bluuebunny/crossref_metadata_2025 huggingface dataset:
p42/mitogen-activated protein kinase as a converging target for different growth factor signaling pathways: use of pertussis toxin as a discrimination factor.
Authors: L'Allemain G , Pouyssegur J , Weber M J
Published: August 2013

Mitogen-activated protein (MAP) kinase is a 42-kDa serine/threonine-specific protein kinase that requires phosphorylation on both tyrosine and threonine residues for activity. This enzyme is rapidly and transiently activated in quiescent cells after addition of various agonists, including insulin, epidermal growth factor, platelet-derived growth factor, and phorbol esters. We show here that addition of the growth factors thrombin or basic fibroblast growth factor to CCL39 fibroblasts rapidly induces tyrosine phosphorylation of the p42 MAP kinase protein and concomitantly stimulates MAP kinase enzymatic activity. To elucidate the signaling pathways utilized in this activation, we took advantage of the sensitivity of CCL39 cells to the toxin of bordetella pertussis, which ADP-ribosylates two Gi proteins in this cell system. We show that pretreatment of cells with the toxin inhibited thrombin stimulation of MAP kinase by greater than 75% but had no detectable effect on the stimulation induced by basic fibroblast growth factor. We also demonstrate that these two growth factors that synergize for mitogenicity are able to cooperate in activation of MAP kinase and that this synergism is partially sensitive to pertussis toxin. Finally, we describe a 44-kDa protein, the tyrosine phosphorylation of which appears to be coregulated with p42 MAP kinase. We conclude that p42 MAP kinase (and the pp44 protein) are at or are downstream from a point of convergence of two different receptor-induced signaling pathways and might well play a key role in integrating those signals.
Selective expression of high molecular weight basic fibroblast growth factor confers a unique phenotype to NIH 3T3 cells.
Authors: Quarto N , Talarico D , Florkiewicz R , Rifkin D B
Published: August 2013

The phenotypes of NIH 3T3 cells transfected with basic fibroblast growth factor (bFGF) cDNAs that express only the high molecular weight (HMW) forms of bFGF, the 18-kDa form, or all forms were examined. Cells producing the 18 kDa or all forms of bFGF were transformed at high levels of growth factor expression but were nontransformed at low levels. Cell producing low levels of HMW forms of bFGF were growth impaired when compared with the parental cells. These cells tended to form multinucleated giant cells, did not grow in soft agar, were nontumorigenic, had a normal bFGF receptor number, and had a nontransformed morphology. Cells expressing high levels of HMW bFGFs had a transformed morphology and were tumorigenic. These data suggest a specific functional role for HMWbFGF.
Potential oncogenic effects of basic fibroblast growth factor requires cooperation between CUG and AUG-initiated forms.
Authors: Couderc B , Prats H , Bayard F , Amalric F
Published: August 2013

Normal adult bovine aortic endothelial cells were infected with various recombinant retroviruses expressing one, two, or three human basic fibroblast growth factor (bFGF) proteins normally synthesized by an alternative use of translation initiation codons. We show here that the constitutive expression of the AUG-initiated from (18 kDa) leads the transfected cells to form colonies in soft agar. The expression of the high molar weight (HMW) forms (22.5 and 21 kDa) initiated at one of the two CUG initiation codons allows cell immortalization, whereas the tumorigenic potential is reached when the three forms are constitutively expressed. Furthermore, we provide evidence that constitutive expression of (HMW) bFGF forms has a down-regulation effect on bFGF synthesis from the gene naturally active in parental endothelial cells.
Basic fibroblast growth factor requires a long-lasting activation of protein kinase C to induce cell proliferation in transformed fetal bovine aortic endothelial cells.
Authors: Presta M , Tiberio L , Rusnati M , Dell'Era P , Ragnotti G
Published: August 2013

Basic fibroblast growth factor (bFGF) induces a protein kinase C (PKC)-dependent mitogenic response in transformed fetal bovine aortic endothelial GM 7373 cells. A long-lasting interaction of bFGF with the cell is required to induce cell proliferation. bFGF-treated cells are in fact committed to proliferate only after they have entered the phase S of the cell cycle, 12-14 h after the beginning of bFGF treatment. Before that time, the mitogenic response to bFGF is abolished by 1) removal of extracellular bFGF by suramin, 2) addition of neutralizing anti-bFGF antibodies to the culture medium, 3) inhibition of PKC activity by the protein kinase inhibitor H-7, and 4) down-regulation of PKC by cotreatment with phorbol ester. Thus the requirement for a prolonged interaction of bFGF with the cell reflects the requirement for a prolonged activation of PKC. Similar conclusions can be drawn for the PKC activators 12-O-tetradecanoyl phorbol 13-acetate and 1,2-dioctanoyl-sn-glycerol. The two molecules require 16 and 6 h, respectively, of activation of PKC to induce 50% of maximal cell proliferation. The requirement for a long-lasting activation of PKC appears to be a mechanism for the control of cell proliferation capable of discriminating among transient nonmitogenic stimuli and long-lasting mitogenic stimuli.
Differential regulation of interleukin-6 receptor and gp130 gene expression in rat hepatocytes.
Authors: Nesbitt J E , Fuller G M
Published: August 2013

