponder and gene-knockout mice, LPS-induced proliferation of CD44hi CD8+ cells appears to operate via an indirect pathway involving LPS stimulation of APC and release of type I (α, β) interferon (IFN-I). Similar selective stimulation of CD44hi CD8+ cells occurs in viral infections and after injection of IFN-I, implying a common mechanism. Hence, intermittent exposure to pathogens (gram-negative bacteria and viruses) could contribute to the high background proliferation of memory–phenotype CD8+ cells found in normal animals.
Quantitative Contribution of CD4 and CD8 to T Cell Antigen Receptor Serial Triggering
Authors: Viola Antonella , Salio Mariolina , Tuosto Loretta , Linkert Susanne , Acuto Oreste , Lanzavecchia Antonio
Published: July 2002

CD4 and CD8 are thought to function as coreceptors by binding to the cognate major histocompatibility complex (MHC) molecules recognized by the T cell antigen receptor (TCR) and initiating the signal transduction cascade. We report that during T cell–antigen-presenting cell interaction, triggered TCRs and coreceptors are downregulated and degraded with identical kinetics. This coordinated disappearance takes place whenever the TCR is triggered, even when the coreceptor does not engage the cognate MHC molecule and is the consequence of binding of the coreceptor-associated Lck to ZAP-70. The interaction of coreceptor and cognate MHC molecules is dispensable when T cells are stimulated by optimal ligands, but becomes crucial when suboptimal ligands are used. In the latter case the coreceptor increases the efficiency of TCR triggering without changing the activation threshold or the quality of the T cell response.
Granule-mediated Killing: Pathways for Granzyme B–initiated Apoptosis
Authors: Talanian Robert V. , Yang XiaoHe , Turbov Jane , Seth Prem , Ghayur Tariq , Casiano Carlos A. , Orth Kim , Froelich Christopher J.
Published: July 2002

We report that the serine protease granzyme B (GrB), which is crucial for granule-mediated cell killing, initiates apoptosis in target cells by first maturing caspase-10. In addition, GrB has a limited capacity to mature other caspases and to cause cell death independently of the caspases. Compared with other members, GrB in vitro most efficiently processes caspase-7 and -10. In a human cell model, full maturation of caspase-7 does not occur unless caspase-10 is present. Furthermore, GrB matured caspase-3 with less efficiency than caspase-7 or caspase-10. With the caspases fully inactivated by peptidic inhibitors, GrB induced in Jurkat cells growth arrest and, over a delayed time period, cell death. Thus, the primary mechanism by which GrB initiates cell death is activation of the caspases through caspase-10. However, under circumstances where caspase-10 is absent or dysfunctional, GrB can act through secondary mechanisms including activation of other caspases and direct cell killing by cleavage of noncaspase substrates. The redundant functions of GrB ensure the effectiveness of granule-mediated cell killing, even in target cells that lack the expression or function (e.g., by mutation or a viral serpin) of one or more of the caspases, providing the host with overlapping safeguards against aberrantly replicating, nonself or virally infected cells.
Mutational Evidence for Control of Cell Adhesion Through Integrin Diffusion/Clustering, Independent of Ligand Binding
Authors: Yauch Robert L. , Felsenfeld Dan P. , Kraeft Stine-Kathrein , Chen Lan Bo , Sheetz Michael P. , Hemler Martin E.
Published: July 2002

Previous studies have shown that integrin α chain tails make strong positive contributions to integrin-mediated cell adhesion. We now show here that integrin α4 tail deletion markedly impairs static cell adhesion by a mechanism that does not involve altered binding of soluble vascular cell adhesion molecule 1 ligand. Instead, truncation of the α4 cytoplasmic domain caused a severe deficiency in integrin accumulation into cell surface clusters, as induced by ligand and/ or antibodies. Furthermore, α4 tail deletion also significantly decreased the membrane diffusivity of α4β1, as determined by a single particle tracking technique. Notably, low doses of cytochalasin D partially restored the deficiency in cell adhesion seen upon α4 tail deletion. Together, these results suggest that α4 tail deletion exposes the β1 cytoplasmic domain, leading to cytoskeletal associations that apparently restrict integrin lateral diffusion and accumulation into clusters, thus causing reduced static cell adhesion. Our demonstration of integrin adhesive activity regulated through receptor diffusion/clustering (rather than through altered ligand binding affinity) may be highly relevant towards the understanding of inside–out signaling mechanisms for β1 integrins.
Interleukin-1β Converting Enzyme–like Protease Involvement in Fas-induced and Activation-induced Peripheral Blood T Cell Apoptosis in HIV Infection. TNF-related Apoptosis-inducing Ligand Can Mediate Activation-induced T Cell Death in HIV Infection
Authors: Katsikis Peter D. , Garcia-Ojeda Marcos E. , Torres-Roca Javier F. , Tijoe Iwan M. , Smith Craig A. , Herzenberg Leonore A. , Herzenberg Leonard A.
Published: July 2002

Apoptosis of peripheral blood T cells has been suggested to play an important role in the pathogenesis of human immunodeficiency virus (HIV) infection. Spontaneous, Fas (CD95)–induced and activation-induced T cell apoptosis have all been described in peripheral blood mononuclear cell cultures of HIV-infected individuals. We have previously shown that activation-induced T cell apoptosis is Fas independent in peripheral blood T cells from HIV+ individuals. In this study, we extend and confirm these observations by using an inhibitor of interleukin-1β converting enzyme (ICE) homologues. We show that z-VAD-fmk, a tripeptide inhibitor of ICE homologues, can inhibit Fas-induced apoptosis of peripheral blood CD4+ and CD8+ T cells from asymptomatic HIV+ individuals. z-VAD-fmk also inhibited activation (anti-CD3)– induced CD4+ and CD8+ T cell apoptosis (AICD) in some but not all asymptomatic HIV+ individuals. Apoptosis was measured by multiparameter flow cytometry. The z-VAD-fmk inhibitor also enhanced survival of T cells in anti-Fas or anti-CD3 antibody-treated cultures and inhibited DNA fragmentation. AICD that could be inhibited by z-VAD-fmk was Fas independent and could be inhibited with a blocking monoclonal antibody to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a recently described member of the TNF/nerve growth factor ligand family. The above findings show that Fas-induced T cell apoptosis is ICE dependent in HIV infection. AICD can be blocked by ICE inhibitors in some patients, and this AICD is mediated by TRAIL. These results show that TRAIL can be a mediator of AICD in T cells. These different mechanisms of peripheral blood T cell apoptosis may play different roles in the pathogenesis of HIV infection.