Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 27
Filter
Add more filters










Publication year range
2.
Theriogenology ; 79(9): 1284-93, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23566670

ABSTRACT

The first successful rabbit SCNT was achieved more than one decade ago, yet rabbits remain one of the most difficult species to clone. The present study was designed to evaluate the effects of two histone deacetylase inhibitors (HDACis), namely trichostatin A (TSA) and scriptaid (SCP), on cloning efficiency in rabbits. The in vitro development, acetylation levels of histone H4 lysine 5 (H4K5), and octamer-binding transcription factor 4 (Oct-4) expression patterns of cloned embryos were systemically examined after various HDACi treatments. Supplementation of TSA (50 nM) or SCP (250 nM) in the culture medium for 6 hours improved blastocyst development rates of cloned embryos compared with the treatment without HDACi. The combined treatment with TSA (50 nM) and SCP (250 nM) further enhanced morula (58.6%) and blastocyst (49.4%) rates in vitro. More importantly, compared with single HDACi treatments, embryos with the combined treatment had a higher level of H4K5 and an increased total cell number (203.7 ± 14.4 vs. 158.9 ± 9.0 or 162.1 ± 8.2; P < 0.05) with a better Oct-4 expression pattern in hatching blastocysts, indicating substantially improved embryo quality. This was apparently the first report regarding Oct-4 expression in cloned rabbit embryos. We inferred that most cloned rabbit embryos had an aberrant inner cell mass (ICM) structure accompanied with abnormal spatial distribution of Oct-4 signals. This study demonstrated a synergistic effect of TSA and SCP treatments on cloned rabbit embryos, which might be useful to improve cloning efficiency in rabbits.


Subject(s)
Embryo Culture Techniques/veterinary , Hydroxamic Acids/pharmacology , Hydroxylamines/pharmacology , Quinolines/pharmacology , Rabbits/embryology , Animals , Cloning, Organism , Female , Gene Expression Regulation, Developmental , Male , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism
3.
Theriogenology ; 75(4): 760-8, 2011 Mar 01.
Article in English | MEDLINE | ID: mdl-21144562

ABSTRACT

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.


Subject(s)
Cryopreservation/veterinary , Embryo, Mammalian , Embryonic Development , Rabbits/embryology , Animals , Cryopreservation/methods , Embryo Culture Techniques , Female , Reproductive Techniques, Assisted/veterinary
4.
Br J Pharmacol ; 161(6): 1329-42, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20735404

ABSTRACT

BACKGROUND AND PURPOSE: Vasoactive intestinal peptide is expressed in the respiratory tract and induces its effects via its receptors, VPAC(1) and VPAC(2). RO5024118 is a selective VPAC(2) receptor agonist derived via chemical modification of an earlier VPAC(2) agonist, RO0251553. In the present studies, we characterized the pharmacological activity of RO5024118. EXPERIMENTAL APPROACH: Stability of RO5024118 to human neutrophil elastase was assessed. Bronchodilatory activity of RO5024118 was investigated in guinea pig and human isolated airway smooth muscle preparations and in a guinea pig bronchoconstriction model. Pulmonary anti-inflammatory activity of RO5024118 was investigated in a lipopolysaccharide mouse model and in a porcine pancreatic elastase (PPE) rat model. KEY RESULTS: RO5024118 demonstrated increased stability to neutrophil elastase compared with RO0251553. In human and guinea pig isolated airway preparations, RO5024118 induced bronchodilatory effects comparable with RO0251553 and the long-acting ß-agonist salmeterol and was significantly more potent than native vasoactive intestinal peptide and the short-acting ß-agonist salbutamol. In 5-HT-induced bronchoconstriction in guinea pigs, RO5024118 exhibited inhibitory activity with similar efficacy as, and longer duration than, RO0251553. In a lipopolysaccharide-mouse model, RO5024118 inhibited neutrophil and CD8(+) cells and myeloperoxidase levels. In rats, intratracheal instillation of PPE induced airway neutrophilia that was resistant to dexamethasone. Pretreatment with RO5024118 significantly inhibited PPE-induced neutrophil accumulation. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that RO5024118 induces dual bronchodilatory and pulmonary anti-inflammatory activity and may be beneficial in treating airway obstructive and inflammatory diseases. LINKED ARTICLES: This article is part of a themed section on Analytical Receptor Pharmacology in Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2010.161.issue-6.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Bronchoconstriction/drug effects , Bronchodilator Agents/pharmacology , Lung/drug effects , Lung/pathology , Receptors, Vasoactive Intestinal Peptide, Type II/agonists , Vasoactive Intestinal Peptide/pharmacology , Amino Acid Sequence , Animals , Anti-Inflammatory Agents, Non-Steroidal/metabolism , Bronchoconstriction/physiology , Bronchodilator Agents/metabolism , Guinea Pigs , HT29 Cells , Humans , Lung/metabolism , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Rats , Receptors, Vasoactive Intestinal Peptide, Type II/metabolism , Swine , Vasoactive Intestinal Peptide/agonists , Vasoactive Intestinal Peptide/metabolism
5.
Curr Top Microbiol Immunol ; 279: 199-213, 2004.
Article in English | MEDLINE | ID: mdl-14560959

