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Alex Toker - One of the best experts on this subject based on the ideXlab platform.
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d3 phosphoinositides and outside in integrin signaling by glycoprotein iib iiia mediate platelet actin assembly and filopodial extension induced by phorbol 12 myristate 13 acetate
Journal of Biological Chemistry, 1996Co-Authors: John H Hartwig, Lewis C Cantley, Sophia Kung, Tibor Kovacsovics, Paul A Janmey, Thomas P Stossel, Alex TokerAbstract:Phorbol 12-myristate 13-acetate (PMA) uncaps a small number of the fast-growing (barbed) ends of actin filaments, thereby eliciting slow actin assembly and extension of filopodia in human blood platelets. These reactions, which also occur in response to immunologic perturbation of the integrin glycoprotein (GP) IIb-IIIa, are sensitive to the phosphoinositide 3-kinase inhibitor Wortmannin. Platelets deficient in GPIIb-IIIa integrins or with GPIIb-IIIa function inhibited by calcium chelation or the peptide RGDS have diminished PMA responsiveness. The effects of PMA contrast with thrombin receptor stimulation by ≥5 μM thrombin receptor-activating peptide (TRAP), which causes rapid and massive Wortmannin-insensitive actin assembly and lamellar and filopodial extension. However, we show here that Wortmannin can inhibit filopod formation if the thrombin receptor is ligated using suboptimal doses (
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d3 phosphoinositides and outside in integrin signaling by glycoprotein iib iiia mediate platelet actin assembly and filopodial extension induced by phorbol 12 myristate 13 acetate
Journal of Biological Chemistry, 1996Co-Authors: John H Hartwig, Lewis C Cantley, Sophia Kung, Tibor Kovacsovics, Paul A Janmey, Thomas P Stossel, Alex TokerAbstract:Abstract Phorbol 12-myristate 13-acetate (PMA) uncaps a small number of the fast-growing (barbed) ends of actin filaments, thereby eliciting slow actin assembly and extension of filopodia in human blood platelets. These reactions, which also occur in response to immunologic perturbation of the integrin glycoprotein (GP) IIb-IIIa, are sensitive to the phosphoinositide 3-kinase inhibitor Wortmannin. Platelets deficient in GPIIb-IIIa integrins or with GPIIb-IIIa function inhibited by calcium chelation or the peptide RGDS have diminished PMA responsiveness. The effects of PMA contrast with thrombin receptor stimulation by ≥5 μM thrombin receptor-activating peptide (TRAP), which causes rapid and massive Wortmannin-insensitive actin assembly and lamellar and filopodial extension. However, we show here that Wortmannin can inhibit filopod formation if the thrombin receptor is ligated using suboptimal doses (<1 μM) of TRAP. Phosphatidylinositol 3,4-bisphosphate inhibits actin filament severing and capping by human gelsolin in vitro. The findings implicate D3 polyphosphoinositides and integrin signaling in PMA-mediated platelet stimulation and implicate D3 containing phosphoinositides generated in response to protein kinase C activation and GPIIb-IIIa signaling as late-acting intermediates leading to filopodial actin assembly.
John H Hartwig - One of the best experts on this subject based on the ideXlab platform.
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d3 phosphoinositides and outside in integrin signaling by glycoprotein iib iiia mediate platelet actin assembly and filopodial extension induced by phorbol 12 myristate 13 acetate
Journal of Biological Chemistry, 1996Co-Authors: John H Hartwig, Lewis C Cantley, Sophia Kung, Tibor Kovacsovics, Paul A Janmey, Thomas P Stossel, Alex TokerAbstract:Phorbol 12-myristate 13-acetate (PMA) uncaps a small number of the fast-growing (barbed) ends of actin filaments, thereby eliciting slow actin assembly and extension of filopodia in human blood platelets. These reactions, which also occur in response to immunologic perturbation of the integrin glycoprotein (GP) IIb-IIIa, are sensitive to the phosphoinositide 3-kinase inhibitor Wortmannin. Platelets deficient in GPIIb-IIIa integrins or with GPIIb-IIIa function inhibited by calcium chelation or the peptide RGDS have diminished PMA responsiveness. The effects of PMA contrast with thrombin receptor stimulation by ≥5 μM thrombin receptor-activating peptide (TRAP), which causes rapid and massive Wortmannin-insensitive actin assembly and lamellar and filopodial extension. However, we show here that Wortmannin can inhibit filopod formation if the thrombin receptor is ligated using suboptimal doses (
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d3 phosphoinositides and outside in integrin signaling by glycoprotein iib iiia mediate platelet actin assembly and filopodial extension induced by phorbol 12 myristate 13 acetate
Journal of Biological Chemistry, 1996Co-Authors: John H Hartwig, Lewis C Cantley, Sophia Kung, Tibor Kovacsovics, Paul A Janmey, Thomas P Stossel, Alex TokerAbstract:Abstract Phorbol 12-myristate 13-acetate (PMA) uncaps a small number of the fast-growing (barbed) ends of actin filaments, thereby eliciting slow actin assembly and extension of filopodia in human blood platelets. These reactions, which also occur in response to immunologic perturbation of the integrin glycoprotein (GP) IIb-IIIa, are sensitive to the phosphoinositide 3-kinase inhibitor Wortmannin. Platelets deficient in GPIIb-IIIa integrins or with GPIIb-IIIa function inhibited by calcium chelation or the peptide RGDS have diminished PMA responsiveness. The effects of PMA contrast with thrombin receptor stimulation by ≥5 μM thrombin receptor-activating peptide (TRAP), which causes rapid and massive Wortmannin-insensitive actin assembly and lamellar and filopodial extension. However, we show here that Wortmannin can inhibit filopod formation if the thrombin receptor is ligated using suboptimal doses (<1 μM) of TRAP. Phosphatidylinositol 3,4-bisphosphate inhibits actin filament severing and capping by human gelsolin in vitro. The findings implicate D3 polyphosphoinositides and integrin signaling in PMA-mediated platelet stimulation and implicate D3 containing phosphoinositides generated in response to protein kinase C activation and GPIIb-IIIa signaling as late-acting intermediates leading to filopodial actin assembly.
