okadaic-acid and herbimycin

In resident mouse peritoneal macrophages, group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) mediates arachidonic acid (AA) release and eicosanoid production in response to diverse agonists such as A23187, phorbol myristate acetate, zymosan, and the enterotoxin, okadaic acid (OA). cPLA(2)alpha is regulated by phosphorylation and by calcium that binds to the C2 domain and induces translocation from the cytosol to membranes. In contrast, OA activates cPLA(2)alpha-induced AA release and translocation to the Golgi in macrophages without an apparent increase in calcium. Inhibitors of heat shock protein 90 (hsp90), geldanamycin, and herbimycin blocked AA release in response to OA but not to A23187, PMA, or zymosan. OA, but not the other agonists, induced activation of a cytosolic serine/threonine 54-kDa kinase (p54), which phosphorylated cPLA(2)alpha in in-gel kinase assays and was associated with cPLA(2)alpha in immunoprecipitates. Activation of the p54 kinase was inhibited by geldanamycin. The kinase coimmunoprecipitated with hsp90 in unstimulated macrophages, and OA induced its loss from hsp90, concomitant with its association with cPLA(2)alpha. The results demonstrate a role for hsp90 in regulating cPLA(2)alpha-mediated AA release that involves association of a p54 kinase with cPLA(2)alpha upon OA stimulation.

Topics: Animals; Arachidonic Acid; Benzoquinones; Calcimycin; Calcium; Carcinogens; Cytosol; Enzyme Activation; Enzyme Inhibitors; Female; Golgi Apparatus; Green Fluorescent Proteins; Group IV Phospholipases A2; HSP90 Heat-Shock Proteins; Immunoblotting; Immunoprecipitation; Ionophores; Lactams, Macrocyclic; Macrophages, Peritoneal; Mice; Mice, Inbred ICR; Mitogen-Activated Protein Kinase 10; NIH 3T3 Cells; Okadaic Acid; Phosphoamino Acids; Phosphorylation; Protein Transport; Rifabutin; Signal Transduction; Tetradecanoylphorbol Acetate; Zymosan

Pulmonary surfactant is secreted by the type II alveolar cells of the lung, and this secretion is induced by secretagogues of several types (e.g., ionomycin, phorbol esters, and terbutaline). Secretagogue-induced secretion is inhibited by surfactant-associated protein A (SP-A), which binds to a specific receptor (SPAR) on the surface of type II cells. The mechanism of SP-A-activated SPAR signaling is completely unknown. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 rescued surfactant secretion from inhibition by SP-A. In order to directly demonstrate a role for PI3K in SPAR signaling, PI3K activity was immunoprecipitated from type II cell extracts. PI3K activity increased rapidly after SP-A addition to type II cells. Since many receptors that activate PI3K do so through tyrosine-specific protein phosphorylation, antisera to phosphotyrosine, insulin-receptor substrate-1 (IRS-1), or SPAR were also examined. These antisera coimmunoprecipitated PI3K activity that was stimulated by SP-A. In addition, the tyrosine-specific protein kinase inhibitors genistein and herbimycin A blocked the action of SP-A on surfactant secretion. We conclude that SP-A signals to regulate surfactant secretion through SPAR, via pathways that involve tyrosine phosphorylation, include IRS-1, and entail activation of PI3K. This activation leads to inhibition of secretagogue-induced secretion of pulmonary surfactant.

Topics: Animals; Benzoquinones; Cells, Cultured; Chromones; Enzyme Activation; Enzyme Inhibitors; Female; Flavonoids; Genistein; Insulin Receptor Substrate Proteins; Lactams, Macrocyclic; Mitogen-Activated Protein Kinases; Morpholines; Okadaic Acid; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein-Tyrosine Kinases; Proteolipids; Pulmonary Alveoli; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; Quinones; Rats; Rats, Sprague-Dawley; Receptors, Cell Surface; Rifabutin; Tyrosine