ABSTRACT

mTOR is a founding member of a family of protein kinases having catalytic domains homologous to those in phosphatidylinositol 3-OH kinase. mTOR participates in the control by insulin of the phosphorylation of lipin, which is required for adipocyte differentiation, and the two translational regulators, p70S6K and PHAS-I. The phosphorylation of mTOR, itself, is stimulated by insulin in Ser2448, a site that is also phosphorylated by protein kinase B (PKB) in vitro and in response to activation of PKB activity in vivo. Ser2448 is located in a short stretch of amino acids not found in the two TOR proteins in yeast. A mutant mTOR lacking this stretch exhibited increased activity, and binding of the antibody, mTAb-1, to this region markedly increased mTOR activity. In contrast, rapamycin-FKBP12 inhibited mTOR activity towards both PHAS-I and p70S6K, although this complex inhibited the phosphorylation of some sites more than that of others. Mutating Ser2035 to Ile in the FKBP12-rapamycin binding domain rendered mTOR resistant to inhibition by rapamycin. Unexpectedly, this mutation markedly decreased the ability of mTOR to phosphorylate certain sites in both PHAS-I and p70S6K. The results support the hypotheses that rapamycin disrupts substrate recognition instead of directly inhibiting phosphotransferase activity and that mTOR activity in cells is controlled by the phosphorylation of an inhibitory regulatory domain containing the mTAb-1 epitope.


Subject(s)
Carrier Proteins/metabolism , Phosphoproteins/metabolism , Protein Kinases/metabolism , Adaptor Proteins, Signal Transducing , Adipocytes/enzymology , Adipocytes/metabolism , Animals , Binding Sites , Cell Cycle Proteins , Eukaryotic Initiation Factors , Insulin/metabolism , Mice , Phosphorylation , TOR Serine-Threonine Kinases
6.
J Biol Chem ; 273(43): 28178-84, 1998 Oct 23.
Article in English | MEDLINE | ID: mdl-9774438

ABSTRACT

Amino acids have been identified as important signaling molecules involved in pancreatic beta-cell proliferation, although the cellular mechanism responsible for this effect is not well defined. We previously reported that amino acids are required for glucose or exogenous insulin to stimulate phosphorylation of PHAS-I (phosphorylated heat- and acid-stable protein regulated by insulin), a recently discovered regulator of translation initiation during cell mitogenesis. Here we demonstrate that essential amino acids, in particular branched-chain amino acids (leucine, valine, and isoleucine), are largely responsible for mediating this effect. The transamination product of leucine, alpha-ketoisocaproic acid, also stimulates PHAS-I phosphorylation although the transamination products of isoleucine and valine are ineffective. Since amino acids are secretagogues for insulin secretion by beta-cells, we investigated whether endogenous insulin secreted by beta-cells is involved. Interestingly, branched-chain amino acids stimulate phosphorylation of PHAS-I independent of endogenous insulin secretion since genistein (10 microM) and herbimycin A (1 microM), two tyrosine kinase inhibitors in the insulin signaling pathway, exert no effect on amino acid-induced phosphorylation of PHAS-I. Furthermore, branched-chain amino acids retain their ability to induce phosphorylation of PHAS-I under conditions that block insulin secretion from beta-cells. In exploring the signaling pathway responsible for these effects, we find that rapamycin (25 nM) inhibits the ability of branched-chain amino acids to stimulate the phosphorylation of PHAS-I and p70(s6) kinase, suggesting that the mammalian target of rapamycin signaling pathway is involved. The branched-chain amino acid, leucine, also exerts similar effects on PHAS-I phosphorylation in isolated pancreatic islets. In addition, we find that amino acids are necessary for insulin-like growth factor (IGF-I) to stimulate the phosphorylation of PHAS-I indicating that a requirement for amino acids may be essential for other beta-cell growth factors in addition to insulin and IGF-I to activate this signaling pathway. We propose that amino acids, in particular branched-chain amino acids, may promote beta-cell proliferation either by stimulating phosphorylation of PHAS-I and p70(s6k) via the mammalian target of rapamycin pathway and/or by facilitating the proliferative effect mediated by growth factors such as insulin and IGF-I.


