-
Journal of Virology Jul 1998Inhibitors of the phosphatidylinositol 3-kinase (PI3 kinase)-FKBP-rapamycin-associated protein (FRAP) pathway, such as rapamycin and wortmannin, induce dephosphorylation...
Inhibitors of the phosphatidylinositol 3-kinase (PI3 kinase)-FKBP-rapamycin-associated protein (FRAP) pathway, such as rapamycin and wortmannin, induce dephosphorylation and activation of the suppressor of cap-dependent translation, 4E-BP1. Encephalomyocarditis virus (EMCV) infection leads to activation of 4E-BP1 at the time of host translation shutoff. Consistent with these data, rapamycin mildly enhances the synthesis of viral proteins and the shutoff of host cell protein synthesis after EMCV infection. In this study, two defective EMCV strains were generated by deleting portions of the 2A coding region of an infectious cDNA clone. These deletions dramatically decreased the efficiency of viral protein synthesis and abolished the virus-induced shutoff of host translation after infection of BHK-21 cells. Both translation and processing of the P1-2A capsid precursor polypeptide are impaired by the deletions in 2A. The translation and yield of mutant viruses were increased significantly by the presence of rapamycin and wortmannin during infection. Thus, inhibition of the PI3 kinase-FRAP signaling pathway partly complements mutations in 2A protein and reverses a slow-virus phenotype.
Topics: Androstadienes; Animals; Cells, Cultured; Cricetinae; Defective Viruses; Encephalomyocarditis virus; Enzyme Inhibitors; Phosphoinositide-3 Kinase Inhibitors; Polyenes; Protein Biosynthesis; Rabbits; Sirolimus; Viral Proteins; Virus Replication; Wortmannin
PubMed: 9621041
DOI: 10.1128/JVI.72.7.5811-5819.1998 -
DNA and Cell Biology Dec 2019Complex human diseases such as metabolic disorders, cancer, neurodegenerative diseases, and mitochondrial dysfunctions arise from the biochemical or genetic defects in...
Complex human diseases such as metabolic disorders, cancer, neurodegenerative diseases, and mitochondrial dysfunctions arise from the biochemical or genetic defects in various cellular processes. Therefore, it is important to understand which metabolic processes are affected by which cellular impairment. Because genome-wide screening of mutant collections (haploid/diploid deletion library) provides important clues for the understanding of conserved biological processes and for finding potential target genes, we screened the haploid mutant collection of with wortmannin that inhibits phosphatidylinositol-3-kinase signaling. Using genome-wide screening, we determined that 52 mutants were resistant to this chemical. When 52 genes that are deleted in these mutants were grouped in 41 different biological processes, we found that 37 of them have human orthologues and 4 genes were associated with human metabolic disorders. In addition, when we examined the pathways in which these 52 genes function, we determined that 9 genes were related to phosphorylation process. These results might provide new insights for better understanding of certain human diseases.
Topics: Antifungal Agents; Drug Resistance, Fungal; Genome, Fungal; Genome-Wide Association Study; Humans; Mutation; Phosphorylation; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Signal Transduction; Wortmannin
PubMed: 31657618
DOI: 10.1089/dna.2019.5003 -
Biochemical and Biophysical Research... Aug 1994Activated phosphoinositide 3-kinase has been suggested to be involved in cytoskeletal reorganization and mitogenesis. Lysophosphatidic acid has been found to trigger...
Activated phosphoinositide 3-kinase has been suggested to be involved in cytoskeletal reorganization and mitogenesis. Lysophosphatidic acid has been found to trigger several "classic" signal transduction pathways and also accounts for the ability of serum to stimulate focal adhesion and stress fiber formation in fibroblasts. We present evidence that serum or lysophosphatidic acid activates phosphoinositide 3-kinase in CHRF-288 cells (a leukemic cell line derived from megakaryoblasts), leading to transient accumulation of phosphatidylinositol(3,4,5)P3 and increased phosphatidylinositol(3,4)P2, and stimulates phospholipase C. Exposure of CHRF cells to serum promotes cell proliferation, whereas exposure to lysophosphatidic acid does not. Wortmannin, a potent inhibitor of phosphoinositide 3-kinase, inhibits 3-phosphorylated phosphoinositide accumulation and cell proliferation without inhibiting phospholipase C. We propose that activation of phosphoinositide 3-kinase is required for the full proliferative response of CHRF cells exposed to serum but, as gauged by our findings for lysophosphatidic acid, not sufficient to induce proliferation.
Topics: Androstadienes; Blood; Cell Division; Enzyme Activation; Humans; Lysophospholipids; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Tumor Cells, Cultured; Wortmannin
PubMed: 8060348
DOI: 10.1006/bbrc.1994.2118 -
Proceedings of the National Academy of... May 1994Wortmannin (WT) and its derivative 17-hydroxywortmannin (HWT) inhibit at nanomolar concentrations superoxide formation and exocytosis in neutrophils stimulated with...
Wortmannin (WT) and its derivative 17-hydroxywortmannin (HWT) inhibit at nanomolar concentrations superoxide formation and exocytosis in neutrophils stimulated with chemotactic agonists. Treatment of neutrophils with radiolabeled [3H]HWT resulted in specific and saturable binding that paralleled the inhibition of the respiratory burst. Both half-maximal binding and half-maximal inhibition were observed at 5 nM, and > 90% of maximal binding and inhibition was observed at 20 nM HWT. Fluorography of subcellular fractions that were separated on NaDodSO4/PAGE showed that [3H]HWT binds covalently to a 110-kDa cytosolic protein. The WT-binding protein was purified from human neutrophils and bovine brain homogenates by column chromatography. The pure protein was eluted from gel filtration columns with an apparent molecular mass of 200 kDa and showed a heterodimeric structure on Coomassie-stained NaDodSO4/PAGE. In addition to the 110 kDa wortmannin binding protein an equally intense band was seen migrating at 85 kDa. This band was identified on Western blots as p85 alpha, the regulatory subunit of phosphatidylinositol (PI) 3-kinase (ATP:1-phosphatidyl-1D-myo-inositol 3-phosphotransferase, EC 2.7.1.137). The purified protein contained PI 3-kinase activity that was enriched > 20,000-fold from human neutrophil cytosol during preparation. The data impose a key role for PI 3-kinase-mediated signal transduction through guanine nucleotide-binding protein-coupled receptors and suggest that 3-phosphorylated inositol phospholipids are important second messengers for immediate responses in neutrophils. Furthermore, the results show that WT is a powerful and selective tool to study the function of PI 3-kinase.
Topics: Androstadienes; Blotting, Western; Exocytosis; GTP-Binding Proteins; Humans; In Vitro Techniques; Neutrophils; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Receptors, Cell Surface; Signal Transduction; Superoxides; Wortmannin
PubMed: 8197165
DOI: 10.1073/pnas.91.11.4960 -
Pharmacological Reports : PR 2011The effects of the sulfhydryl reducing agents 2-mercaptoethanol and dithiothreitol on wortmannin-induced inhibition of phosphoinositide 3-kinase (PI3K) were studied in...
The effects of the sulfhydryl reducing agents 2-mercaptoethanol and dithiothreitol on wortmannin-induced inhibition of phosphoinositide 3-kinase (PI3K) were studied in order to examine whether the sulfhydryl reducing agents directly affect the wortmannin inhibition of PI3K. These reducing agents are commonly used to stabilize enzyme structures by maintaining protein sulfhydryl groups in the reduced state. Preincubation of wortmannin with millimolar levels of 2-mercaptoethanol, a sulfhydryl derivative of ethanol, markedly prevented subsequent wortmannin-induced inhibition of PI3K. In contrast, ethanol, 2-mercaptoethanol lacking sulfhydryl group, and 2-(methylthio)ethanol, a methyl derivative of the sulfhydryl group of 2-mercaptoethanol, had little effect on the wortmannin-induced inhibition of PI3K, which suggests that the prevention of wortmannin-induced inhibition by 2-mercaptoethanol occurs through the sulfhydryl group of this agent. Moreover, dithiothreitol, a second sulfhydryl reducing agent, also markedly prevented wortmannin-induced inhibition of PI3K. These results indicate that the wortmannin-induced inhibition of PI3K is markedly prevented by millimolar concentrations of sulfhydryl reducing agents such as 2-mercaptoethanol and dithiothreitol in the medium, presumably by the binding of wortmannin to the agents.
Topics: Androstadienes; Dithiothreitol; Drug Interactions; Enzyme Inhibitors; Humans; Mercaptoethanol; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Sulfhydryl Reagents; Wortmannin
PubMed: 21857084
DOI: 10.1016/s1734-1140(11)70585-0 -
Biochemical and Biophysical Research... Mar 1997P19 EC cells undergoes apoptosis during neuronal differentiation induced by retinoic acid. Two CPP32-like proteases, CPP32 and Mch-3, are expressed in untreated and...
P19 EC cells undergoes apoptosis during neuronal differentiation induced by retinoic acid. Two CPP32-like proteases, CPP32 and Mch-3, are expressed in untreated and retinoic acid-treated P19 EC cells. CPP32-like activity is remarkably increased in apoptosis during neuronal differentiation of P19 EC cells. Inhibition of CPP32-like proteases prevents apoptosis, suggesting that activation of CPP32-like proteases play central roles in the apoptosis during neuronal differentiation of P19 EC cells. Wortmannin, PI-3K inhibitor, enhances the CPP32-like activity of the retinoic acid-treated P19 EC cells. PI-3K may be involved in the apoptosis during neuronal differentiation as negative regulator.
Topics: Androstadienes; Apoptosis; Carcinoma, Embryonal; Caspase 3; Caspases; Cell Differentiation; Cysteine Endopeptidases; DNA Fragmentation; Molecular Sequence Data; Neurons; Tretinoin; Tumor Cells, Cultured; Wortmannin
PubMed: 9125129
DOI: 10.1006/bbrc.1997.6234 -
Drug Metabolism and Disposition: the... May 2004The phosphatidylinositol 3-kinase inhibitor, wortmannin, is extensively used in molecular signaling studies and has been proposed as a potential antineoplastic agent.... (Comparative Study)
Comparative Study
The phosphatidylinositol 3-kinase inhibitor, wortmannin, is extensively used in molecular signaling studies and has been proposed as a potential antineoplastic agent. The failure to detect wortmannin in mouse plasma after i.v. administration prompted in vitro studies of wortmannin metabolism. Wortmannin was incubated with mouse tissue homogenates, homogenate fractions, or purified, recombinant human carbonyl reductase in the presence of specified cofactors and inhibitors. Reaction products were characterized and quantified with liquid chromatography (LC)/mass spectrometry. Reaction rates were characterized using Michaelis-Menten kinetics. Wortmannin was metabolized to a material 2 atomic mass units greater than wortmannin. Liver homogenate had the highest metabolic activity. Some metabolism occurred in kidney and lung homogenates. Very little metabolism occurred in brain or red blood cell homogenates. Liver S9 fraction and cytosol metabolized wortmannin in the presence of NADPH and, to a much lesser extent, in the presence of NADH. Microsomal metabolism of wortmannin was minimal. Purified, recombinant human carbonyl reductase metabolized wortmannin. Quercetin, a carbonyl reductase inhibitor, greatly decreased wortmannin metabolism by S9, cytosol, and carbonyl reductase. The K(M) for wortmannin metabolism by purified, recombinant human carbonyl reductase was 119 +/- 9 microM, and the V(max) was 58 +/- 9 nmol/min/mg of protein. LC-tandem mass spectrometry spectra indicated that carbonyl reductase metabolized wortmannin to 17-OH-wortmannin. Wortmannin reduction by carbonyl reductase may partly explain why wortmannin is not detected in plasma after being administered to mice. Metabolism of wortmannin to 17-OH-wortmannin has mechanistic, and possibly toxicologic, implications because 17-OH-wortmannin is 10-fold more potent an inhibitor of phosphatidylinositol 3-kinase than is wortmannin.
Topics: Alcohol Oxidoreductases; Aldehyde Reductase; Aldo-Keto Reductases; Androstadienes; Animals; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Humans; Mice; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Wortmannin
PubMed: 15100170
DOI: 10.1124/dmd.32.5.490 -
Methods in Molecular Biology (Clifton,... 2018Eukaryotic cells comprise various organelles surrounded by the membrane. Each organelle is characterized by unique proteins and lipids and has its own specific...
Eukaryotic cells comprise various organelles surrounded by the membrane. Each organelle is characterized by unique proteins and lipids and has its own specific functions. Single membrane-bounded organelles, including the Golgi apparatus, endosomes, and vacuoles are connected by membrane trafficking. Identifying the organelle localization of a protein of interest is essential for determining the proteins physiological functions. Here, we describe methods for determining protein subcellular localization using the inhibitors brefeldin A and wortmannin in Arabidopsis thaliana.
Topics: Arabidopsis; Arabidopsis Proteins; Brefeldin A; Endosomes; Golgi Apparatus; Protein Kinase Inhibitors; Protein Synthesis Inhibitors; Protein Transport; Vacuoles; Wortmannin; trans-Golgi Network
PubMed: 29916078
DOI: 10.1007/978-1-4939-7856-4_12 -
The Journal of Biological Chemistry Dec 1993The surface engagement of high affinity immunoglobulin E receptor (Fc epsilon RI) of rat basophilic leukemia 2H3 (RBL-2H3) cells induced histamine secretion and...
The surface engagement of high affinity immunoglobulin E receptor (Fc epsilon RI) of rat basophilic leukemia 2H3 (RBL-2H3) cells induced histamine secretion and leukotriene release following activation of the tyrosine kinase Lyn together with phosphatidylinositol 3-kinase (PI3-kinase). Wortmannin inhibited the activity of partially purified PI3-kinase from calf thymus, as well as the PI3-kinase activity in anti-PI3-kinase p85 immunoprecipitates from RBL-2H3 cells, at a concentration as low as 1.0 nM and with IC50 values of 3.0 nM, but did not inhibit PI4-kinase activity. The inhibition of PI3-kinase by wortmannin was irreversible. Wortmannin inhibited both Fc epsilon RI-mediated histamine secretion and leukotriene release up to 80% with IC50 values of 2.0 and 3.0 nM, respectively. Wortmannin inhibited PI3-kinase activity in intact cells up to 80% with an IC50 value of 2.0 nM, which is almost equal to those for PI3-kinase in vitro and for histamine secretion and leukotriene release. With anti-wortmannin antibody, we have shown that wortmannin binds to the 110-kDa protein, but not to PI3-kinase 85-kDa regulatory subunit both in vitro and in whole cells. Furthermore, there was a positive correlation between the potencies of wortmannin derivatives as inhibitors of PI3-kinase and as inhibitors of histamine secretion. Wortmannin had no effect on the activation of the tyrosine kinase Lyn. These results suggest that PI3-kinase is involved in the signal transduction pathway responsible for histamine secretion following stimulation of Fc epsilon RI and that wortmannin blocks these responses through direct interaction with the catalytic subunit of this enzyme.
Topics: Androstadienes; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Line; Cell Membrane; Dose-Response Relationship, Drug; Histamine Release; Kinetics; Leukemia, Basophilic, Acute; Leukotrienes; Models, Biological; Molecular Structure; Mycotoxins; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Rats; Receptors, IgE; Structure-Activity Relationship; Tumor Cells, Cultured; Wortmannin
PubMed: 7503989
DOI: No ID Found -
Acta Pharmacologica Sinica Jun 2005To investigate the effect of dishevelled-1 (DVL-1) on wortmannin-induced Alzheimer-like hyperphosphorylation of cytoskeletal proteins in mouse neuroblastoma 2a (N2a)...
AIM
To investigate the effect of dishevelled-1 (DVL-1) on wortmannin-induced Alzheimer-like hyperphosphorylation of cytoskeletal proteins in mouse neuroblastoma 2a (N2a) cells.
METHODS
Cultured N2a cells were transitorily transfected with DVL-1 expression plasmid using Lipofectamine 2000. Western blot and immunofluorescence microscopy were used to measure the phosphorylation of neurofilament and tau.
RESULTS
Level of phosphorylated neurofilament at SMI31 epitope and phosphorylated tau determined by PHF-1 was increased at 1 h and 3 h and back to normal at 6 h after wortmannin 1 micromol/L treatment. The highest level of phosphorylated neurofilament and phosphorylated tau was seen at 1 h and 3 h after wortmannin treatment, respectively. When DVL-1 protein was overexpressed, the hyperphosphorylation of neurofilament at SMI31 and SMI32 epitopes and tau at PHF-1 (Ser-396/404), M4 (Thr-231/Ser-235), and Tau-1 (Ser-198/199/202) epitopes was attenuated.
CONCLUSION
Overexpression of mouse DVL-1 protein inhibits wortmannin-induced hyperphosphorylation of neurofilament and tau in N2a cells.
Topics: Adaptor Proteins, Signal Transducing; Androstadienes; Animals; Dishevelled Proteins; Mice; Neuroblastoma; Neurofilament Proteins; Phosphoproteins; Phosphorylation; Proteins; Transfection; Tumor Cells, Cultured; Wortmannin; tau Proteins
PubMed: 15916733
DOI: 10.1111/j.1745-7254.2005.00131.x