Interleukin-6 (IL-6) relays an important signal to hepatocytes during the early stages of an acute inflammatory response, causing an alteration in the expression of several major defense proteins. Additional regulation of this signal could occur either by altering the number of IL-6 receptors (IL-6-R) or of the signal transducing protein, gp130. We employed ribonuclease protection assays to measure the expression of IL-6-R and gp130 mRNA in primary rat hepatocytes in response to IL-6, interleukin-1, dexamethasone, and combinations thereof. Dexamethasone increases receptor mRNA levels 2.7-fold above controls but has no detectable effect on that of gp130. Such treatment increased surface expression of IL-6-R from 600 receptors per cell to greater than 6000, without a change in Kd (2.5-4.6 x 10(-10) M). In contrast to the stimulatory effect of the steroid signal, the inflammatory cytokines, individually and together, down-modulated both the mRNA and the cell surface expression of IL-6-R. These findings demonstrate for the first time that a sensitive control system exists between inflammatory mediators and IL-6-R.
Coordination of hormone-induced calcium signals in isolated rat hepatocyte couplets: demonstration with confocal microscopy.
Authors: Nathanson M H , Burgstahler A D
Published: August 2013

Excitable cells often display rapid coordination of hormone-induced intracellular calcium signals. Calcium elevations that begin in a single epithelial cell also may spread to adjacent cells, but coordination of hormone-induced signals among epithelial cells has not been described. We report the use of confocal microscopy to determine the inter- and intracellular distribution of cytosolic calcium in isolated rat hepatocyte couplets, an isolated epithelial cell system in which functional polarity is maintained. Both vasopressin and phenylephrine evoked sequential coordinated calcium signals in the couplets, even during cytosolic calcium oscillations. The coupling was abolished by closure of intercellular gap junction channels by treatment with octanol. These observations demonstrate that hormone-induced intracellular calcium signals are coordinated among hepatocytes and suggest that gap junction channels mediate this intercellular integration of tissue responsiveness.
Role of phosphorylation in p34cdc2 activation: identification of an activating kinase.
Authors: Solomon M J , Lee T , Kirschner M W
Published: August 2013

Phosphorylation of p34cdc2 can both positively and negatively regulate its kinase activity. We have mapped two phosphorylation sites in Xenopus p34cdc2 to Thr-14 and Tyr-15 within the putative ATP-binding region of p34cdc2. Mutation of these sites to Ala-14 and Phe-15 has no effect on the final histone H1 kinase activity of the cyclin/p34cdc2 complex. Phosphopeptide analysis shows that there is at least one more site of phosphorylation on p34cdc2. When Thr-161 is changed to Ala, two phosphopeptide spots disappear and it is no longer possible to activate the H1 kinase activity of p34cdc2. We suggest that Thr-161 is a third site of phosphorylation, which is required for kinase activity. All three phosphorylations are induced by cyclin. None of the phosphorylations appears to be required for binding to cyclin, as indicated by the ability of the triple mutant, Ala-14, Phe-15, Ala-161, to bind cyclin. The activating phosphorylation that requires Thr- or Ser-161 occurs even in a catalytically inactive K33R mutant of p34cdc2 and hence does not appear to be the result of intramolecular autophosphorylation. We have detected an activity in Xenopus extracts required for activation of p34cdc2 and present evidence that this is a p34cdc2 activating kinase which, in a cyclin-dependent manner, probably directly phosphorylates Thr-161.
Functional expression and growth factor activation of an epitope-tagged p44 mitogen-activated protein kinase, p44mapk.
Authors: Meloche S , Pagès G , Pouysségur J
Published: August 2013

Mitogen-activated protein kinases (MAPKs) or extracellular signal-regulated kinases (ERKs) are serine/threonine kinases of apparent Mr 42-44 kDa that are rapidly activated by a variety of extracellular signals in many cell types. This activation coincides with their phosphorylation on tyrosine and threonine residues, and these covalent modifications are required for full activity of the enzymes. They are thought to play a pivotal role in integrating and transmitting transmembrane signals for growth and differentiation. Here, we report the cloning, sequence, and functional expression in fibroblasts of the hamster p44 MAP kinase (p44mapk). The protein deduced from the nucleotide sequence of an almost full-length cDNA is 98.6% homologous to the rat p44mapk (ERK1). To distinguish the expression of the cloned cDNA from the endogenous p44mapk, we fused to the 5' end of the cDNA an initiating codon followed by an influenza hemagglutinin 9-residue peptide epitope (HAP). The chimeric kinase HAP/p44mapk, under transcriptional control of the cytomegalovirus promoter, was stably expressed in Chinese hamster lung fibroblasts in a functional form. We show that its basal activity, measured by phosphorylation of the substrate myelin basic protein, is activated severalfold (up to 25) by the mitogens alpha-thrombin, platelet-derived growth factor, and fetal calf serum. In addition, we report that in response to alpha-thrombin, this activation is rapid (6-fold in 1 min), biphasic (first peak at 5 min, second broader peak at 1-2 h), persistent (for greater than or equal to 4 h), and parallel to an increased phosphorylation on tyrosine.We conclude that the constructed and stably expressed chimera, HAP/p44mapk, has retained apparently all the hormonal regulation features of the endogenous form. This system now offers the possibility to study structure-function relationships and to determine the role of this kinase in growth control.
Activation of a muscle-specific actin gene promoter in serum-stimulated fibroblasts.
Authors: Stoflet E S , Schmidt L J , Elder P K , Korf G M , Foster D N , Strauch A R , Getz M J
Published: August 2013