Subject(s)
Amino Acids, Branched-Chain/pharmacology , Carrier Proteins , Islets of Langerhans/drug effects , Phosphoproteins/metabolism , Ribosomal Protein S6 Kinases/metabolism , Animals , Cell Division , Drug Synergism , Gene Expression Regulation , Growth Substances/pharmacology , Insulin/pharmacology , Insulin-Like Growth Factor I/pharmacology , Intracellular Signaling Peptides and Proteins , Male , Phosphorylation/drug effects , Protein Biosynthesis , Rats , Rats, Sprague-Dawley , Signal Transduction , Sirolimus/pharmacology
7.
J Biol Chem ; 273(8): 4485-91, 1998 Feb 20.
Article in English | MEDLINE | ID: mdl-9468502

ABSTRACT

Although glucose regulates the biosynthesis of a variety of beta cell proteins at the level of translation, the mechanism responsible for this effect is unknown. We demonstrate that incubation of pancreatic islets with elevated glucose levels results in rapid and concentration-dependent phosphorylation of PHAS-I, an inhibitor of mRNA cap-binding protein, eukaryotic initiation factor (eIF)-4E. Our initial approach was to determine if this effect is mediated by the metabolism of glucose and activation of islet cell protein kinases, or whether insulin secreted from the beta cell stimulates phosphorylation of PHAS-I via an insulin-receptor mechanism as described for insulin-sensitive cells. In support of the latter mechanism, inhibitors of islet cell protein kinases A and C exert no effect on glucose-stimulated phosphorylation of PHAS-I, whereas the phosphatidylinositol 3-kinase inhibitor, wortmannin, the immunosuppressant, rapamycin, and theophylline, a phosphodiesterase inhibitor, promote marked dephosphorylation of PHAS-I. In addition, exogenous insulin and endogenous insulin secreted by the beta cell line, betaTC6-F7, increase phosphorylation of PHAS-I, suggesting that beta cells of the islet, in part, mediate this effect. Studies with beta cell lines and islets indicate that amino acids are required for glucose or exogenous insulin to stimulate the phosphorylation of PHAS-I, and amino acids alone dose-dependently stimulate the phosphorylation of PHAS-I, which is further enhanced by insulin. Furthermore, rapamycin inhibits by approximately 62% the increase in total protein synthesis stimulated by high glucose concentrations. These results indicate that glucose stimulates PHAS-I phosphorylation via insulin interacting with its own receptor on the beta cell which may serve as an important mechanism for autoregulation of protein synthesis by translation.


Subject(s)
Carrier Proteins , Glucose/metabolism , Insulin/pharmacology , Islets of Langerhans/drug effects , Phosphoproteins/metabolism , Repressor Proteins/metabolism , Animals , Cells, Cultured , Eukaryotic Initiation Factor-4E , Intracellular Signaling Peptides and Proteins , Islets of Langerhans/metabolism , Male , Peptide Initiation Factors/metabolism , Phosphorylation , Protein Binding , Protein Biosynthesis , Rats , Rats, Sprague-Dawley , Signal Transduction
8.
Diabetologia ; 40 Suppl 2: S18-24, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9248697

ABSTRACT

PHAS-I is a recently discovered regulator of translation initiation. Non-phosphorylated PHAS-I binds and inhibits eukaryotic initiation factor-4E, the mRNA cap-binding protein that mediates a rate-limiting step in translation initiation. When PHAS-I is phosphorylated in response to insulin, the PHAS-I/eukaryotic initiation factor-4E complex dissociates. The present study was conducted to investigate mechanisms involved in the control of PHAS-I. Phosphorylation of PHAS-I was monitored by immunoblotting after subjecting extracts to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This was possible because phosphorylation markedly decreases the electrophoretic mobility of PHAS-I. Incubating 3T3-L1 adipocytes with rapamycin and wortmannin inhibited insulin-stimulated phosphorylation of PHAS-I at concentrations similar to those that inhibited activation of p70S6K. Both agents increased the amount of PHAS-I that co-purified with eukaryotic initiation factor-4E when extracts were fractionated using a cap affinity resin, indicating that PHAS-I binding to the initiation factor was increased. Incubating adipocytes with the protein phosphatase inhibitors, calyculin A and okadaic acid, increased PHAS-I phosphorylation and opposed the effects of rapamycin on decreasing PHAS-I phosphorylation. However, neither okadaic acid nor calyculin A abolished the effects of rapamycin on PHAS-I. These results suggest that the phosphorylation of PHAS-I in response to insulin occurs via the p70S6K signalling pathway. By regulating eukaryotic initiation factor-4E, PHAS-I may have important roles in the control of both protein synthesis and mitogenesis.


Subject(s)
Carrier Proteins , Enzyme Inhibitors/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoproteins/metabolism , Repressor Proteins/metabolism , Ribosomal Protein S6 Kinases/metabolism , 3T3 Cells , Adaptor Proteins, Signal Transducing , Androstadienes/pharmacology , Animals , Cell Cycle Proteins , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Enzyme Activation/drug effects , Eukaryotic Initiation Factor-4E , Eukaryotic Initiation Factors , Fibroblasts , Immune Sera/immunology , Immunoblotting , Insulin/pharmacology , Marine Toxins , Mice , Okadaic Acid/pharmacology , Oxazoles/pharmacology , Peptide Initiation Factors/drug effects , Peptide Initiation Factors/metabolism , Phosphoproteins/drug effects , Phosphoproteins/immunology , Phosphorylation , Polyenes/pharmacology , Rabbits , Repressor Proteins/drug effects , Ribosomal Protein S6 Kinases/drug effects , Sirolimus , Wortmannin
9.
Adv Enzyme Regul ; 37: 239-67, 1997.
Article in English | MEDLINE | ID: mdl-9381973