Robert T Abraham - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of Phosphoinositide 3-Kinase Related Kinases by the Radiosensitizing Agent Wortmannin
Cancer research, 1998Co-Authors: Jann N. Sarkaria, Randal S. Tibbetts, Ericka C. Busby, Amy Kennedy, David E. Hill, Robert T AbrahamAbstract:Members of the phosphatidylinositol-3 kinase related kinase (PIKK) family function in both cell cycle progression and DNA damage-induced cell cycle checkpoints. The fungal metabolite, Wortmannin, is an effective radiosensitizer that irreversibly inhibits certain members of the PIKK family. Based on their roles in DNA damage responses, several PIKKs, DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) and the ataxia- and Rad3-related protein (ATR), are potential targets for the radiosensitizing effect of Wortmannin. In this report, we demonstrate that Wortmannin is a relatively potent inhibitor of DNA-PK (IC50, 16 nM) and ATM (IC50, 150 nM) activities, whereas ATR activity is significantly less sensitive to this drug (IC50, 1.8 microM). In intact A549 lung adenocarcinoma cells, Wortmannin inhibited both DNA-PK and ATM at concentrations that correlated closely with those required for radiosensitization. Furthermore, pretreatment of A549 cells with Wortmannin resulted in radioresistant DNA synthesis, a characteristic abnormality of ATM-deficient cells. These results identify Wortmannin as an inhibitor of ATM activity and suggest that ATM and DNA-PK are relevant targets for the radiosensitizing effect of this drug in cancer cells.
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direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3 kinase inhibitors Wortmannin and ly294002
The EMBO Journal, 1996Co-Authors: Gregory J Brunn, Josie M Williams, Candace J Sabers, Gregory J Wiederrecht, John C Lawrence, Robert T AbrahamAbstract:The immunosuppressant, rapamycin, inhibits cell growth by interfering with the function of a novel kinase, termed mammalian target of rapamycin (mTOR). The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3-kinases. This study demonstrates that mTOR is a component of a cytokine-triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor-4E (eIF-4E) binding protein, PHAS-1, in activated T lymphocytes. This event promotes G1 phase progression by stimulating eIF-4E-dependent translation initiation. A mutant YAC-1 T lymphoma cell line, which was selected for resistance to the growth-inhibitory action of rapamycin, was correspondingly resistant to the suppressive effect of this drug on PHAS-1 phosphorylation. In contrast, the PI 3-kinase inhibitor, Wortmannin, reduced the phosphorylation of PHAS-1 in both rapamycin-sensitive and -resistant T cells. At similar drug concentrations (0.1-1 microM), Wortmannin irreversibly inhibited the serine-specific autokinase activity of mTOR. The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3-kinase inhibitor, LY294002, at concentrations (1-30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85-p110 heterodimer. These studies indicate that the signaling functions of mTOR, and potentially those of other high molecular weight PI 3-kinase homologs, are directly affected by cellular treatment with Wortmannin or LY294002.