Treatment of AKR-2B mouse fibroblasts with serum growth factors or inhibitors of protein synthesis, such as cycloheximide, results in a stimulation of cytoskeletal beta-actin transcription but has no effect on transcription of muscle-specific isotypes, such as the vascular smooth muscle (VSM) alpha-actin gene. Deletion mapping and site-specific mutagenesis studies demonstrated that a single "CArG" element of the general form CC(A/T)6GG was necessary and possibly sufficient to impart serum and cycloheximide-inducibility to the beta-actin promoter. Although the VSM alpha-actin promoter exhibits at least three similar sequence elements, it remained refractory to serum and cycloheximide induction. However, deletion of a 33 base pair sequence between -191 and -224 relative to the transcription start site resulted in the transcriptional activation of this muscle-specific promoter in rapidly growing or serum-stimulated fibroblasts. Although the activity of this truncated promoter was potentiated by cycloheximide in a manner indistinguishable from that of the beta-actin promoter, this was dependent on a more complex array of interacting elements. These included at least one CArG box and a putative upstream activating element closely associated with the -191 to -224 inhibitory sequences. These results demonstrate that the expression of a muscle-specific actin gene in fibroblasts is suppressed by a cis-acting negative control element and that in the absence of this element, the promoter is responsive to growth factor-induced signal transduction pathways.
Mapping of RNA polymerase on mammalian genes in cells and nuclei.
Authors: Mirkovitch J , Darnell J E
Published: August 2013

The assembly of an RNA polymerase II initiation complex at a promoter is associated with the melting of the DNA template to allow the polymerase to read the DNA sequence and synthesize the corresponding RNA. Using the specific single-stranded modifying reagent KMnO4 and a new genomic sequencing technique, we have explored the melted regions of specific genes in genomic DNA of whole cells or of isolated nuclei. We have demonstrated for the first time in vivo the melting in the promoter proximal transcribed region that is associated with the presence of RNA polymerase II complexes. An interferon-inducible gene, ISG-54, exhibited KMnO4 sensitivity over approximately 300 nucleotides downstream of the RNA initiation site in interferon-treated cells when the gene was actively transcribed but not in untreated cells where the gene was not transcribed. The extent of KMnO4 modification was proportional to transcription levels. The KMnO4 sensitivity was retained when nuclei were isolated from induced cells but was lost if the engaged polymerases were further allowed to elongate the nascent RNA chains ("run-on"). The sensitivity to KMnO4 in isolated nuclei was retained if the run-on incubation was performed in the presence of alpha-amanitin, which blocks progress of engaged polymerases. A similar analysis identified an open sequence of only approximately 30 bases just downstream of the start site of the transthyretin (TTR) gene in nuclei isolated from mouse liver, a tissue where TTR is actively transcribed. This abrupt boundary of KMnO4 sensitivity, which was removed completely by allowing engaged polymerases to elongate RNA chains, suggests that most polymerases transcribing this gene paused at about position +20. The possibility of mapping at the nucleotide level the position of actively transcribing RNA polymerases in whole cells or isolated nuclei opens new prospects in the study of transcription initiation and elongation.
Escherichia coli ribonucleotide reductase expression is cell cycle regulated.
Authors: Sun L , Fuchs J A
Published: August 2013

The expression of the genes encoding ribonucleotide reductase in Escherichia coli was investigated in cultures synchronized by obtaining the smallest cells in a population after sucrose gradient centrifugation. Specific activity of ribonucleotide reductase and DNA initiation were found to increase in parallel, periodically as a function of the cell cycle. The expression of nrd was also determined in cells synchronized by periodic repeated doubling in a phosphate limited medium. Antibodies directed against the B2 subunit of ribonucleotide reductase were raised in a rabbit and purified. Immunoprecipitation of the B2 subunit and RNA-DNA dot blot hybridization assays were developed and employed to determine the expression of ribonucleotide reductase translational and transcriptional products during the cell cycle. Both of nrd-mRNA and B2 subunit expression were found to increase each generation at approximately the same time DNA synthesis was initiated and then to decrease back to the basal level shortly after DNA initiation. These results provided evidence of cell cycle dependent regulation of ribonucleotide reductase in E. coli. When the upstream regulatory region of nrd was fused to a promoterless lacZ gene on a single copy plasmid, lac-mRNA and beta-galactosidase were found to be synthesized in parallel to nrd expression from the chromosomal operon. When nrd sequences surrounding the promoter were removed from this construct, lac-mRNA and beta-galactosidase synthesis were no longer cell cycle regulated.
Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau.
Authors: Drechsel D N , Hyman A A , Cobb M H , Kirschner M W
Published: August 2013

Microtubule-associated proteins (MAP), such as tau, modulate the extent and rate of microtubule assembly and play an essential role in morphogenetic processes, such as axonal growth. We have examined the mechanism by which tau affects microtubule polymerization by examining the kinetics of microtubule assembly and disassembly through direct observation of microtubules using dark-field microscopy. Tau increases the rate of polymerization, decreases the rate of transit into the shrinking phase (catastrophe), and inhibits the rate of depolymerization. Tau strongly suppresses the catastrophe rate, and its ability to do so is independent of its ability to increase the elongation rate. Thus, tau generates a partially stable but still dynamic state in microtubules. This state is perturbed by phosphorylation by MAP2 kinase, which affects all three activities by lowering the affinity of tau for the microtubule lattice.
Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP.
Authors: Hyman A A , Salser S , Drechsel D N , Unwin N , Mitchison T J
Published: August 2013

