A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002).

Intracellular phosphorylations polymorphonuclear neutrophils are mediated by kinases, including mitogen activated-protein (MAP) kinases and phosphatidylinositol 3-kinase. In the present study we demonstrate their effector functions upon both ligation of cell-surface seven-transmembrane-spanning receptors by bacterial peptide formylmethionyl-leucylphenylalanine as well as in the process of destruction of Staphylococcus aureus. To regulate neutrophil MAP kinases p38 and p44/42, specifically, we made use of their specific inhibitors 10 µM SK&F 86002 (for p38) and PD 098059 (for activating kinase of p44/42). SK&F 86002 was a potent inhibitor (by 70%) of induced antimicrobial oxygen-radical generation compared with PD 098059 (by 20%). SK&F 86002 and PD 098059 inhibited mobilization of a dominant neutrophil adhesion molecule, β2 integrin, from cytoplasmic granules to the plasma membrane by 40 and 10% respectively, and the combination of the two drugs resulted in a 90% effect. The combined effect of both drugs was moderate inhibition of bacterial destruction, despite the fact that neither compound had detectable effect on bactericidal activity if applied individually. Bacterial destruction was also inhibited by wortmannin (0.1 µM), the specific inhibitor of phosphatidylinositol 3-kinase, which had previously been described to target various other activations of the neutrophil, including oxygen-radical generation. Although the relative contribution of p38 and p44/42 MAP kinases varied, the marked effects of the combined inhibition of the kinases revealed their concerted actions to be critical for normal neutrophil function.

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c-Cbl Is Tyrosine-Phosphorylated by Interleukin-4 and Enhances Mitogenic and Survival Signals of Interleukin-4 Receptor by Linking With the Phosphatidylinositol 3′-Kinase Pathway

Blood ◽

10.1182/blood.v91.1.46.46_46_53 ◽

1998 ◽

Vol 91 (1) ◽

pp. 46-53 ◽

Cited By ~ 7

Author(s):

Hiroo Ueno ◽

Ko Sasaki ◽

Hiroaki Honda ◽

Tetsuya Nakamoto ◽

Tetsuya Yamagata ◽

...

Keyword(s):

Specific Inhibitor ◽

Pi3 Kinase ◽

Interleukin 4 ◽

Phosphatidylinositol 3 Kinase ◽

Proliferation And Differentiation ◽

Interleukin 4 Receptor ◽

Pi3 Kinase Pathway ◽

Kinase Pathway ◽

Phosphatidylinositol 3 ◽

Survival Signals

Interleukin-4 (IL-4) is a cytokine that induces both proliferation and differentiation and suppresses apoptosis of B cells. Although IL-4 has been shown to activate the phosphatidylinositol 3′ (PI3)-kinase pathway, the role of PI3 kinase in the IL-4 receptor (IL-4R) signaling remains unclear. In this study, we demonstrated that c-Cbl proto-oncogene product is inducibly phosphorylated on tyrosine residues and is associated with the p85 subunit of PI3-kinase by IL-4 stimulation. Overexpression of c-Cbl enhances the PI3-kinase activity and, at the same time, mitogenic activity and survival of cells in the presence of IL-4. However, these effects of c-Cbl were abolished by wortmannin, a specific inhibitor for the PI3 kinase pathway, or by a point mutation at tyrosine 731 of c-Cbl, which is a major binding site for p85. These results indicate that c-Cbl plays a role in linking IL-4R with the PI3 kinase pathway and thus enhancing the mitogenic and survival signals.

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Phosphatidylinositol 3-Kinase and Prostaglandylinositol Cyclic Phosphate (Cyclic PIP), a Mediator of Insulin Action, in the Signal Transduction of Insulin

Biological Chemistry ◽

10.1515/bc.2000.140 ◽

2000 ◽

Vol 381 (11) ◽

pp. 1139-1141 ◽

Cited By ~ 1

Author(s):

A. Gypakis ◽

H.K. Wasner

Keyword(s):