In the capping of cell-surface receptors two stages can be distinguished: 1) clustering of the receptors (patching) induced by cross-linking with specific antibodies and 2) subsequent assembly of patches into a cap which is driven by the actin-based cytoskeleton. We found that patching of Fcgamma receptor II in U937 cells was correlated with tyrosine phosphorylation of certain proteins, most prominently those of 130, 110, 75 and 28 kDa. The phosphotyrosine-bearing proteins were accumulated at the receptor patches. Formation of the receptor caps was coincident with dephosphorylation of these proteins. Inhibition of protein tyrosine kinases with herbimycin A and genistein attenuated the protein tyrosine hyperphosphorylation and blocked capping in a dose-dependent manner. Phenylarsine oxide and pervanadate, inhibitors of protein tyrosine phosphatases, also suppressed capping of Fcgamma receptor II in a concentration-dependent fashion. Simultaneously, tyrosine hyperphosphorylation of proteins occurred. In the presence of the tyrosine kinase and phosphatase inhibitors the receptors were arrested at the patching stage. In contrast, okadaic acid, a serine/threonine phosphatase blocker, did not affect assembly of the receptor caps. The inhibitory effect of phenylarsine oxide was rapidly reversed by dithiols, 2,3-dimercapto-1-propanoldithiol and dithiotreitol, and was coincident with dephosphorylation of protein tyrosine residues. Extensive washing of pervanadate-exposed cells also resulted in progressive restoration of the cap assembly. Using streptolysin O-permeabilized cells we confirmed regulatory function played by dephosphorylation of tyrosine residues in capping of Fcgamma receptor II. Exogenous phosphatases, applied to permeabilized cells in which activity of endogenous tyrosine phosphatases was blocked, evoked dephosphorylation of protein tyrosine residues that was accompanied by recovery of capping ability in the cells.

Topics: Arsenicals; Bacterial Proteins; Benzoquinones; Cell Membrane Permeability; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genistein; Humans; Immunoblotting; Lactams, Macrocyclic; Microscopy, Fluorescence; Okadaic Acid; Phosphorylation; Quinones; Receptor Aggregation; Receptors, IgG; Rifabutin; Streptolysins; Temperature; Time Factors; Tyrosine; U937 Cells; Vanadates

It is important to understand the mechanisms by which phosphorylation-dependent events play a role in regulation of apoptosis in toxicant-metabolizing organs such as the kidney. Our previous work demonstrated that the toxicant and phosphatase inhibitor okadaic acid induces apoptosis of renal epithelial cells via a mechanism that appears to involve the modulation of c-raf-1, p38 kinase, and extracellular regulatory kinase (ERK) cascades. Using the benzoquinone ansamycins and tyrosine kinase inhibitors geldanamycin and herbimycin A, we examined the contribution of tyrosine phosphorylation and c-raf-1 activities to okadaic acid-induced apoptosis. In this report we show that both geldanamycin and herbimycin A protected NRK-52E cells from okadaic acid-induced apoptosis, abrogated the overall okadaic acid-induced kinase activation, and specifically inhibited activation of p38 kinase by okadaic acid. Herbimycin A and geldanamycin also abrogated okadaic-acid induced morphologic changes such as cell rounding and cell membrane blebbing. Herbimycin A and geldanamycin caused pronounced cell spreading, cell flattening, and a decrease in okadaic acid-induced loss of actin filaments. Interestingly, herbimycin A showed more potent inhibitory effect than geldanamycin, and herbimycin A alone inhibited okadaic acid-induced movement of p38 kinase into the cytosol. These results imply that decreased p38 activity and its cytosolic translocation together with cellular resistance to cytoskeletal disorganization may play a significant role in resistance to phosphorylation-dependent apoptosis. Furthermore, the results imply that changes in cell shape may partially modulate the observed alterations in signal transduction induced by okadaic acid.

Topics: Actins; Activating Transcription Factor 2; Animals; Apoptosis; Benzoquinones; Cell Line; Cell Size; Chromatin; Cyclic AMP Response Element-Binding Protein; Cytosol; Enzyme Activation; Enzyme Inhibitors; Epithelial Cells; Imidazoles; Kidney; Lactams, Macrocyclic; Mitogen-Activated Protein Kinases; Okadaic Acid; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins c-raf; Pyridines; Quinones; Rats; Rifabutin; Signal Transduction; Transcription Factors; Transfection

Prostaglandin H2 synthase (PGHS)-1 and PGHS-2 expression was examined in primary cultures of human amnion cells, an in vitro model of amnion tissue. Epidermal growth factor (EGF), the protein kinase C (PKC) activating phorbol ester TPA, and the protein phosphatase inhibitor, okadaic acid (OA), stimulated PGHS activity and the level of PGHS-2 mRNA, but did not affect the level of PGHS-1 mRNA. In situ hybridization suggested that the same population of cells responded to EGF, TPA and OA. Okadaic acid promoted PGHS activity independently of PKC. EGF stimulated the activity of extracellular signal-regulated protein kinase (Erk) and N-terminal c-Jun kinase (Jnk). OA increased Jnk activity but had no effect on Erk activity, while TPA had no influence on either Erk or Jnk activity. PD098059, a selective inhibitor of the Erk-activating kinase MEK, blocked the stimulation of PGHS expression by EGF, but did not decrease stimulation in response to OA. Herbimycin A, a tyrosine kinase inhibitor, suppressed the stimulation of PGHS activity and PGHS-2 mRNA abundance by all three stimulants, and blocked signalling via the Erk and Jnk mitogen-activated protein kinase pathways. Thus, growth factor stimulation, PKC activation and protein phosphatase inhibition induced the expression of PGHS-2 in primary amnion cells by distinct regulatory mechanisms involving tyrosine kinase(s). Tyrosine kinase inhibitors may constitute a new category of PGHS-2 inhibitors that act by blocking the expression of the enzyme.