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Wortmannin a potent and selective inhibitor of phosphatidylinositol 3 kinase
Cancer Research, 1994Co-Authors: Garth Powis, Margareta M Berggren, Alfred Gallegos, Rosanne Bonjouklian, Robert T Abraham, Curtis L. Ashendel, Leon H. ZalkowAbstract:Abstract Phosphatidylinositol-3-kinase is an important enzyme for intracellular signaling. The microbial product worthmannin and some of its analogues have been shown to be potent inhibitors of phosphatidylinositol-3-kinase. The 50% inhibitory concentration for inhibition by Wortmannin is 2 to 4 nm. Kinetic analysis demonstrates that Wortmannin is a noncompetitive, irreversible inhibitor of phosphatidylinositol-3-kinase, with inactivation being both time- and concentration-dependent. Wortmannin has previously been reported to be an inhibitor of myosin light chain kinase but with an inhibitory concentration of 0.2 µm. Wortmannin was found not to be an inhibitor of phosphatidylinositol-4-kinase, protein kinase C, or protein tyrosine kinase. Wortmannin inhibited the formation of phosphatidylinositol-3-phosphates in intact cells. The results of the study suggest that Wortmannin and its analogues may have utility as pharmacological probes for studying the actions of phosphatidylinositol-3-kinase.
Osamu Hazeki - One of the best experts on this subject based on the ideXlab platform.
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Wortmannin as a unique probe for an intracellular signalling protein phosphoinositide 3 kinase
Trends in Biochemical Sciences, 1995Co-Authors: Taro Okada, Kaoru Hazeki, Osamu HazekiAbstract:Wortmannin is a fungal metabolite that so far has been shown to act as a selective inhibitor of phosphoinositide 3-kinase. It can therefore be used to investigate the convergence between two major cellular signalling systems: those involving G-protein-coupled receptors and those involving receptor tyrosine kinases. Importantly, Wortmannin can enter intact cells, making whole-cell studies of the above signalling pathways possible.
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blockage of chemotactic peptide induced stimulation of neutrophils by Wortmannin as a result of selective inhibition of phosphatidylinositol 3 kinase
Journal of Biological Chemistry, 1994Co-Authors: Tomoyo Okada, Yasuhisa Fukui, L Sakuma, Osamu HazekiAbstract:Wortmannin, a fungal metabolite, inhibited 32P labeling of phosphatidylinositol trisphosphate, a product of phosphatidylinositol 3-kinase (PI 3-kinase), selectively in formyl peptide-stimulated 32P-loaded guinea pig neutrophils. The inhibition was of the same concentration dependence (with the half-maximal inhibition around 50 nM) as was observed for the simultaneous inhibition of formyl peptide-induced superoxide anion production. Wortmannin inhibited all three of the PI 3-kinase activities found in the cytosol fraction of guinea pig neutrophils, with a similar dose dependence (the half-maximal effects at 5 nM). Wortmannin was also effective on an immunologically purified preparation of the enzyme. The inhibition was of a noncompetitive type with regard to ATP and was observed consistently when PI, PI monophosphate, or PI bisphosphate was used as substrate. PI 4-kinase activity was not affected. It is concluded, therefore, that Wortmannin abolished the formyl peptide-induced stimulation of neutrophils as a result of the inhibition of PI 3-kinase. An essential role of PI 3-kinase in receptor-mediated signaling in neutrophils thus evidenced with the use of Wortmannin will be expanded to other cellular signaling systems.
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essential role of phosphatidylinositol 3 kinase in insulin induced glucose transport and antilipolysis in rat adipocytes studies with a selective inhibitor Wortmannin
Journal of Biological Chemistry, 1994Co-Authors: Tomoyo Okada, Y Kawano, T Sakakibara, Osamu HazekiAbstract:Significant activity of phosphatidylinositol 3-kinase (PI 3-kinase) was detected in the membrane fractions, or in the immunoprecipitates prepared with anti-phosphotyrosine antibodies, from rat adipocytes that had been incubated with insulin for 20 min. The PI 3-kinase activity in these preparations as well as in the whole cell lysates of adipocytes not treated with insulin was inhibited by the addition of Wortmannin, a fungal metabolite, to the enzyme assay mixture. The inhibition was dependent on the inhibitor concentration with IC50 being less than 10 nM and perfect inhibition at 100 nM. The effect of insulin to induce membrane PI 3-kinase activity was mostly abolished, but its effects to tyrosine-phosphorylate the beta-subunit of the insulin receptor or other cellular substrate proteins including insulin-receptor-substrate-1 were not at all antagonized, by Wortmannin added to the cell incubation medium. Insulin stimulation of cellular 2-deoxyglucose uptake and inhibition of isoproterenol-induced lipolysis observable in adipocytes under the same conditions were also antagonized by Wortmannin added in the same concentration range as used for the inhibition of insulin-susceptible PI 3-kinase. It is concluded, therefore, that activation of Wortmannin-sensitive PI 3-kinase plays a pivotal role in the intracellular signaling pathways arising from the insulin receptor autophosphorylation and leading to certain metabolic responses.
Lewis C Cantley - One of the best experts on this subject based on the ideXlab platform.