Insulin Receptor ◽

Glucose Transport ◽

Functional Role ◽

Specific Inhibitor ◽

Insulin Receptor Substrate ◽

Phosphatidylinositol 3 Kinase ◽

Downstream Signaling ◽

Cyclic Phosphate ◽

Phosphatidylinositol 3

Abstract It has been suggested that downstream signaling from the insulin receptor to the level of the protein kinases and protein phosphatases is accomplished by prostaglandylinositol cyclic phosphate (cyclic PIP), a proposed second messenger of insulin. However, evidence points also to both phosphatidylinositol 3-kinase, which binds to the tyrosine phosphorylated insulin receptor substrate-1, and the Ras complex in insulin's downstream signaling. We have examined whether a correlation exists between these various observations. It was found that wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, prevented insulin-induced, as well as cyclic PIP-induced activation of glucose transport, indicating that PI 3-kinase action on glucose transport involves downstream signaling of both insulin and cyclic PIP. Wortmannin has no effect on cyclic PIP synthase activity nor on the substrate production for cyclic PIP synthesis either, indicating that the functional role of PI 3-kinase is exclusively downstream of cyclic PIP.

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Wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, induces accumulation of DNA double-strand breaks

Journal of Radiation Research ◽

10.1093/jrr/rrz102 ◽

2020 ◽

Vol 61 (2) ◽

pp. 171-176 ◽

Cited By ~ 1

Author(s):

Makoto Ihara ◽

Kazuko Shichijo ◽

Satoshi Takeshita ◽

Takashi Kudo

Keyword(s):

Dna Damage ◽

Specific Inhibitor ◽

Double Strand Breaks ◽

Dependent Manner ◽

Dna Double Strand Breaks ◽

Phosphatidylinositol 3 Kinase ◽

Strand Breaks ◽

Γ Ray ◽

Scid Cells ◽

Phosphatidylinositol 3

Abstract Wortmannin, a fungal metabolite, is a specific inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, which includes double-stranded DNA dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated kinase (ATM). We investigated the effects of wortmannin on DNA damage in DNA-PK-deficient cells obtained from severe combined immunodeficient mice (SCID cells). Survival of wortmannin-treated cells decreased in a concentration-dependent manner. After treatment with 50 μM wortmannin, survival decreased to 60% of that of untreated cells. We observed that treatment with 20 and 50 μM wortmannin induced DNA damage equivalent to that by 0.37 and 0.69 Gy, respectively, of γ-ray radiation. The accumulation of DNA double-strand breaks (DSBs) in wortmannin-treated SCID cells was assessed using pulsed-field gel electrophoresis. The maximal accumulation was observed 4 h after treatment. Moreover, the presence of DSBs was confirmed by the ability of nuclear extracts from γ-ray-irradiated SCID cells to produce in vitro phosphorylation of histone H2AX. These results suggest that wortmannin induces cellular toxicity by accumulation of spontaneous DSBs through inhibition of ATM.

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Dehydroepiandrosterone Protects Vascular Endothelial Cells against Apoptosis through a Gαi Protein-Dependent Activation of Phosphatidylinositol 3-Kinase/Akt and Regulation of Antiapoptotic Bcl-2 Expression

Endocrinology ◽

10.1210/en.2006-1378 ◽

2007 ◽

Vol 148 (7) ◽

pp. 3068-3076 ◽

Cited By ~ 74

Author(s):

Dongmin Liu ◽

Hongwei Si ◽

Kathryn A. Reynolds ◽

Wei Zhen ◽

Zhenquan Jia ◽

...

Keyword(s):

Endothelial Cells ◽

Estrogen Receptor ◽

Vascular Function ◽

Vascular Endothelial Cells ◽

Specific Inhibitor ◽

Vascular Endothelial ◽

Phosphatidylinositol 3 Kinase ◽

Antiapoptotic Effect ◽

Dose Dependent ◽

Phosphatidylinositol 3

The adrenal steroid dehydroepiandrosterone (DHEA) may improve vascular function, but the mechanism is unclear. In the present study, we show that DHEA significantly increased cell viability, reduced caspase-3 activity, and protected both bovine and human vascular endothelial cells against serum deprivation-induced apoptosis. This effect was dose dependent and maximal at physiological concentrations (0.1–10 nm). DHEA stimulation of bovine aortic endothelial cells resulted in rapid and dose-dependent phosphorylation of Akt, which was blocked by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), the upstream kinase of Akt. Accordingly, inhibition of PI3K or transfection of the cells with dominant-negative Akt ablated the antiapoptotic effect of DHEA. The induced Akt phosphorylation and subsequent cytoprotective effect of DHEA were dependent on activation of Gαi proteins, but were estrogen receptor independent, because these effects were blocked by pertussis toxin but not by the estrogen receptor inhibitor ICI182,780 or the aromatase inhibitor aminoglutethimide. Finally, DHEA enhanced antiapoptotic Bcl-2 protein expression, its promoter activity, and gene transcription attributable to the activation of the PI3K/Akt pathway. Neutralization of Bcl-2 by antibody transfection significantly decreased the antiapoptotic effect of DHEA. These findings provide the first evidence that DHEA acts as a survival factor for endothelial cells by triggering the Gαi-PI3K/Akt-Bcl-2 pathway to protect cells against apoptosis. This may represent an important mechanism underlying the vascular protective effect of DHEA.

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Phosphatidylinositol-3-kinase activity is required for the anti-ig- mediated growth inhibition of a human B-lymphoma cell line

Blood ◽

10.1182/blood.v87.1.202.bloodjournal871202 ◽

1996 ◽

Vol 87 (1) ◽

pp. 202-210 ◽

Cited By ~ 1

Author(s):

M Beckwith ◽

RG Fenton ◽

IM Katona ◽

DL Longo

Keyword(s):

Growth Inhibition ◽

Tyrosine Kinases ◽

Specific Inhibitor ◽

Phosphatidylinositol 3 Kinase ◽

Intact Cells ◽

B Lymphoma ◽

Cycle Arrest ◽

Pi3k Activity ◽

B Lymphoma Cells ◽

Phosphatidylinositol 3

Stimulation of B lymphocytes through the Ig receptor initiates a cascade of biochemical changes, which can ultimately lead to either activation and growth, or cell-cycle arrest and cell death. One of the critical events that occurs in both cases is the activation of tyrosine kinases, and the resulting phosphorylation of a variety of proteins on tyrosine residues. In this report we identify one of the substrates of phosphorylation as the 85-kD subunit of the enzyme phosphatidylinositol-3 kinase (PI3K), and show that both anti-IgM and anti-IgD stimulation results in an increase in the anti-phosphotyrosine-precipitable PI3K activity. Furthermore, we show that the potent and specific inhibitor of PI3K, Wortmannin, can completely abrogate anti-Ig-mediated growth inhibition without affecting tyrosine kinase induction or protein kinase C (PKC) activation. Treatment of intact cells with Wortmannin results in an irreversible decrease in anti-Ig-induced PI3K activity, suggesting that the effect of Wortmannin on anti-Ig-mediated growth inhibition is caused by its inactivation of PI3K activity. Taken together, these data show that activation of PI3K is a critical component of the anti-Ig-initiated signaling cascade that leads to growth inhibition of human B lymphoma cells.

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Activation of Phosphatidylinositol 3-Kinase Signaling Promotes Aberrant Pituitary Growth in a Mouse Model of Thyroid-Stimulating Hormone-Secreting Pituitary Tumors

Endocrinology ◽

10.1210/en.2007-1696 ◽

2008 ◽

Vol 149 (7) ◽

pp. 3339-3345 ◽

Cited By ~ 33

Author(s):

Changxue Lu ◽

Mark C. Willingham ◽

Fumihiko Furuya ◽

Sheue-yann Cheng

Keyword(s):

Cell Proliferation ◽

Mouse Model ◽

Signaling Pathway ◽

Molecular Mechanisms ◽

Thyroid Hormone Receptor ◽

Specific Inhibitor ◽

Pituitary Tumors ◽

Akt Pathway ◽

Phosphatidylinositol 3 Kinase ◽

Phosphatidylinositol 3

TSH-secreting pituitary tumors (TSHomas) are pituitary tumors that constitutively secrete TSH. Molecular mechanisms underlying this abnormality are largely undefined. We recently created a knock-in mutant mouse harboring a mutation (denoted as PV) in the thyroid hormone receptor-β gene (TRβPV/PV mouse). As these mice age, they spontaneously develop TSHomas. Using this mouse model, we investigated the role of the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway in the pathogenesis of TSHomas. Concurrent with aberrant growth of pituitaries, AKT and its downstream effectors, mammalian target rapamycin and p70S6K, were activated to contribute to increased cell proliferation and pituitary growth. In addition, activation of AKT led to decreased apoptosis by inhibiting proapoptotic activity of Bcl-2-associated death promoter, further contributing to the aberrant cell proliferation. These results suggest an activated PI3K-AKT pathway could underscore tumorigenesis, raising the possibility that this pathway could be a potential therapeutic target in TSHomas. Indeed, TRβPV/PV mice treated with a PI3K-specific inhibitor, LY294002, showed a significant decrease in pituitary growth. The progrowth signaling via AKT-mammalian target rapamycin-p70S6K and cyclin D1/cyclin-dependent kinase were inhibited, and proapoptotic activity of Bcl-2-associated death promoter was increased by LY294002 treatment. Thus, activation of the PI3K-AKT pathway mediates, at least in part, the aberrant pituitary growth, and the intervention of this signaling pathway presents a novel therapeutic opportunity for TSHomas.

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Phosphatidylinositol 3-kinase activation is required for insulin-stimulated sodium transport in A6 cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1998.274.4.e611 ◽

1998 ◽

Vol 274 (4) ◽

pp. E611-E617 ◽

Cited By ~ 46

Author(s):

Rae D. Record ◽

Larry L. Froelich ◽

Chris J. Vlahos ◽

Bonnie L. Blazer-Yost

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Sodium Transport ◽

Specific Inhibitor ◽

Dependent Manner ◽

Phosphatidylinositol 3 Kinase ◽

Insulin Receptor Tyrosine Kinase ◽

A6 Cells ◽

Phosphatidylinositol 3

Insulin stimulates amiloride-sensitive sodium transport in models of the distal nephron. Here we demonstrate that, in the A6 cell line, this action is mediated by the insulin receptor tyrosine kinase and that activation of phosphatidylinositol 3-kinase (PI 3-kinase) lies downstream of the receptor tyrosine kinase. Functionally, a specific inhibitor of PI 3-kinase, LY-294002, blocks basal as well as insulin-stimulated sodium transport in a dose-dependent manner (IC50 ≈ 6 μM). Biochemically, PI 3-kinase is present in A6 cells and is inhibited both in vivo and in vitro by LY-294002. Furthermore, a subsequent potential downstream signaling element, pp70 S6 kinase, is activated in response to insulin but does not appear to be part of the pathway involved in insulin-stimulated sodium transport. Together with previous reports, these results suggest that insulin may induce the exocytotic insertion of sodium channels into the apical membrane of A6 cells in a PI 3-kinase-mediated manner.

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c-Cbl Is Tyrosine-Phosphorylated by Interleukin-4 and Enhances Mitogenic and Survival Signals of Interleukin-4 Receptor by Linking With the Phosphatidylinositol 3′-Kinase Pathway

Blood ◽

10.1182/blood.v91.1.46 ◽

1998 ◽

Vol 91 (1) ◽

pp. 46-53 ◽

Cited By ~ 56

Author(s):

Hiroo Ueno ◽

Ko Sasaki ◽

Hiroaki Honda ◽

Tetsuya Nakamoto ◽

Tetsuya Yamagata ◽

...

Keyword(s):

Specific Inhibitor ◽

Pi3 Kinase ◽

Interleukin 4 ◽

Phosphatidylinositol 3 Kinase ◽

Proliferation And Differentiation ◽

Interleukin 4 Receptor ◽

Pi3 Kinase Pathway ◽

Kinase Pathway ◽

Phosphatidylinositol 3 ◽

Survival Signals

Abstract Interleukin-4 (IL-4) is a cytokine that induces both proliferation and differentiation and suppresses apoptosis of B cells. Although IL-4 has been shown to activate the phosphatidylinositol 3′ (PI3)-kinase pathway, the role of PI3 kinase in the IL-4 receptor (IL-4R) signaling remains unclear. In this study, we demonstrated that c-Cbl proto-oncogene product is inducibly phosphorylated on tyrosine residues and is associated with the p85 subunit of PI3-kinase by IL-4 stimulation. Overexpression of c-Cbl enhances the PI3-kinase activity and, at the same time, mitogenic activity and survival of cells in the presence of IL-4. However, these effects of c-Cbl were abolished by wortmannin, a specific inhibitor for the PI3 kinase pathway, or by a point mutation at tyrosine 731 of c-Cbl, which is a major binding site for p85. These results indicate that c-Cbl plays a role in linking IL-4R with the PI3 kinase pathway and thus enhancing the mitogenic and survival signals.

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