ABSTRACT

PHAS-I and PHAS-II are members of a newly discovered family of proteins that regulate translation initiation. PHAS-I is expressed in a wide variety of cell types, but it is highest in adipocytes, where protein synthesis is markedly increased by insulin. PHAS-II is highest in liver and kidney, where very little PHAS-I is found. PHAS proteins bind to eIF-4E, the mRNA cap-binding protein, and inhibit translation of capped mRNA in vitro and in cells. In rat adipocytes PHAS-I is phosphorylated in at least five sites, all of which conform to the consensus, (Ser/Thr)-Pro. Both PHAS proteins are phosphorylated in response to insulin or growth factors, such as EGF, PDGF and IGF-1. Phosphorylation in the appropriate site(s) promotes dissociation of PHAS/eIF-4E complexes. This allows eIF-4E to bind to eIF-4G (p220), thereby increasing the amount of the eIF-4F complex and the rate of translation initiation. Increasing cAMP promotes PHAS-I dephosphorylation and increases binding to eIF-4E. Unlike PHAS-I, PHAS-II is readily phosphorylated by PKA in vitro, suggesting that regulation of the two proteins differs. However, increasing cAMP in cells also promotes dephosphorylation of PHAS-II. Thus, PHAS proteins appear to be key mediators not only of the stimulatory effects of insulin and growth factors on protein synthesis, but also of the inhibitory effects of cAMP. Moreover, by controlling eIF-4E PHAS proteins may be involved in the control of cell proliferation, as increasing eIF-4E is mitogenic and can even cause malignant transformation of cells. MAP kinase readily phosphorylates both PHAS-I and PHAS-II in vitro, but inhibiting activation of MAP kinase does not attenuate the effects of insulin on increasing phosphorylation of the PHAS proteins in adipocytes or skeletal muscle. MAP kinase phosphorylates neither PHAS-I nor PHAS-II at a significant rate when the proteins are bound to eIF-4E. Therefore, the role of MAP kinase in promoting the dissociation of PHAS/eIF-4E complexes is not clear. Of several protein kinases tested, only casein kinase-II phosphorylated PHAS-I when it was bound eIF-4E. Indeed, the bound form of PHAS-I was phosphorylated more rapidly than the free form. However, it is unlikely that casein kinase II regulates either PHAS protein, as the major site (Ser111) in PHAS-I phosphorylated by casein kinase II in vitro is not phosphorylated in adipocytes, and PHAS-II is not a substrate for casein kinase-II. Pharmacological and genetic evidence indicates that the mTOR/p70S6K pathway is involved in the control of PHAS-I and -II. Thus, PHAS proteins may be mediators of the effects of this pathway on protein synthesis and cell proliferation.


Subject(s)
Carrier Proteins , Cell Division , Cyclic AMP/metabolism , Eukaryotic Initiation Factors , Growth Substances/pharmacology , Insulin/metabolism , Phosphoproteins/metabolism , Protein Biosynthesis , RNA-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Cell Cycle Proteins , Cell Division/drug effects , Humans , Insulin/pharmacology , Molecular Sequence Data , Peptide Initiation Factors/metabolism , Phosphoproteins/chemistry , Protein Kinases/metabolism , RNA-Binding Proteins/chemistry , Signal Transduction/physiology
10.
J Biol Chem ; 271(47): 30199-204, 1996 Nov 22.
Article in English | MEDLINE | ID: mdl-8939971

ABSTRACT

The eukaryotic initiation factor 4E (eIF-4E)-binding proteins PHAS-I and PHAS-II were found to have overlapping but different patterns of expression in tissues. Both PHAS proteins were expressed in 3T3-L1 adipocytes, in which insulin stimulated their phosphorylation, promoted dissociation of PHAS.eIF-4E complexes, and decreased the ability of both to bind exogenous eIF-4E. The effects of insulin were attenuated by rapamycin and wortmannin, two agents that block activation of p70(S6K). Unlike PHAS-I, PHAS-II was readily phosphorylated by cAMP-dependent protein kinase in vitro; however, the effects of insulin on both PHAS proteins were attenuated by agents that increase intracellular cAMP, by cAMP derivatives, and by phosphodiesterase inhibitors. These agents also markedly inhibited the activation of p70(S6K). In summary, our results indicate that PHAS-I and -II are controlled by the mammalian target of rapamycin and p70(S6K) signaling pathway and that in 3T3-L1 adipocytes this pathway is inhibited by increased cAMP.


Subject(s)
Adipocytes/drug effects , Carrier Proteins , Cyclic AMP/pharmacology , Insulin/pharmacology , Phosphoproteins/metabolism , Protein Biosynthesis , Repressor Proteins/metabolism , 3T3 Cells , Adaptor Proteins, Signal Transducing , Adipocytes/metabolism , Animals , Cell Cycle Proteins , Eukaryotic Initiation Factors , Kinetics , Mice , Phosphorylation
11.
Stroke ; 26(10): 1893-900, 1995 Oct.
Article in English | MEDLINE | ID: mdl-7570745

ABSTRACT

BACKGROUND AND PURPOSE: Although the signaling pathway involving polyphosphoinositide (poly-PI) hydrolysis and release of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is an important mechanism for regulation of neuronal calcium homeostasis, the effect of cerebral ischemia-reperfusion on this calcium signaling pathway is not well understood. Because activity of this pathway is dependent on availability of ATP, this study is aimed at examining the poly-PI signaling pathway and high-energy metabolites in a rat stroke model. METHODS: Focal cerebral ischemia in rats was induced by temporary occlusion of the right middle cerebral artery and both common carotid arteries. Levels of Ins(1,4,5)P3 were determined by use of the radioreceptor binding assay. Poly-PI turnover in rat cortex was assessed with an in vivo protocol involving intracerebral injection of [3H] inositol and systemic administration of lithium. High-energy metabolites (ATP, ADP, and AMP) were analyzed by high-performance liquid chromatography. RESULTS: Ischemia induced an increase in poly-PI turnover in the right middle cerebral artery cortex, but reperfusion led to a decline in this signaling activity. However, Ins(1,4,5)P3 levels decreased during ischemia, and these levels were not restored if ischemic insults were longer than 30 minutes. ATP levels decreased to 26% of control during ischemia and recovered to 80% of control during the initial 4 hours of reperfusion; these changes were followed by a second phase of decline. CONCLUSIONS: Results show an important relationship between ischemia-induced depletion of high-energy metabolites and poly-PI signaling activity. However, the uncoupling between Ins(1,4,5)P3 and ATP during reperfusion after severe ischemia suggests that metabolism of Ins(1,4,5)P3 is more stringently regulated than ATP.


Subject(s)
Adenosine Triphosphate/metabolism , Brain/metabolism , Inositol 1,4,5-Trisphosphate/metabolism , Ischemic Attack, Transient/metabolism , Phosphatidylinositol Phosphates/metabolism , Reperfusion , Adenosine Diphosphate/metabolism , Adenosine Monophosphate/metabolism , Animals , Calcium/metabolism , Carotid Artery, Common , Cerebral Arteries , Cerebral Cortex/metabolism , Chromatography, High Pressure Liquid , Disease Models, Animal , Homeostasis , Hydrolysis , Neurons/metabolism , Rats , Signal Transduction , Tritium
12.
J Biol Chem ; 270(31): 18531-8, 1995 Aug 04.
Article in English | MEDLINE | ID: mdl-7629182

ABSTRACT

PHAS-I levels increased 8-fold as 3T3-L1 fibroblasts differentiated into adipocytes and acquired sensitivity to insulin. Insulin increased PHAS-I protein (3.3-fold after 2 days), the rate of PHAS-I synthesis (3-fold after 1 h), and the half-life of the protein (from 1.5 to 2.5 days). Insulin also increased the phosphorylation of PHAS-I and promoted dissociation of the PHAS-I eukaryotic initiation factor-4E (eIF-4E) complex, effects that were maximal within 10 min. With recombinant [H6]PHAS-I as substrate, mitogen-activated protein (MAP) kinase was the only insulin-stimulated PHAS-I kinase detected after fractionation of extracts by Mono Q chromatography; however, MAP kinase did not readily phosphorylate [H6]PHAS-I when the [H6]PHAS-I.eIF-4E complex was the substrate. Thus, while MAP kinase may phosphorylate free PHAS-I, it is not sufficient to dissociate the complex. Moreover, rapamycin attenuated the stimulation of PHAS-I phosphorylation by insulin and markedly inhibited dissociation of PHAS-I.eIF-4E, without decreasing MAP kinase activity. Rapamycin abolished the effects of insulin on increasing phosphorylation of ribosomal protein S6 and on activating p70S6K. The MAP kinase kinase inhibitor, PD 098059, markedly decreased MAP kinase activation by insulin, but it did not change PHAS-I phosphorylation or the association of PHAS-I with eIF-4E. In summary, insulin increases the expression of PHAS-I and promotes phosphorylation of multiple sites in the protein via multiple transduction pathways, one of which is rapamycin-sensitive and independent of MAP kinase. Rapamycin may inhibit translation initiation by increasing PHAS-I binding to eIF-4E.


Subject(s)
Adipocytes/metabolism , Carrier Proteins , Insulin/pharmacology , Muscle Proteins , Phosphoproteins/biosynthesis , Signal Transduction , 3T3 Cells , Adaptor Proteins, Signal Transducing , Adipocytes/drug effects , Amino Acid Sequence , Animals , Base Sequence , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cell Cycle Proteins , Cell Differentiation , Eukaryotic Initiation Factors , Glucose Transporter Type 4 , Mice , Molecular Sequence Data , Monosaccharide Transport Proteins/biosynthesis , Phosphorylation/drug effects , Polyenes/pharmacology , Protein Kinase Inhibitors , Protein Kinases/metabolism , Protein Processing, Post-Translational , RNA, Messenger/analysis , Sequence Homology, Amino Acid , Sirolimus
13.
Proc Natl Acad Sci U S A ; 92(16): 7222-6, 1995 Aug 01.
Article in English | MEDLINE | ID: mdl-7638171

ABSTRACT

Incubating rat aortic smooth muscle cells with either platelet-derived growth factor BB (PDGF) or insulin-like growth factor I (IGF-I) increased the phosphorylation of PHAS-I, an inhibitor of the mRNA cap binding protein, eukaryotic initiation factor (eIF) 4E. Phosphorylation of PHAS-I promoted dissociation of the PHAS-I-eIF-4E complex, an effect that could partly explain the stimulation of protein synthesis by the two growth factors. Increasing cAMP with forskolin decreased PHAS-I phosphorylation and markedly increased the amount of eIF-4E bound to PHAS-I, effects consistent with an action of cAMP to inhibit protein synthesis. Both PDGF and IGF-I activated p70S6K, but only PDGF increased mitogen-activated protein kinase activity. Forskolin decreased by 50% the effect of PDGF on increasing p70S6K, and forskolin abolished the effect of IGF-I on the kinase. The effects of PDGF and IGF-I on increasing PHAS-I phosphorylation, on dissociating the PHAS-I-eIF-4E complex, and on increasing p70S6K were abolished by rapamycin. The results indicate that IGF-I and PDGF increase PHAS-I phosphorylation in smooth muscle cells by the same rapamycin-sensitive pathway that leads to activation of p70S6K.


Subject(s)
Carrier Proteins , Cyclic AMP/pharmacology , Peptide Initiation Factors/metabolism , Phosphoproteins/metabolism , Polyenes/pharmacology , 3T3 Cells , Adaptor Proteins, Signal Transducing , Animals , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Cycle Proteins , Cells, Cultured , Colforsin/pharmacology , Cyclic AMP/metabolism , Eukaryotic Initiation Factor-4E , Eukaryotic Initiation Factors , Insulin-Like Growth Factor I/pharmacology , Intracellular Signaling Peptides and Proteins , Mice , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Phosphorylation , Platelet-Derived Growth Factor/pharmacology , Protein Serine-Threonine Kinases/metabolism , Rats , Ribosomal Protein S6 Kinases , Sirolimus
14.
J Neurochem ; 64(2): 761-6, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7830070

ABSTRACT

An alteration in signal transduction systems in Alzheimer's disease (AD) would likely be of pathophysiological significance, because these processes control normal brain functions. Previously, a diminished beta-adrenergic-mediated cyclic AMP response was found in cultured fibroblasts from AD patients. Because cross-talk between the phosphoinositide and cyclic AMP pathways exists, the phosphoinositide cascade was studied under conditions that were similar to those for studying the cyclic AMP response. Cells from AD patients and age-matched controls responded to bradykinin (BK) and released inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in a time- and dose-dependent manner. The level of Ins(1,4,5)P3 increased rapidly and transiently in response to BK, peaked at 5 s, but still remained 116-132% above the basal level by 30 s. Although the temporal patterns were similar in both groups, the Ins(1,4,5)P3 concentrations in AD fibroblasts were 73 and 89% above levels in the age-matched controls at 5 and 10 s, respectively. Prostaglandin E1 also increased Ins(1,4,5)P3 formation, but this response was not different between the two groups. Although KD (affinity) values for the BK receptor were similar in both control and AD cells, the number of BK receptors (Bmax) was significantly elevated in AD fibroblasts (186.8 +/- 0.8 fmol/mg of protein) as compared with control fibroblasts (57.2 +/- 15.3 fmol/mg of protein). These results indicate that the elevated Ins(1,4,5)P3 production in response to BK in AD fibroblasts is positively correlated with an increase in the receptor numbers.


Subject(s)
Alzheimer Disease/metabolism , Inositol 1,4,5-Trisphosphate/metabolism , Receptors, Bradykinin/metabolism , Up-Regulation , Aged , Bradykinin/pharmacology , Dose-Response Relationship, Drug , Fibroblasts/metabolism , Humans , Middle Aged , Reference Values , Skin/metabolism , Time Factors
15.
Science ; 266(5185): 653-6, 1994 Oct 28.
Article in English | MEDLINE | ID: mdl-7939721

ABSTRACT

PHAS-I is a heat-stable protein (relative molecular mass approximately 12,400) found in many tissues. It is rapidly phosphorylated in rat adipocytes incubated with insulin or growth factors. Nonphosphorylated PHAS-I bound to initiation factor 4E (eIF-4E) and inhibited protein synthesis. Serine-64 in PHAS-I was rapidly phosphorylated by mitogen-activated (MAP) kinase, the major insulin-stimulated PHAS-I kinase in adipocyte extracts. Results obtained with antibodies, immobilized PHAS-I, and a messenger RNA cap affinity resin indicated that PHAS-I did not bind eIF-4E when serine-64 was phosphorylated. Thus, PHAS-I may be a key mediator of the stimulation of protein synthesis by the diverse group of agents and stimuli that activate MAP kinase.


Subject(s)
Carrier Proteins , Insulin/pharmacology , Peptide Initiation Factors/metabolism , Phosphoproteins/metabolism , Protein Biosynthesis , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , 3T3 Cells , Adaptor Proteins, Signal Transducing , Adipocytes/metabolism , Animals , Cell Cycle Proteins , Eukaryotic Initiation Factors , Intracellular Signaling Peptides and Proteins , Mice , Mitogen-Activated Protein Kinase 1 , Peptide Initiation Factors/isolation & purification , Phosphorylation , Rats , Recombinant Proteins/metabolism , Serine/metabolism
16.
Nature ; 371(6500): 762-7, 1994 Oct 27.
Article in English | MEDLINE | ID: mdl-7935836

ABSTRACT

The cloning is described of two related human complementary DNAs encoding polypeptides that interact specifically with the translation initiation factor eIF-4E, which binds to the messenger RNA 5'-cap structure. Interaction of these proteins with eIF-4E inhibits translation but treatment of cells with insulin causes one of them to become hyperphosphorylated and dissociate from eIF-4E, thereby relieving the translational inhibition. The action of this new regulator of protein synthesis is therefore modulated by insulin, which acts to stimulate the overall rate of translation and promote cell growth.


Subject(s)
Carrier Proteins , Insulin/physiology , Peptide Initiation Factors/metabolism , Phosphoproteins/metabolism , Protein Biosynthesis/physiology , RNA Caps/metabolism , Acid Phosphatase/metabolism , Adaptor Proteins, Signal Transducing , Adipose Tissue/metabolism , Amino Acid Sequence , Animals , Cell Cycle Proteins , Cell Line , Cloning, Molecular , DNA, Complementary , Escherichia coli , Eukaryotic Initiation Factor-4E , Humans , Intracellular Signaling Peptides and Proteins , Male , Molecular Sequence Data , Phosphorylation , Protein Binding , Rabbits , Rats , Rats, Sprague-Dawley , Sequence Homology, Amino Acid
17.
J Biol Chem ; 269(37): 23185-91, 1994 Sep 16.
Article in English | MEDLINE | ID: mdl-8083223

ABSTRACT

PHAS-I is a heat- and acid-stable protein that is phosphorylated on Ser/Thr residues in response to insulin and growth factors. To investigate the phosphorylation of PHAS-I, the protein was expressed in bacteria and purified for use as substrate in protein kinase reactions in vitro. Recombinant PHAS-I was rapidly and stoichiometrically phosphorylated by mitogen-activated protein (MAP) kinase. At saturating MgATP, the Km and Vmax observed with PHAS-I were almost identical to those obtained with myelin basic protein, one of the best MAP kinase substrates. PHAS-I was also phosphorylated at a significant rate by casein kinase II and protein kinase C. To investigate sites of phosphorylation, PHAS-I was digested with collagenase and phosphopeptides were resolved by reverse phase high performance liquid chromatography. Almost all of the phosphate introduced by MAP kinase was recovered in the peptide, Leu-Met-Glu-Cys-Arg-Asn-Ser-Pro-Val-Ala-Lys-Thr. 32P was released in the seventh cycle of Edman degradation, identifying the Ser (Ser64) as the phosphorylated residue. Ser64 was also phosphorylated in response to insulin in rat adipocytes. We conclude that PHAS-I is a substrate for MAP kinase both in vivo and in vitro. As PHAS-I is one of the most prominent insulin-stimulated phosphoproteins in adipocytes, it may qualify as the major MAP kinase substrate in these cells.


Subject(s)
Adipocytes/metabolism , Carrier Proteins , Insulin/pharmacology , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Adipocytes/drug effects , Amino Acid Sequence , Animals , Intracellular Signaling Peptides and Proteins , Male , Mitogen-Activated Protein Kinase 1 , Molecular Sequence Data , Phosphorylation , Rats , Rats, Sprague-Dawley , Recombinant Proteins/metabolism
18.
J Biol Chem ; 269(33): 21255-61, 1994 Aug 19.
Article in English | MEDLINE | ID: mdl-8063749

ABSTRACT

Rat adipocytes were incubated with insulin or epidermal growth factor (EGF) before the mitogen-activated protein (MAP) kinases, ERK-1 and ERK-2, and the ribosomal protein S6 kinases, Rsk-2 and p70S6K, were resolved by ion exchange chromatography and identified by immunoblotting. EGF was more effective than insulin in increasing the activity of two kinases that reacted with Rsk-2 antibody (2- and 2.5-fold with EGF versus 1.6- and 1.2-fold with insulin). EGF was also more effective than insulin in increasing the activity of ERK-1 (5-fold versus 2-fold) and ERK-2 (2.5-fold versus 1.5 fold). The activity of p70S6K was increased by approximately the same extent by EGF and insulin (1.7-fold versus 2-fold). Rapamycin blocked activation of p70S6K by insulin, but it did not attenuate the effect (2-fold) of insulin on increasing the glycogen synthase activity ratio (+/-glucose-6-P). Insulin increased glucose incorporation into glycogen and 2-deoxyglucose uptake by approximately 5-fold, whereas EGF and phorbol 12-myristate were without effect. Thus, activation of MAP kinases and ribosomal protein S6 kinases appears insufficient to activate glycogen synthase or glucose transport, the two key components in the stimulation of glycogen synthesis by insulin.


Subject(s)
Adipocytes/metabolism , Glucose/metabolism , Glycogen/biosynthesis , Protein Serine-Threonine Kinases/metabolism , 3T3 Cells , Adipocytes/drug effects , Adipocytes/enzymology , Amino Acid Sequence , Animals , Biological Transport , Enzyme Activation/drug effects , Epidermal Growth Factor/pharmacology , Glycogen Synthase/metabolism , Insulin/pharmacology , Kinetics , Mice , Molecular Sequence Data , Polyenes/pharmacology , Rats , Rats, Sprague-Dawley , Ribosomal Protein S6 Kinases , Sirolimus , Tetradecanoylphorbol Acetate/pharmacology
19.
Brain Res ; 622(1-2): 169-76, 1993 Sep 17.
Article in English | MEDLINE | ID: mdl-8242355

ABSTRACT

The cholinergic receptor-linked poly-phosphoinositide hydrolysis was studied in mouse cerebrum and cerebellum after prelabeling the brain with [3H]inositol. I.p. injection of Li (8 meq/kg) to C57Bl/6J mice for 4 h resulted in 14- and five-fold increases in [3H]inositol-labeled inositol monophosphate (IP1) in cerebrum and cerebellum, respectively. The labeled inositol bisphosphate (IP2) was also increased 83 and 19% in cerebrum and cerebellum, respectively. Prior injection of atropine (100 mg/kg) resulted in inhibition of Li-induced increases in labeled IP1 by 74 and 56% in cerebrum and cerebellum, respectively. Administration of pilocarpine (20 mg/kg) to the Li-treated mice for 30 min resulted in further increases in labeled IP1 and IP2 and a concomitant decrease in labeled inositol in cerebrum but not in cerebellum. Mass measurements of IP1 and IP2 isomers by HPLC revealed that inositol 1-monophosphate (Ins(1)P), inositol 4-monophosphate (Ins(4)P) and inositol 1,4-bisphosphate (Ins(1,4)P2) were all increased by pilocarpine administration in the Li-treated mouse cerebrum. The effects of pilocarpine administration in mouse cerebrum (increases in IP1 and IP2) could be completely inhibited by preinjection of atropine. Atropine injection also decreased the levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Surprisingly, a decrease in Ins(1,4,5)P3 level was also found in non-Li-treated mice after pilocarpine administration (30 mg/kg, 10-40 min). Except for the increase (20%) in [32P]-labeled PIP in the cerebrum, Li or Li together with pilocarpine administration did not alter the levels of [3H]inositol or [32P]phosphate-labeled phosphoinositides.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Brain/metabolism , Cerebellum/metabolism , Phosphatidylinositols/metabolism , Receptors, Cholinergic/metabolism , Animals , Atropine , Basal Metabolism , Chromatography, High Pressure Liquid , Inositol 1,4,5-Trisphosphate/metabolism , Inositol Phosphates/analysis , Male , Mice , Mice, Inbred C57BL , Phosphorus Radioisotopes , Pilocarpine , Stereoisomerism
20.
Ann N Y Acad Sci ; 679: 382-7, 1993 May 28.
Article in English | MEDLINE | ID: mdl-8390148

ABSTRACT

Results from this study clearly indicate that Ins(1,4,5)P3 3-kinase is a target enzyme of cerebral ischemia insult. This enzyme is responsible for removal of Ins(1,4,5)P3 which, in turn, plays an important role in the maintenance of intracellular Ca2+ homeostasis. Not only did a time-dependent decrease in enzyme activity occur due to the focal cerebral ischemic insult, but there was also a second phase for the decline in enzyme activity around 6 h after the insult. Examination of the mRNA for the 3-kinase in frozen brain sections suggested an increase in message at a time (around 8 h) prior to development of tissue infarct. Since the initial decline in enzyme activity during ligation correlated well with the time for development of an infarct, assay of this enzyme could be used as a biochemical marker of cerebral ischemic insult.


Subject(s)
Cerebral Cortex/enzymology , Ischemic Attack, Transient/enzymology , Phosphotransferases (Alcohol Group Acceptor) , Phosphotransferases/metabolism , Animals , Biomarkers , Brain Stem/enzymology , Cerebellum/enzymology , Hippocampus/enzymology , Inositol Polyphosphate 5-Phosphatases , Phosphoric Monoester Hydrolases/metabolism , Rats , Reperfusion , Time Factors
SELECTION OF CITATIONS
SEARCH DETAIL
...