The role of GTP hydrolysis in microtubule dynamics has been reinvestigated using an analogue of GTP, guanylyl-(alpha, beta)-methylene-diphosphonate (GMPCPP). This analogue binds to the tubulin exchangeable nucleotide binding site (E-site) with an affinity four to eightfold lower than GTP and promotes the polymerization of normal microtubules. The polymerization rate of microtubules with GMPCPP-tubulin is very similar to that of GTP-tubulin. However, in contrast to microtubules polymerized with GTP, GMPCPP-microtubules do not depolymerize rapidly after isothermal dilution. The depolymerization rate of GMPCPP-microtubules is 0.1 s-1 compared with 500 s-1 for GDP-microtubules. GMPCPP also completely suppresses dynamic instability. Contrary to previous work, we find that the beta--gamma bond of GMPCPP is hydrolyzed extremely slowly after incorporation into the microtubule lattice, with a rate constant of 4 x 10(-7) s-1. Because GMPCPP hydrolysis is negligible over the course of a polymerization experiment, it can be used to test the role of hydrolysis in microtubule dynamics. Our results provide strong new evidence for the idea that GTP hydrolysis by tubulin is not required for normal polymerization but is essential for depolymerization and thus for dynamic instability. Because GMPCPP strongly promotes spontaneous nucleation of microtubules, we propose that GTP hydrolysis by tubulin also plays the important biological role of inhibiting spontaneous microtubule nucleation.
Expression and distribution of osteopontin in human tissues: widespread association with luminal epithelial surfaces.
Authors: Brown L F , Berse B , Van de Water L , Papadopoulos-Sergiou A , Perruzzi C A , Manseau E J , Dvorak H F , Senger D R
Published: August 2013

Osteopontin, a glycoprotein with a glycine-arginine-glycine-aspartate-serine (GRGDS) cell-binding domain, has been described in bone and is also known to be expressed in other organs, particularly kidney. The goal of the present work was to define the distribution of osteopontin synthesis and deposition in a wide variety of normal adult human tissues using a multifaceted approach that included immunohistochemistry, in situ hybridization, and Northern analysis. Immunohistochemical studies have revealed the unexpected finding that osteopontin is deposited as a prominent layer at the luminal surfaces of specific populations of epithelial cells of the gastrointestinal tract, gall bladder, pancreas, urinary and reproductive tracts, lung, breast, salivary glands, and sweat glands. Northern analyses identified gallbladder as a major site of osteopontin gene transcription comparable in magnitude with that of kidney, and immunoblotting identified osteopontin in bile. In situ hybridization localized osteopontin gene transcripts predominantly to the epithelium of a variety of organs as well as to ganglion cells of bowel wall. Osteopontin of epithelial cell origin, like bone-derived osteopontin, promoted GRGDS-dependent cell spreading in attachment assays. We postulate that osteopontin secreted by epithelium binds integrins on luminal surfaces. Collectively, these findings suggest an important role for osteopontin on many luminal epithelial surfaces communicating with the external environment.
Mutation of a tyrosine localization signal in the cytosolic tail of yeast Kex2 protease disrupts Golgi retention and results in default transport to the vacuole.
Authors: Wilcox C A , Redding K , Wright R , Fuller R S
Published: August 2013

Kex2 protease processes pro-alpha-factor in a late Golgi compartment in Saccharomyces cerevisiae. The first approximately 30 residues of the 115 amino acid CO2H-terminal cytosolic tail (C-tail) of the Kex2 protein (Kex2p) contain a Golgi retention signal that resembles coated-pit localization signals in mammalian cell surface receptors. Mutation of one (Tyr713) of two tyrosine residues in the C-tail or deletion of sequences adjacent to Tyr713 results in loss of normal Golgi localization. Surprisingly, loss of the Golgi retention signal resulted in transport of C-tail mutant Kex2p to the vacuole (yeast lysosome), as judged by kinetics of degradation and by indirect immunofluorescence. Analysis of the loss of Kex2 function in vivo after shutting off expression of wild-type or mutant forms proved that mutations that cause rapid vacuolar turnover do so by increasing the rate of exit of the enzyme from the pro-alpha-factor processing compartment. The most likely explanation for these results is that mutation of the Golgi retention signal in the C-tail results in transport of Kex2p to the vacuole by default. Wild-type Kex2p also was transported to the vacuole at an increased rate when overproduced, although apparently not due to saturation of a Golgi-retention mechanism. Instead, the wild-type and C-tail mutant forms of Kex2p may follow distinct paths to the vacuole.
Activation of Ca(2+)-dependent currents in cultured rat dorsal root ganglion neurones by a sperm factor and cyclic ADP-ribose.
Authors: Currie K P , Swann K , Galione A , Scott R H
Published: August 2013

The effects of intracellular application of two novel Ca2+ releasing agents have been studied in cultured rat dorsal root ganglion (DRG) neurones by monitoring Ca(2+)-dependent currents as a physiological index of raised free cytosolic Ca2+ ([Ca2+]i). A protein based sperm factor (SF) extracted from mammalian sperm, has been found to trigger Ca2+ oscillations and to sensitize unfertilized mammalian eggs to calcium induced calcium release (CICR). In this study intracellular application of SF activated Ca(2+)-dependent currents in approximately two-thirds of DRG neurones. The SF induced activity was abolished by heat treatment, attenuated by increasing the intracellular Ca2+ buffering capacity of the cells and persisted when extracellular Ca2+ was replaced by Ba2+. In addition, activity could be triggered or potentiated by loading the cells with Ca2+ by activating a series of voltage-gated Ca2+ currents. Ca(2+)-activated inward current activity was also generated by intracellular application of cyclic ADP-ribose (cADPR), a metabolite of NAD+, which causes Ca2+ release in sea urchin eggs. This activity could also be enhanced by loading the cells with Ca2+. The cADPR induced activity, but not the SF induced activity, was abolished by depleting the caffeine sensitive Ca2+ store. Ruthenium red markedly attenuated SF induced activity but had little action on cADPR induced activity or caffeine induced activity. Our results indicate that both SF and cADPR release intracellular Ca2+ pools in DRG neurones and that they appear to act on subtly distinct stores or distinct intracellular Ca2+ release mechanisms, possibly by modulating CICR.
A Dictyostelium myosin II lacking a proximal 58-kDa portion of the tail is functional in vitro and in vivo.
Authors: Kubalek E W , Uyeda T Q , Spudich J A
Published: August 2013

We used molecular genetic approaches to delete 521 amino acid residues from the proximal portion of the Dictyostelium myosin II tail. The deletion encompasses approximately 40% of the tail, including the S2-LMM junction, a region that in muscle myosin II has been proposed to be important for contraction. The functions of the mutant myosin II are indistinguishable from the wild-type myosin II in our in vitro assays. It binds to actin in a typical rigor configuration in the absence of ATP and it forms filaments in a normal salt-dependent manner. In an in vitro motility assay, both monomeric and filamentous forms of the mutant myosin II translocate actin filaments at 2.4 microns/s at 30 degrees C, similar to that of wild-type myosin II. The mutant myosin II is also functional in vivo. Cells expressing the mutant myosin II in place of the native myosin II perform myosin II-dependent activities such as cytokinesis and formation of fruiting bodies, albeit inefficiently. Growth of the mutant cells in suspension gives rise to many large multinucleated cells, demonstrating that cytokinesis often fails. The majority of the fruiting bodies are also morphologically abnormal. These results demonstrate that this region of the myosin II tail is not required for motile activities but its presence is necessary for optimum function in vivo.
Membrane potential modulates release of tumor necrosis factor in lipopolysaccharide-stimulated mouse macrophages.
Authors: Haslberger A , Romanin C , Koerber R
Published: August 2013

Lipopolysaccharide (LPS)-mediated synthesis of macrophage gene products such as tumor necrosis factor (TNF) is controlled by different signaling pathways. We investigated intracellular free Ca2+ (Ca2+ic) and the membrane potential as early cellular responses to LPS and their role in the synthesis and release of TNF. In peritoneal macrophages and in the RAW 269 mouse macrophage cell line, LPS and its biologically active moiety lipid A stimulated TNF synthesis but exerted no significant effects on these early cellular responses using Fura-2/Indo-1 to measure Ca2+ic and bis-oxonol, as well as the patch-clamp technique to monitor membrane potential. In contrast, the platelet-activating factor transiently induced both an increase in Ca2+ic and cell membrane depolarization but no significant TNF release. Increased extracellular K+ concentrations or K(+)-channel blockers, such as quinine, tetraethylammonium, or barium chloride, inhibited the LPS-stimulated release of TNF alpha, as well as the accumulation of cell-associated TNF alpha as found by enzyme-linked immunosorbent assay analysis, but did not inhibit TNF alpha mRNA accumulation. Concentrations of quinine (greater than 125 microM) or of enhanced extracellular K+ (25-85 mM) required to inhibit TNF production both significantly depolarized macrophages. These results indicate a lack of ion transport activities as early cellular responses of macrophages to LPS but suggest an important regulatory role of the membrane potential on the posttranscriptional synthesis and release of TNF in macrophages.
The beta-PDGF receptor induces neuronal differentiation of PC12 cells.
Authors: Heasley L E , Johnson G L
Published: August 2013

Expression of the mouse beta-PDGF receptor by gene transfer confers PDGF-dependent and reversible neuronal differentiation of PC12 pheochromocytoma cells similar to that observed in response to NGF and basic FGF. A common property of the PDGF, NGF, and basic FGF-induced differentiation response is the requirement for constant exposure of cells to the growth factor. To test the hypothesis that a persistent level of growth factor receptor signaling is required for the maintenance of the neuronal phenotype, we examined the regulation of the serine/threonine-specific MAP kinases after either short- (10 min) or long-term (24 h) stimulation with growth factors. Mono Q FPLC resolved two peaks of growth factor-stimulated MAP kinase activity that coeluted with tyrosine phosphorylated 41- and 43-kDa polypeptides. MAP kinase activity was markedly stimulated (approximately 30-fold) within 5 min of exposure to several growth factors (PDGF, NGF, basic FGF, EGF, and IGF-I), but was persistently maintained at 10-fold above basal activity after 24 h only by the growth factors that also induce PC12 cell differentiation (PDGF, NGF, and basic FGF). Thus the beta-PDGF receptor is in a subset of tyrosine kinase-encoded growth factor receptors that are capable of maintaining continuous signals required for differentiation of PC12 cells. These signals include the constitutive activation of cytoplasmic serine/threonine protein kinases.
Biologically active MK-801 and SKF-10,047 binding sites distinct from those in rat brain are expressed on human lung cancer cells.
Authors: Maneckjee R , Minna J D
Published: August 2013

We have shown previously that cultured human lung cancer cells of different histologic types express multiple opioid receptors that can regulate their growth. In this report, we show that these cells also express specific, saturable, and high-affinity binding sites (Kd approximately 1 nM) for the non-opioid phencyclidine (PCP), [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine hydrogen maleate] (MK-801) and sigma N-allylnormetazocine (SKF-10,047) receptor ligands. Characterization of these binding sites showed them to be protein in nature and sensitive to the guanine nucleotide GTP. Pharmacological studies showed that (+) MK-801 and (+) SKF-10,047 competed with each other for their binding sites and also for the methadone binding site present in these cells. However, the mu and delta opioid ligands did not compete for (+) MK-801 and (+) SKF-10,047 binding sites. In addition, these binding sites on lung cancer cells appear to be distinct from the N-methyl D-aspartate/PCP receptor ionophore complex reported to be present in rat brain. MK-801 and SKF-10,047, at nM concentrations, were found to inhibit the growth of these cells in culture within a few hours of exposure, and this effect was irreversible after 24 h. The growth effects of these ligands could not be reversed by the opioid antagonist naloxone, suggesting involvement of nonopioid type receptors in the actions of these ligands. The abundant expression of biologically active MK-801 and SKF-10 047 binding sites in these cell lines, distinct from those in rat brain, suggests that these cell lines may prove to be a valuable source for further characterization and purification of these binding sites.
Multiple roles for protein phosphatase 1 in regulating the Xenopus early embryonic cell cycle.
Authors: Walker D H , DePaoli-Roach A A , Maller J L
Published: August 2013

Using cytostatic factor metaphase II-arrested extracts as a model system, we show that protein phosphatase 1 is regulated during early embryonic cell cycles in Xenopus. Phosphatase 1 activity peaks during interphase and decreases shortly before the onset of mitosis. A second peak of activity appears in mitosis at about the same time that cdc2 becomes active. If extracts are inhibited in S-phase with aphidicolin, then phosphatase 1 activity remains high. The activity of phosphatase 1 appears to determine the timing of exit from S-phase and entry into M-phase; inhibition of phosphatase 1 by the specific inhibitor, inhibitor 2 (Inh-2), causes premature entry into mitosis, whereas exogenously added phosphatase 1 lengthens the interphase period. Analysis of DNA synthesis in extracts treated with Inh-2, but lacking the A- and B-type cyclins, shows that phosphatase 1 is also required for the process of DNA replication. These data indicate that phosphatase 1 is a component of the signaling pathway that ensures that M-phase is not initiated until DNA synthesis is complete.
A novel transcriptional enhancer is involved in the prolactin- and extracellular matrix-dependent regulation of beta-casein gene expression.
Authors: Schmidhauser C , Casperson G F , Myers C A , Sanzo K T , Bolten S , Bissell M J
Published: August 2013

Lactogenic hormones and extracellular matrix (ECM) act synergistically to regulate beta-casein expression in culture. We have developed a functional subpopulation of the mouse mammary epithelial cell strain COMMA-1D (designated CID 9), which expresses high level of beta-casein, forms alveolar-like structures when plated onto the EHS tumor-derived matrix, and secretes beta-casein unidirectionally into a lumen. We have further shown that ECM- and prolactin-dependent regulations of beta-casein occur mainly at the transcriptional level and that 5' sequences play an important role in these regulations. To address the question of the nature of the DNA sequence requirements for such regulation, we analyzed the bovine beta-casein gene promoter in these cells. We now have located a 160-bp transcriptional enhancer (BCE1) within the 5' flanking region of the beta-casein gene. Using functional assays, we show that BCE1 contains responsive elements for prolactin- and ECM-dependent regulation. BCE1 placed upstream of a truncated and inactive beta-casein promoter (the shortest extending from -89 to +42 bp with regard to the transcription start site) reconstitutes a promoter even more potent than the intact promoter, which contains BCE1 in its normal context more than 1.5 kb upstream. This small fusion promoter also reconstitutes the normal pattern of regulation, including a requirement for both prolactin and ECM and a synergistic action of prolactin and hydrocortisone. By replacing the milk promoter with a heterologous viral promoter, we show that BCE1 participates in the prolactin- and ECM-mediated regulation.
A gene encoding a protein with seven zinc finger domains acts on the sexual differentiation pathways of Schizosaccharomyces pombe.
Authors: Xu H P , Rajavashisth T , Grewal N , Jung V , Riggs M , Rodgers L , Wigler M
Published: August 2013

Byr3 was selected as a multicopy suppressor of the sporulation defects of diploid Schizosaccharomyces pombe cells that lack ras1. Like cells mutant at byr1 and byr2, two genes that encode putative protein kinases and that in multiple copies are also suppressors of the sporulation defects of ras1 null diploid cells, cells mutant at byr3 are viable but defective in conjugation. Nucleic acid sequence indicates byr3 has the capacity to encode a protein with seven zinc finger binding domains, similar in structure to the cellular nucleic acid binding protein (CNBP), a human protein that was identified on the basis of its ability to bind DNA. Expression of CNBP in yeast can partially suppress conjugation defects of cells lacking byr3.
Amino acid substitutions in the Dictyostelium G alpha subunit G alpha 2 produce dominant negative phenotypes and inhibit the activation of adenylyl cyclase, guanylyl cyclase, and phospholipase C.
Authors: Okaichi K , Cubitt A B , Pitt G S , Firtel R A
Published: August 2013

Previous studies have demonstrated that the Dictyostelium G alpha subunit G alpha 2 is essential for the cAMP-activation of adenylyl cyclase and guanylyl cyclase and that g alpha 2 null mutants do not aggregate. In this manuscript, we extend the analysis of the function of G alpha 2 in regulating downstream effectors by examining the in vivo developmental and physiological phenotypes of both wild-type and g alpha 2 null cells carrying a series of mutant G alpha 2 subunits expressed from the cloned G alpha 2 promoter. Our results show that wild-type cells expressing G alpha 2 subunits carrying mutations G40V and Q208L in the highly conserved GAGESG (residues 38-43) and GGQRS (residues 206-210) domains, which are expected to reduce the intrinsic GTPase activity, are blocked in multicellular development. Analysis of down-stream effector pathways essential for mediating aggregation indicates that cAMP-mediated activation of guanylyl cyclase and phosphatidylinositol-phospholipase C (PI-PLC) is almost completely inhibited and that there is a substantial reduction of cAMP-mediated activation of adenylyl cyclase. Moreover, neither mutant G alpha 2 subunit can complement g alpha 2 null mutants. Expression of G alpha 2(G43V) and G alpha 2(G207V) have little or no effect on the effector pathways and can partially complement g alpha 2 null cells. Our results suggest a model in which the dominant negative phenotypes resulting from the expression of G alpha 2(G40V) and G alpha 2(Q208L) are due to a constitutive adaptation of the effectors through a G alpha 2-mediated pathway. Analysis of PI-PLC in g alpha 2 null mutants and in cell lines expressing mutant G alpha 2 proteins also strongly suggests that G alpha 2 is the G alpha subunit that directly activates PI-PLC during aggregation. Moreover, overexpression of wild-type G alpha 2 results in the ability to precociously activate guanylyl cyclase by cAMP in vegetative cells, suggesting that G alpha 2 may be rate limiting in the developmental regulation of guanylyl cyclase activation. In agreement with previous results, the activation of adenylyl cyclase, while requiring G alpha 2 function in vivo, does not appear to be directly carried out by the G alpha 2 subunit. Our data are consistent with adenylyl cyclase being directly activated by either another G alpha subunit or by beta gamma subunits released on activation of the G protein containing G alpha 2.
Immunological characterization of avian MAP kinases: evidence for nuclear localization.
Authors: Sanghera J S , Peter M , Nigg E A , Pelech S L
Published: August 2013

The subcellular distribution and regulation of MAP kinase isoforms in chicken hepatoma DU249 cells was investigated with antibodies directed against peptides patterned after sequences in the mitogen-activated protein (MAP) kinases, sea star p44mpk, and rat p44erk1. MonoQ chromatography of cytosol from these cells afforded the resolution of at least four peaks of myelin basic protein (MBP) phosphotransferase activity, but only one of these (peak II) was stimulated in extracts from phorbol ester-treated cells. A 40- to 41-kDa (p41) doublet on Western blots detected with three different MAP kinase antibodies was coincident with peak II, and it probably corresponded to the avian homolog of p42mapk/erk2. Immunofluorescent studies with DU249 cells and chicken embryo fibroblasts revealed that most of the cross-reactive protein with at least two different MAP kinase antibodies was distributed in the nucleus. Subcellular fractionation studies confirmed a predominantly nuclear localization for p41 MAP kinase. Nocodazole arrest of DU249 cells was exploited for the detection of an M-phase-activated MBP kinase that was resolved from p41 MAP kinase by phenyl-Superose chromatography. Western blotting analysis with antibodies for the cdc2-encoded protein kinase and p13suc1-agarose binding studies allowed positive identification of this MBP kinase as p34cdc2.
Transcription of the gene for the gap junctional protein connexin43 and expression of functional cell-to-cell channels are regulated by cAMP.
Authors: Mehta P P , Yamamoto M , Rose B
Published: August 2013

We investigated the mechanism by which cyclic AMP (cAMP) induces gap junctional communication via cell-to-cell channels in a communication-deficient rat Morris hepatoma cell line. We found that under basal conditions, the cells transcribe cx43 at a low level but do not transcribe cx26 or cx32. Elevation of intracellular cAMP, which induced communication, increased cx43 mRNA 15- to 40-fold and the rate of cx43 transcription 6-fold. Cx43 protein was detected by immunostaining in junctions of only those cells in which communication had been induced. We found the regulation by cAMP also in other cell lines; namely, in those with a low basal level of cx43 mRNA.
Epidermal growth factor disrupts gap-junctional communication and induces phosphorylation of connexin43 on serine.
Authors: Lau A F , Kanemitsu M Y , Kurata W E , Danesh S , Boynton A L
Published: August 2013

Growth factors regulate cellular proliferation and differentiation by activating plasma membrane tyrosine kinase receptors and triggering a cascade of events mediated by intracellular signaling proteins. The mechanism underlying growth factor modification of cellular functions, such as gap-junctional communication (gjc), has not been established clearly. Addition of epidermal growth factor (EGF) to T51B rat liver epithelial cells resulted in the rapid activation of EGF receptor tyrosine kinase activity followed by a transient dose-dependent disruption of gjc. This change did not result from the gross disturbance of membrane gap junction plaques as measured by immunofluorescence microscopy, but instead correlated with markedly elevated phosphorylation of the connexin43 (cx43) gap junction protein, a profound shift to predominantly phosphorylated forms of cx43, and the appearance of a novel phosphorylated cx43 protein. These changes in cx43 phosphorylation involved only serine residues. On restoration of gjc, these alterations in cx43 phosphorylation reverted to the pre-EGF treatment state. Both events were inhibited by the serine/threonine protein phosphatase inhibitor, okadaic acid. Therefore, unlike the case for pp60v-src, EGF-induced disruption of gjc is not associated with tyrosine phosphorylation of cx43, but instead may result from phosphorylation of cx43 by activated intracellular signaling serine protein kinase(s).
A developmentally regulated Caulobacter flagellar promoter is activated by 3' enhancer and IHF binding elements.
Authors: Gober J W , Shapiro L
Published: August 2013