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Phosphatidylinositol 3-kinase-dependent activation of trypsinogen modulates the severity of acute pancreatitis
Journal of Clinical Investigation, 2001Co-Authors: Vijay Prem Singh, Gijs Jd Van Acker, Albert M. Song, Ajay Kumar Saluja, Stephen P. Soltoff, Lakshmi Bhagat, Lewis C Cantley, Michael L SteerAbstract:Intra-acinar cell activation of digestive enzyme zymogens including trypsinogen is generally believed to be an early and critical event in acute pancreatitis. We have found that the phosphatidylinositol 3-kinase inhibitor Wortmannin can reduce the intrapancreatic activation of trypsinogen that occurs during two dissimilar experimental models of rodent acute pancreatitis, secretagogue- and duct injection-induced pancreatitis. The severity of both models was also reduced by Wortmannin administration. In contrast, the NF-κB activation that occurs during the early stages of secretagogue-induced pancreatitis is not altered by administration of Wortmannin. Ex vivo, caerulein-induced trypsinogen activation is inhibited by Wortmannin and LY294002. However, the cytoskeletal changes induced by caerulein were not affected by Wortmannin. Concentrations of caerulein that induced ex vivo trypsinogen activation do not significantly increase phosphatidylinositol-3,4-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate levels or induce phosphorylation of Akt/PKB, suggesting that class I phosphatidylinositol 3-kinases are not involved. The concentration of Wortmannin that inhibits trypsinogen activation causes a 75% decrease in phosphatidylinositol 3-phosphate, which is implicated in vesicle trafficking and fusion. We conclude that a Wortmannin-inhibitable phosphatidylinositol 3-kinase is necessary for intrapancreatic activation of trypsinogen and regulating the severity of acute pancreatitis. Our observations suggest that phosphatidylinositol 3-kinase inhibition might be of benefit in preventing acute pancreatitis.
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d3 phosphoinositides and outside in integrin signaling by glycoprotein iib iiia mediate platelet actin assembly and filopodial extension induced by phorbol 12 myristate 13 acetate
Journal of Biological Chemistry, 1996Co-Authors: John H Hartwig, Lewis C Cantley, Sophia Kung, Tibor Kovacsovics, Paul A Janmey, Thomas P Stossel, Alex TokerAbstract:Phorbol 12-myristate 13-acetate (PMA) uncaps a small number of the fast-growing (barbed) ends of actin filaments, thereby eliciting slow actin assembly and extension of filopodia in human blood platelets. These reactions, which also occur in response to immunologic perturbation of the integrin glycoprotein (GP) IIb-IIIa, are sensitive to the phosphoinositide 3-kinase inhibitor Wortmannin. Platelets deficient in GPIIb-IIIa integrins or with GPIIb-IIIa function inhibited by calcium chelation or the peptide RGDS have diminished PMA responsiveness. The effects of PMA contrast with thrombin receptor stimulation by ≥5 μM thrombin receptor-activating peptide (TRAP), which causes rapid and massive Wortmannin-insensitive actin assembly and lamellar and filopodial extension. However, we show here that Wortmannin can inhibit filopod formation if the thrombin receptor is ligated using suboptimal doses (
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d3 phosphoinositides and outside in integrin signaling by glycoprotein iib iiia mediate platelet actin assembly and filopodial extension induced by phorbol 12 myristate 13 acetate
Journal of Biological Chemistry, 1996Co-Authors: John H Hartwig, Lewis C Cantley, Sophia Kung, Tibor Kovacsovics, Paul A Janmey, Thomas P Stossel, Alex TokerAbstract:Abstract Phorbol 12-myristate 13-acetate (PMA) uncaps a small number of the fast-growing (barbed) ends of actin filaments, thereby eliciting slow actin assembly and extension of filopodia in human blood platelets. These reactions, which also occur in response to immunologic perturbation of the integrin glycoprotein (GP) IIb-IIIa, are sensitive to the phosphoinositide 3-kinase inhibitor Wortmannin. Platelets deficient in GPIIb-IIIa integrins or with GPIIb-IIIa function inhibited by calcium chelation or the peptide RGDS have diminished PMA responsiveness. The effects of PMA contrast with thrombin receptor stimulation by ≥5 μM thrombin receptor-activating peptide (TRAP), which causes rapid and massive Wortmannin-insensitive actin assembly and lamellar and filopodial extension. However, we show here that Wortmannin can inhibit filopod formation if the thrombin receptor is ligated using suboptimal doses (<1 μM) of TRAP. Phosphatidylinositol 3,4-bisphosphate inhibits actin filament severing and capping by human gelsolin in vitro. The findings implicate D3 polyphosphoinositides and integrin signaling in PMA-mediated platelet stimulation and implicate D3 containing phosphoinositides generated in response to protein kinase C activation and GPIIb-IIIa signaling as late-acting intermediates leading to filopodial actin assembly.