Topics: Amnion; Benzoquinones; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Flavonoids; Gene Expression Regulation; Humans; In Situ Hybridization; JNK Mitogen-Activated Protein Kinases; Lactams, Macrocyclic; Mitogen-Activated Protein Kinases; Okadaic Acid; Prostaglandin-Endoperoxide Synthases; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rifabutin; RNA, Messenger; Tetradecanoylphorbol Acetate

Tissue factor (TF) is a cell-surface glycoprotein responsible for initiating the extrinsic pathway of coagulation. The overexpression of TF in human malignancy has been correlated with the angiogenic phenotype, poor prognosis, and thromboembolic complications. The mechanisms underlying constitutive expression of TF in cancer cells are poorly defined. We cloned TF cDNA on the basis of its strong expression in metastatic MDA-MB-231 breast carcinoma cells in contrast to its weak expression in non-metastatic MCF-7 cells. Transient transfection analysis showed that TF promoter activity in MCF-7 cells could be stimulated by expression of a membrane-targeted raf kinase (raf-CAAX). raf-induced activity was dependent on the presence of an AP-1/NF-kappaB motif in the TF promoter and was inhibited by dominant-negative mutants of jun and by I-kappaB alpha. MDA-MB-231 cells were found to contain higher levels of ERK1/2 kinase activity than did MCF-7 cells. Electrophoretic mobility shift assays showed that MDA-MB-231 nuclear proteins bound strongly to an oligonucleotide corresponding to the AP-1/NF-kappaB sequence, whereas MCF-7 nuclear extracts showed weak binding to this element. Finally, we showed that TF mRNA levels in MDA-MB-231 cells declined after addition of the mitogen-activated protein kinase kinase inhibitor PD98059. Our data showed that activation of the raf-ERK pathway led to activation of TF expression in breast carcinoma cells and suggested that constitutive activation of this pathway leads to high TF expression in MDA-MB-231 cells.

Topics: Base Sequence; Benzoquinones; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Dactinomycin; DNA, Complementary; Enzyme Activation; Enzyme Induction; Enzyme Inhibitors; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Genistein; Humans; Hydroquinones; Lactams, Macrocyclic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neovascularization, Pathologic; NF-kappa B; Okadaic Acid; Phenols; Proto-Oncogene Proteins c-raf; Quinones; Rifabutin; Signal Transduction; Tetradecanoylphorbol Acetate; Thromboplastin; Transcription Factor AP-1; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Topics: Amnion; Benzoquinones; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Cycloheximide; Cyclooxygenase 2; Enzyme Induction; Epidermal Growth Factor; Female; Humans; Isoenzymes; JNK Mitogen-Activated Protein Kinases; Lactams, Macrocyclic; Membrane Proteins; Mitogen-Activated Protein Kinases; Okadaic Acid; Pregnancy; Prostaglandin-Endoperoxide Synthases; Protein-Tyrosine Kinases; Quinones; Rifabutin; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription, Genetic

The transcription factor AP-1 contributes significantly to the regulation of interleukin-2 gene transcription during T-cell activation and may play a role in thymocyte development. To study the regulation of AP-1 transcriptional activity in primary T-cells, reporter transgenic mice were generated that express luciferase gene under the control of AP-1 binding sites. Here, we demonstrate that while protein kinase C activation is sufficient to induce DNA-binding activity, an additional intracellular calcium increase is required to induce transcriptional activity of AP-1 in primary mouse T-cells. Furthermore, transcriptional, but not DNA-binding, activity of AP-1 is cyclosporin sensitive and requires tyrosine phosphorylation. This dissociation between DNA-binding and transcriptional activity is likely due, at least partially, to post-translational modifications of the AP-1 complex required for transcriptional activity. Moreover, in addition to these two signals delivered by ligand binding to the T-cell receptor (TcR) AP-1 transcriptional activity absolutely requires the presence of a co-stimulatory signal that can be mediated by the interaction of CD28 with its ligands B7-1 and B7-2. Thus, TcR-mediated and co-stimulatory signals required for T-cell activation appear to be integrated, in part, at the level of the regulation of transcriptional activity of AP-1.