The transcription of a group of flagellar genes is temporally and spatially regulated during the Caulobacter crescentus cell cycle. These genes all share the same 5' cis-regulatory elements: a sigma 54 promoter, a binding site for integration host factor (IHF), and an enhancer sequence, known as the ftr element. We have partially purified the ftr-binding proteins, and we show that they require the same enhancer sequences for binding as are required for transcriptional activation. We have also partially purified the Caulobacter homolog of IHF and demonstrate that it can facilitate in vitro integrase-mediated lambda recombination. Using site-directed mutagenesis, we provide the first demonstration that natural enhancer sequences and IHF binding elements that reside 3' to the sigma 54 promoter of a bacterial gene, flaNQ, are required for transcription of the operon, in vivo. The IHF protein and the ftr-binding protein is primarily restricted to the predivisional cell, the cell type in which these promoters are transcribed. flaNQ promoter expression is localized to the swarmer pole of the predivisional cell, as are other flagellar promoters that possess these regulatory sequences 5' to the start site. The requirement for an IHF binding site and an ftr-enhancer element in spatially transcribed flagellar promoters indicates that a common mechanism may be responsible for both temporal and polar transcription.
Specification of sites for polarized growth in Saccharomyces cerevisiae and the influence of external factors on site selection.
Authors: Madden K , Snyder M
Published: August 2013

Many eucaryotic cell types exhibit polarized cell growth and polarized cell division at nonrandom sites. The sites of polarized growth were investigated in G1 arrested haploid Saccharomyces cerevisiae cells. When yeast cells are arrested during G1 either by treatment with alpha-factor or by shifting temperature-sensitive cdc28-1 cells to the restrictive temperature, the cells form a projection. Staining with Calcofluor reveals that in both cases the projection usually forms at axial sites (i.e., next to the previous bud scar); these are the same sites where bud formation is expected to occur. These results indicate that sites of polarized growth are specified before the end of G1. Sites of polarized growth can be influenced by external conditions. Cells grown to stationary phase and diluted into fresh medium preferentially select sites for polarized growth opposite the previous bud scar (i.e., distal sites). Incubation of cells in a mating mixture results in projection formation at nonaxial sites: presumably cells form projections toward their mating partner. These observations have important implications in understanding three aspects of cell polarity in yeast: 1) how yeast cell shape is influenced by growth conditions 2) how sites of polarized growth are chosen, and 3) the pathway by which polarity is affected and redirected during the mating process.
Activation of 5-HT1A receptors expressed in NIH-3T3 cells induces focus formation and potentiates EGF effect on DNA synthesis.
Authors: Varrault A , Bockaert J , Waeber C
Published: August 2013

NIH-3T3 fibroblasts have been transfected with human serotonin 5-HT1A receptors. Clonal cell lines expressed between 40 and 500 fmol receptor/mg. 5-HT1A agonists strongly inhibited nonstimulated- as well as forskolin- or isoproterenol-stimulated adenylyl cyclase. The effects of 5-HT1A receptor activation on cell growth were investigated. 5-HT1A agonists accelerated cell division, generated foci, and increased DNA synthesis. The stimulation of [3H]thymidine incorporation was much stronger when tyrosine kinase receptors were activated concomitantly. Cyclic AMP (cAMP) elevating agents inhibited DNA synthesis induced by all mitogens tested. The mitogenic activity of 5-HT1A agonists did not seem to be linked to adenylyl cyclase inhibition because 1) we were not able to measure any decrease in intracellular cAMP levels under the conditions of DNA synthesis assay and 2) 2',5'-dideoxyadenosine, which strongly inhibited adenylyl cyclase, was not mitogenic and did not modify the mitogenic effects of 5-HT1A agonists. Pertussis toxin completely blocked potentiation of epidermal growth factor effect induced by 8-hydroxy-di-(n-propyl)aminotetralin, a 5-HT1A agonist, but only partially blocked the one induced by insulin. In conclusion, in transfected NIH-3T3 cells, transforming and mitogenic effects of 5-HT1A agonists involve a pertussis toxin-sensitive G protein but do not seem to be linked to adenylyl cyclase inhibition.
Signal transduction by the platelet integrin alpha IIb beta 3: induction of calcium oscillations required for protein-tyrosine phosphorylation and ligand-induced spreading of stably transfected cells.
Authors: Pelletier A J , Bodary S C , Levinson A D
Published: August 2013

We demonstrate an example of signal transduction by an integrin and have begun to define the pathway through which this signaling is achieved. We constructed a stably transfected derivative of 293 cells (ATCC 1573) that expresses the platelet integrin GPIIbIIIa (alpha IIb beta 3). This cell line, clone B, adheres to and spreads on fibrinogen, a ligand for alpha IIb beta 3, while the parent cell line does not. Stimulation of these cells either by adhesion to fibrinogen or with antiserum directed against alpha IIb beta 3 results in induction of calcium oscillations, followed by tyrosine phosphorylation of at least one protein of molecular weight approximately 125 kDa. We establish that this phosphorylation, as well as the morphological rearrangements, requires the mobilization of calcium.