Topics: Animals; Base Sequence; Benzoquinones; Calcium; CD28 Antigens; Cyclosporine; Ethers, Cyclic; Genes, Reporter; Interleukin-2; Lactams, Macrocyclic; Lymph Nodes; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Transgenic; Molecular Sequence Data; Okadaic Acid; Phosphorylation; Protein Binding; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Receptors, Antigen, T-Cell; Rifabutin; Signal Transduction; Spleen; T-Lymphocytes; Transcription Factor AP-1; Transcription, Genetic

In resting DDT1MF-2 smooth muscle cells, the cytosolic free Ca2+ concentration ([Ca2+]c) was higher than the free Ca2+ concentration in the nucleus ([Ca2+]n). However, this nucleocytosolic [Ca2+] gradient was reversed by Ca2+ agonists like ATP or, as is shown here, by the epidermal growth factor (EGF). The ATP-induced reversal of the nucleocytosolic [Ca2+] gradient was blocked by stimulation of protein kinase C with phorbol 12-myristate 13-acetate or with the diacylglycerol kinase inhibitor R59949, or by inhibition of the Ser/Thr-specific protein phosphatases-1 and -2A with okadaic acid or calyculin A. Moreover, the magnitude of the ATP-induced reversal of the [Ca2+] gradient diminished during prolonged culture of the cells. The EGF-induced [Ca2+] rise in the cytosol and nucleus was blocked by okadaic acid and by the tyrosine kinase inhibitors herbimycin A and psi-tectorigenin. Our data suggest that the nucleocytosolic [Ca2+] gradient is modulated by (de)phosphorylation processes catalyzed by tyrosine protein kinases, by protein kinase C, and by Ser/Thr protein phosphatases-1 and -2A.

Topics: Adenosine Triphosphate; Animals; Benzoquinones; Calcium; Cell Line; Cell Nucleus; Colforsin; Cricetinae; Cytosol; Diacylglycerol Kinase; Epidermal Growth Factor; Ethers, Cyclic; Homeostasis; Kinetics; Lactams, Macrocyclic; Leiomyosarcoma; Marine Toxins; Mesocricetus; Models, Biological; Muscle, Smooth; Okadaic Acid; Oxazoles; Phosphoproteins; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Piperidines; Protein Kinase C; Protein Kinases; Protein Tyrosine Phosphatases; Quinazolines; Quinazolinones; Quinones; Rifabutin; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured

The extracellular acidification rate of the human bone marrow cell line, TF-1, increases rapidly in response to a bolus of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF). Extracellular acidification rates were measured using a silicon microphysiometer. This instrument contains micro-flow chambers equipped with potentiometric sensors to monitor pH. The cells are immobilized in a fibrin clot sandwiched between two porous polycarbonate membranes. The membranes are part of a disposable plastic "cell capsule" that fits into the microphysiometer flow chamber. The GM-CSF activated acidification burst is dose dependent and can be neutralized by pretreating the cytokine with anti-GM-CSF antibody. The acidification burst can be resolved kinetically into at least two components. A rapid component of the burst is due to activation of the sodium/proton antiporter as evidenced by its elimination in sodium-free medium and in the presence of amiloride. A slower component of the GM-CSF response is a consequence of increased glycolytic metabolism as demonstrated by its dependence on D-glucose as a medium nutrient. Okadaic acid (a phospho-serine/threonine phosphatase inhibitor), phorbol 12-myristate 13-acetate (PMA, a protein kinase C (PKC) activator), and ionomycin (a calcium ionophore) all produce metabolic bursts in TF-1 cells similar to the GM-CSF response. Pretreatment of TF-1 cells with PMA for 18 h resulted in loss of the GM-CSF acidification response. Although this treatment is reported to destroy protein kinase activity, we demonstrate here that it also down-regulates expression of high-affinity GM-CSF receptors on the surface of TF-1 cells. In addition, GM-CSF driven TF-1 cell proliferation was decreased after the 18 h PMA treatment. Short-term treatment with PMA (1-2 h) again resulted in loss of the GM-CSF acidification response, but without a decrease in expression of high-affinity GM-CSF receptors. Evidence for involvement of PKC in GM-CSF signal transduction was obtained using calphostin C, a specific inhibitor of PKC, which inhibited the GM-CSF metabolic burst at a subtoxic concentration. Genistein and herbimycin A, tyrosine kinase inhibitors, both inhibited the GM-CSF response of TF-1 cells, but only at levels high enough to also inhibit stimulation by PMA. These results indicate that GM-CSF activated extracellular acidification of TF-1 cells is caused by increases in sodium/proton antiporter activity and glycolysis, through protein ki

Topics: Benzoquinones; Bone Marrow; Bone Marrow Cells; Carrier Proteins; Cell Division; Cell Line; Ethers, Cyclic; Genistein; Glucose; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Hydrogen-Ion Concentration; Ionomycin; Isoflavones; Kinetics; Lactams, Macrocyclic; Naphthalenes; Okadaic Acid; Phosphoprotein Phosphatases; Polycyclic Compounds; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rifabutin; Signal Transduction; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate