okadaic-acid and tautomycin

Host cell protein phosphatase-1 (PP1) is an important regulator of human immunodeficiency virus-1 (HIV-1) transcription. PP1 is involved in the regulation of HIV-1 transcription, and dephosphorylates RNA polymerase II C-terminal domain (RNAPII CTD) or CycT1-dependent kinase 9 (CDK9) to increase Tat-dependent HIV-1 transcription. In this review, we discuss the action of PP1 in Tat-induced HIV-1 transcription and related to PP1 inhibitors.

Topics: Anti-HIV Agents; Enzyme Inhibitors; HIV-1; Humans; Okadaic Acid; Protein Phosphatase 1; Pyrans; Spiro Compounds; tat Gene Products, Human Immunodeficiency Virus; Transcription, Genetic

Topics: Alkenes; Antibiotics, Antineoplastic; Antifungal Agents; Cantharidin; Crystallography, X-Ray; Cyclosporine; Enzyme Inhibitors; Humans; Microcystins; Models, Molecular; Okadaic Acid; Peptides, Cyclic; Phosphoprotein Phosphatases; Polyenes; Pyrans; Pyrones; Spiro Compounds; Structure-Activity Relationship

Topics: Animals; Antifungal Agents; Magnetic Resonance Spectroscopy; Molecular Conformation; Okadaic Acid; Phosphoprotein Phosphatases; Pyrans; Spiro Compounds

Topics: Animals; Antifungal Agents; Marine Toxins; Microcystins; Neoplasms, Experimental; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Phosphatase 1; Pyrans; Spiro Compounds

Topics: Alkenes; Animals; Antifungal Agents; Cantharidin; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Polyenes; Pyrans; Pyrones; Spiro Compounds

Topics: Animals; Antifungal Agents; Enzyme Inhibitors; Humans; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Conformation; Molecular Structure; Okadaic Acid; Phosphoprotein Phosphatases; Pyrans; Spiro Compounds

Topics: Animals; Antifungal Agents; Carcinogens; Ethers, Cyclic; Humans; Microcystins; Neoplasms; Okadaic Acid; Peptides, Cyclic; Phosphoprotein Phosphatases; Pyrans; Spiro Compounds; Structure-Activity Relationship

Topics: Animals; Antifungal Agents; Carcinogens; Ethers, Cyclic; Liver Neoplasms; Marine Toxins; Mice; Molecular Structure; Okadaic Acid; Oxazoles; Peptides, Cyclic; Pyrans; Rats; Skin Neoplasms; Spiro Compounds; Stomach Neoplasms; Structure-Activity Relationship

Sphingolipid mediators such as ceramide are pleiotropic regulators of cellular growth, differentiation and apoptosis. We investigated the role of ceramide biosynthesis, metabolism and actions in term human cytotrophoblasts syncytialized over 7 days in culture. Intracellular C16 ceramide levels increased modestly after 3 days in culture, then declined. Ceramidase was present at particularly high levels in syncytialized trophoblasts; inhibition of ceramidase reduced the degree of cell fusion. Exposure to short chain C8 ceramide or aSMase enhanced secretion of the differentiation marker hCG without affecting fusion or cell viability. In contrast, pharmacological inhibition of ceramidase reduced the extent of fusion. Inhibition of the ceramide-responsive JNK and PP2A pathways did not abolish the effects of ceramide, and JNK phosphorylation was unresponsive to ceramide; however, ceramide significantly inhibited phosphorylation of Akt. This study suggests that changes in ceramide biosynthesis and metabolism play a differential role in the biochemical and morphological features of trophoblast differentiation.

Topics: Anthracenes; Antigens, Differentiation; Caspase 8; Cell Differentiation; Cell Fusion; Cells, Cultured; Ceramidases; Ceramides; Chorionic Gonadotropin; DNA-Binding Proteins; Enzyme Inhibitors; Female; Gene Expression; Giant Cells; Humans; Nuclear Proteins; Okadaic Acid; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Processing, Post-Translational; Pyrans; Sphingomyelin Phosphodiesterase; Spiro Compounds; Transcription Factors; Trophoblasts

Sphingosine (SPH) is an important bioactive lipid involved in mediating a variety of cell functions including apoptosis. However, the signaling mechanism of SPH-induced apoptosis remains unclear. We have investigated whether SPH inhibits survival signaling in cells by inhibiting Akt kinase activity. This study demonstrates that treatment of Jurkat cells with SPH leads to Akt dephosphorylation as early as 15  min, and the cells undergo apoptosis after 6  h. This Akt dephosphorylation is not mediated through deactivation of upstream kinases, since SPH does not inhibit the upstream phosphoinositide-dependent kinase 1 (PDK1) phosphorylation. Rather, sensitivity to the Ser/Thr protein phosphatase inhibitors (calyculin A, phosphatidic acid, tautomycin, and okadaic acid) indicates an important role for protein phosphatase 1 (PP1) in this process. In vitro phosphatase assay, using Akt immunoprecipitate following treatment with SPH, reveals an increase in Akt-PP1 association as determined by immunoprecipitation analysis. Moreover, SPH-induced dephosphorylation of Akt at Ser(473) subsequently leads to the activation of GSK-3β, caspase 3, PARP cleavage, and ultimately apoptosis. Pre-treatment with caspase 3 inhibitor z-VAD-fmk and Ser/Thr phosphatase inhibitor abrogates the effect of SPH on facilitating apoptosis. Altogether, these results demonstrate that PP1-mediated inhibition of the key anti-apoptotic protein, Akt, plays an important role in SPH-mediated apoptosis in Jurkat cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Caspase 3; Cell Survival; Enzyme Activation; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Immunoprecipitation; Jurkat Cells; Marine Toxins; Okadaic Acid; Oxazoles; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Binding; Protein Phosphatase 1; Proto-Oncogene Proteins c-akt; Pyrans; Serine; Signal Transduction; Sphingosine; Spiro Compounds

A central question in Wnt signaling is the regulation of beta-catenin phosphorylation and degradation. Multiple kinases, including CKI alpha and GSK3, are involved in beta-catenin phosphorylation. Protein phosphatases such as PP2A and PP1 have been implicated in the regulation of beta-catenin. However, which phosphatase dephosphorylates beta-catenin in vivo and how the specificity of beta-catenin dephosphorylation is regulated are not clear. In this study, we show that PP2A regulates beta-catenin phosphorylation and degradation in vivo. We demonstrate that PP2A is required for Wnt/beta-catenin signaling in Drosophila. Moreover, we have identified PR55 alpha as the regulatory subunit of PP2A that controls beta-catenin phosphorylation and degradation. PR55 alpha, but not the catalytic subunit, PP2Ac, directly interacts with beta-catenin. RNA interference knockdown of PR55 alpha elevates beta-catenin phosphorylation and decreases Wnt signaling, whereas overexpressing PR55 alpha enhances Wnt signaling. Taken together, our results suggest that PR55 alpha specifically regulates PP2A-mediated beta-catenin dephosphorylation and plays an essential role in Wnt signaling.

Topics: Animals; Axin Protein; beta Catenin; Blotting, Western; Cell Line; Cell Line, Tumor; Drosophila melanogaster; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Humans; Immunoprecipitation; Lithium Chloride; Okadaic Acid; Phosphorylation; Protein Binding; Protein Phosphatase 2; Protein Subunits; Pyrans; Repressor Proteins; RNA, Small Interfering; Spiro Compounds

Natural product extracts have proven to be a rich source of small molecules that potently inhibit the catalytic activity of certain PPP-family ser/thr protein phosphatases. To date, the list of inhibitors includes okadaic acid (produced by marine dinoflagelates, Prorocentrum sp. and Dinophysis sp.), calyculin A, dragmacidins (isolated from marine sponges), microcystins, nodularins (cyanobacteria, Microcystis sp. and Nodularia sp.), tautomycin, tautomycetin, cytostatins, phospholine, leustroducsins, phoslactomycins, fostriecin (soil bacteria, Streptomyces sp.), and cantharidin (blister beetles, approx 1500 species). Many of these compounds share structural similarities, and several have become readily available for research purposes. Here we will review the specificity of available inhibitors and present methods for their use in studying sensitive phosphatases. Common mistakes in the employment of these compounds will also be addressed briefly, notably the widespread misconception that they only inhibit the activity of PP1 and PP2A. Inhibitors of PP2B (calcineurin) will only be mentioned in passing, except to state that, in our hands, cypermethrin, deltamethrin, and fenvalerate, which are sold as potent inhibitors of PP2B, do not inhibit the catalytic activity of PP2B.

Topics: Cantharidin; Enzyme Inhibitors; Furans; Indole Alkaloids; Lactones; Lipids; Marine Toxins; Microcystins; Okadaic Acid; Organophosphates; Organophosphorus Compounds; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Pyrans; Pyrones; Spiro Compounds

Human enhancer of filamentation 1 (HEF1), a multifunctional docking protein of the Cas family, participates in integrin and growth factor signaling pathways that regulate global cellular processes including growth, motility and apoptosis. HEF1 consists of two isoforms, p105 and p115, the larger molecular weight form resulting from Ser/Thr phosphorylation of p105HEF1. The molecular mechanisms that regulate the interconversion of the two HEF1 species as well as the function of HEF1 Ser/Thr phosphorylation are unknown. Our study reveals that cell adhesion and detachment regulate the interconversion of the two HEF1 isoforms. Experiments using various inhibitors of cytoskeletal organization indicated that disruption of actin microfilaments but not intermediate filaments or microtubules resulted in a complete conversion of p115HEF1 to p105HEF1. The conversion of p115HEF1 to p105HEF1 was prevented by inhibition of protein phosphatase 2A (PP2A), suggesting that cytoskeletal regulation of PP2A activity controlled the dephosphorylation of p115HEF1. Degradation of endogenous HEF1 was dependent on proteasomes with the p115 species of HEF1 being preferentially targeted for turnover. Dephosphorylation of HEF1 by suspending cells or disrupting actin filaments protected HEF1 from degradation. These results suggest that the adhesion-dependent actin organization regulates proteasomal turnover of HEF1 through the activity of PP2A.

Topics: Actin Cytoskeleton; Actins; Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Cell Adhesion; Cell Line; Colchicine; Cytochalasin D; Enzyme Inhibitors; Humans; Intermediate Filaments; Marine Toxins; Microtubules; Nocodazole; Nucleic Acid Synthesis Inhibitors; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Isoforms; Protein Phosphatase 2; Pyrans; Serine; Signal Transduction; Spiro Compounds; Threonine; Tubulin Modulators

Despite advances in understanding the role of histone deacetylases (HDACs) in tumorigenesis, the mechanism by which HDAC inhibitors mediate antineoplastic effects remains elusive. Modifications of the histone code alone are not sufficient to account for the antitumor effect of HDAC inhibitors. The present study demonstrates a novel histone acetylation-independent mechanism by which HDAC inhibitors cause Akt dephosphorylation in U87MG glioblastoma and PC-3 prostate cancer cells by disrupting HDAC-protein phosphatase 1 (PP1) complexes. Of four HDAC inhibitors examined, trichostatin A (TSA) and HDAC42 exhibit the highest activity in down-regulating phospho-Akt, followed by suberoylanilide hydroxamic acid, whereas MS-275 shows only a marginal effect at 5 microm. This differential potency parallels the respective activities in inducing tubulin acetylation, a non-histone substrate for HDAC6. Evidence indicates that this Akt dephosphorylation is not mediated through deactivation of upstream kinases or activation of downstream phosphatases. However, the effect of TSA on phospho-Akt can be rescued by PP1 inhibition but not that of protein phosphatase 2A. Immunochemical analyses reveal that TSA blocks specific interactions of PP1 with HDACs 1 and 6, resulting in increased PP1-Akt association. Moreover, we used isozyme-specific small interfering RNAs to confirm the role of HDACs 1 and 6 as key mediators in facilitating Akt dephosphorylation. The selective action of HDAC inhibitors on HDAC-PP1 complexes represents the first example of modulating specific PP1 interactions by small molecule agents. From a clinical perspective, identification of this PP1-facilitated dephosphorylation mechanism underscores the potential use of HDAC inhibitors in lowering the apoptosis threshold for other therapeutic agents through Akt down-regulation.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Immunoblotting; Immunohistochemistry; Immunoprecipitation; Marine Toxins; Okadaic Acid; Oxazoles; Phosphatidylinositols; Phosphoprotein Phosphatases; Phosphorylation; Protein Binding; Protein Isoforms; Protein Phosphatase 1; Protein Phosphatase 2; Protein Structure, Tertiary; Proto-Oncogene Proteins c-akt; Pyrans; RNA, Small Interfering; Serine; Signal Transduction; Spiro Compounds; Subcellular Fractions; Threonine; Time Factors

Axon pathfinding depends on attractive and repulsive turning of growth cones to extracellular cues. Localized cytosolic Ca2+ signals are known to mediate the bidirectional responses, but downstream mechanisms remain elusive. Here, we report that calcium-calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) phosphatase provide a switch-like mechanism to control the direction of Ca(2+)-dependent growth cone turning. A relatively large local Ca2+ elevation preferentially activates CaMKII to induce attraction, while a modest local Ca2+ signal predominantly acts through CaN and phosphatase-1 (PP1) to produce repulsion. The resting level of intracellular Ca2+ concentrations also affects CaMKII/CaN operation: a normal baseline allows distinct turning responses to different local Ca2+ signals, while a low baseline favors CaN-PP1 activation for repulsion. Moreover, the cAMP pathway negatively regulates CaN-PP1 signaling to inhibit repulsion. Finally, CaMKII/CaN-PP1 also mediates netrin-1 guidance. Together, these findings establish a complex Ca2+ mechanism that targets the balance of CaMKII/CaN-PP1 activation to control distinct growth cone responses.

Topics: Animals; Benzylamines; Calcineurin; Calcium; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Chlorocebus aethiops; COS Cells; Cyclic AMP; Cyclic GMP; Cyclosporine; Dose-Response Relationship, Drug; Drug Interactions; Egtazic Acid; Embryo, Mammalian; Embryo, Nonmammalian; Enzyme Inhibitors; Growth Cones; Humans; Models, Neurological; Nerve Growth Factors; Netrin-1; Neurons; Nitriles; Okadaic Acid; Phosphoprotein Phosphatases; Photolysis; Protein Phosphatase 1; Pyrans; Pyrethrins; Semaphorin-3A; Spinal Cord; Spiro Compounds; Sulfonamides; Time Factors; Tumor Suppressor Proteins; Xenopus

Inhibitors of serine/threonine protein phosphatases can inhibit apoptosis. We investigated which protein phosphatases are critical for this protection using calyculin A, okadaic acid, and tautomycin. All three phosphatase inhibitors prevented anisomycin-induced apoptosis in leukemia cell models. In vitro, calyculin A does not discriminate between PP1 and PP2A, while okadaic acid and tautomycin are more selective for PP2A and PP1, respectively. Increased phosphorylation of endogenous marker proteins was used to define concentrations that inhibited each phosphatase in cells. Concentrations of each inhibitor that prevented anisomycin-induced apoptosis correlated with inhibition of PP2A. The inhibitors prevented Bax translocation to mitochondria, indicating inhibition upstream of mitochondria. Tautomycin and calyculin A, but not okadaic acid, also prevented apoptosis induced through the CD95/Fas death receptor, and this protection correlated with inhibition of PP1. The inhibitors prevented Fas receptor oligomerization, FADD recruitment, and caspase 8 activation. The differential effects of PP1 and PP2A in protection from death receptor and mitochondrial-mediated pathways of death, respectively, may help one to define critical steps in each pathway, and regulatory roles for serine/threonine phosphatases in apoptosis.

Topics: Apoptosis; Burkitt Lymphoma; Cell Line, Tumor; Enzyme Inhibitors; Humans; Jurkat Cells; Marine Toxins; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Pyrans; Signal Transduction; Spiro Compounds; U937 Cells

A revised model of PP1-tautomycin (TM) complex suggests that this toxin does not bind in a conformation analogous to its structural cousin okadaic acid (OA), as has been assumed, but instead more resembles the mode of binding adopted by calyculin. This model rationalizes the unexpected potency of a truncated TM analogue lacking the bicyclic ketal common to TM and OA.

Topics: Enzyme Inhibitors; Models, Molecular; Molecular Conformation; Okadaic Acid; Phosphoprotein Phosphatases; Pyrans; Spiro Compounds

In adipocytes, protein kinase B (PKB) has been suggested to be the enzyme that phosphorylates phosphodiesterase 3B (PDE3B), a key enzyme in insulin's antilipolytic signalling pathway. In order to screen for PKB phosphatases, adipocyte homogenates were fractionated using ion-exchange chromatography and analysed for PKB phosphatase activities. PKB phosphatase activity eluted as one main peak, which coeluted with serine/threonine phosphatases (PP)2A. In addition, adipocytes were incubated with inhibitors of PP. Incubation of adipocytes with 1 microM okadaic acid inhibited PP2A by 75% and PP1 activity by only 17%, while 1 microM tautomycin inhibited PP1 activity by 54% and PP2A by only 7%. Okadaic acid, but not tautomycin, induced the activation of both PKBalpha and PKBbeta. Finally, PP2A subunits were found in several subcellular compartments, including plasma membranes (PM) where the phosphorylation of PKB is thought to occur. In summary, our results suggest that PP2A is the principal phosphatase that dephosphorylates PKB in adipocytes.

Topics: Adipocytes; Animals; Antifungal Agents; Cells, Cultured; Enzyme Inhibitors; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Pyrans; Rats; Spiro Compounds; Subcellular Fractions

Previous studies have shown that the Bacillus anthracis lethal toxin can induce both necrosis and apoptosis in mouse macrophage-like J774A.1 cells depending on both the toxin concentration and the phosphatase activity. In this study several protein kinase or phosphatase inhibitors were employed to evaluate the hypothesis that the lethal toxin induces cell death via protein phosphorylation processes. Pretreatment with a serine/threonine phosphatase inhibitor Calyculin A (300 nM) could inhibit about 78% of cell death induced by the lethal toxin, whereas inhibitors of kinases, such as H7, HA, Sphingosine, and Genestein, but other inhibitors of phosphatases, such as Okadaic acid, Tautomycin, and Cyclosporin A, did not. In addition, recent reports have demonstrated that the MEK1 protein may serve as a proteolytic target within its N-terminus for lethal factor cleavage. In this study, Calyculin A is shown to enhance the phosphorylation of the MEK1 protein. This prevents the cleavage of the MEK1 by lethal factor. These results suggest that a putative Calyculin A-sensitive protein phosphatase is involved in anthrax toxin induced cytotoxicity and that the blocking effect of Calyculin A on lethal factor cytotoxicity may be mediated through the MEK signaling pathway.

Topics: Animals; Antifungal Agents; Antigens, Bacterial; Bacillus anthracis; Bacterial Toxins; Blotting, Western; Cells, Cultured; Cyclosporine; Enzyme Inhibitors; Genistein; MAP Kinase Kinase Kinase 1; Marine Toxins; Mice; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Protein Serine-Threonine Kinases; Pyrans; Signal Transduction; Sphingosine; Spiro Compounds

The function of cyclin G, a commonly induced p53 target, has remained elusive. We show that cyclin G forms a quaternary complex in vivo and in vitro with enzymatically active phosphatase 2A (PP2A) holoenzymes containing B' subunits. Interestingly, cyclin G also binds in vivo and in vitro to Mdm2 and markedly stimulates the ability of PP2A to dephosphorylate Mdm2 at T216. Consistent with these data, cyclin G null cells have both Mdm2 that is hyperphosphorylated at T216 and markedly higher levels of p53 protein when compared to wild-type cells. Cyclin G expression also results in reduced phosphorylation of human Hdm2 at S166. Thus, our data suggest that cyclin G recruits PP2A in order to modulate the phosphorylation of Mdm2 and thereby to regulate both Mdm2 and p53.

Topics: Animals; Antifungal Agents; Catalysis; Chlorocebus aethiops; COS Cells; Cyclin G; Cyclin G1; Cyclins; Cyclosporine; Enzyme Inhibitors; Humans; Macromolecular Substances; Mice; Nuclear Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Phosphotyrosine; Protein Binding; Protein Interaction Mapping; Protein Phosphatase 2; Protein Processing, Post-Translational; Protein Subunits; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Pyrans; Recombinant Fusion Proteins; Species Specificity; Spiro Compounds; Transfection; Tumor Suppressor Protein p53

The mechanisms underlying T cell receptor (TCR) down-regulation have been extensively studied during the last decade. Whereas the importance of phosphorylation in this process has been established, it is less certain whether dephosphorylation plays a role in TCR down-regulation. In this study, we show that inhibition of the serine/threonine protein phosphatase PP2A family had a biphasic effect on TCR expression. Thus, low concentrations of PP2A inhibitors induced TCR down-regulation, whereas higher concentrations of PP2A inhibitors induced TCR up-regulation. The effect of PP2A inhibition was independent of phosphorylation of the CD3gamma endocytosis motif. Whereas TCR down-regulation was caused by a partial inhibition of exocytosis, TCR up-regulation was caused by an inhibition of endocytosis. The effects on exocytosis and endocytosis were not restricted to the TCR, indicating a more general regulatory role for PP2A in both exocytosis and endocytosis.

Topics: Amino Acid Sequence; Antifungal Agents; CD3 Complex; Down-Regulation; Endocytosis; Enzyme Inhibitors; Exocytosis; Humans; Isoenzymes; Jurkat Cells; Marine Toxins; Molecular Sequence Data; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2; Pyrans; Receptors, Antigen, T-Cell; Receptors, Antigen, T-Cell, gamma-delta; Receptors, Transferrin; Spiro Compounds

A wide range of serine/threonine protein phosphatase (PP) inhibitors were studied for effects on the immunoglobulin E (IgE)-mediated release of histamine from human lung mast cells, human skin mast cells and basophils. Okadaic acid (OA) inhibited the release of histamine from all three cell types in a concentration-dependent manner. Two structural analogues of okadaic acid, okadaol and okadaone, known to be less active than the parent molecule as inhibitors of PP, were less active than okadaic acid as inhibitors of histamine release in these three cell types. A number of PP inhibitors, showing differences in selectivity for PP1 and PP2A, were also evaluated. Calyculin, which is roughly equipotent as a PP1 and PP2A inhibitor, attenuated the release of histamine from all three cell types. Similarly, tautomycin (TAU), which shows greater selectivity for PP1 over PP2A, was also effective at inhibiting histamine release in all three cell types. In contrast, fostriecin, which is very much more potent as an inhibitor of PP2A over PP1, was ineffective as an inhibitor in all three cell types. These data indicate that the regulation of mediator release by PPs is similar in lung mast cells, skin mast cells and basophils. Moreover, the data suggest that PP1 is important in the control of cellular activity.

Topics: Alkenes; Antifungal Agents; Basophils; Enzyme Inhibitors; Histamine Release; Humans; Immunoglobulin E; Lung; Marine Toxins; Mast Cells; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Polyenes; Pyrans; Pyrones; Skin; Spiro Compounds

Growth cones generate spontaneous transient elevations of intracellular Ca(2+) that regulate the rate of neurite outgrowth. Here we report that these Ca(2+) waves inhibit neurite extension via the Ca(2+)-dependent phosphatase calcineurin (CN) in Xenopus spinal neurons. Pharmacological blockers of CN (cyclosporin A and deltamethrin) and peptide inhibitors of CN [the Xenopus CN (xCN) autoinhibitory domain and African swine fever virus protein A238L] block the Ca(2+)-dependent reduction of neurite outgrowth in cultured neurons. Time-lapse microscopy of growing neurites demonstrates directly that the reduction in the rate of outgrowth by Ca(2+) transients is blocked by cyclosporin A. In contrast, expression of a constitutively active form of xCN in the absence of waves results in shorter neurite lengths similar to those seen in the presence of waves. The developmental expression pattern of xCN transcripts in vivo coincides temporally with axonal pathfinding by spinal neurons, supporting a role of CN in regulating Ca(2+)-dependent neurite extension in the spinal cord. Ca(2+) wave frequency and Ca(2+)-dependent expression of GABA are not affected by inhibition or activation of CN. However, phosphorylation of the cytoskeletal element GAP-43, which promotes actin polymerization, is reduced by Ca(2+) waves and enhanced by suppression of CN activity. CN ultimately acts on the growth cone actin cytoskeleton, because disrupting actin microfilaments with cytochalasin D or stabilizing them with jasplakinolide negates the effects of suppressing or activating CN. Destabilization or stabilization of microtubules with colcemide or taxol results in Ca(2+)-independent inhibition of neurite outgrowth. The results identify components of the cascade by which Ca(2+) waves act to regulate neurite extension.

Topics: Actins; Animals; Antifungal Agents; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Calcineurin; Calcium; Calcium Signaling; Cells, Cultured; Cloning, Molecular; Cyclosporine; Cytochalasin D; Demecolcine; Depsipeptides; Embryonic Development; Enzyme Inhibitors; Female; gamma-Aminobutyric Acid; GAP-43 Protein; Gene Expression Regulation, Developmental; Growth Cones; Molecular Sequence Data; Neurites; Neurons; Nucleic Acid Synthesis Inhibitors; Okadaic Acid; Paclitaxel; Peptides, Cyclic; Phosphorylation; Pyrans; Spinal Cord; Spiro Compounds; Xenopus laevis

In the present work we studied the effect of protein phosphatase inhibitors on the phosphorylation state and function of alpha(1b)-adrenoceptors. Okadaic acid increased receptor phosphorylation in a time- and concentration-dependent fashion (maximum at 30 min, EC(50) of 30 nM). Other inhibitors of protein phosphatases (calyculin A, tautomycin and cypermethrin) mimicked this effect. Staurosporine and Ro 31-8220, inhibitors of protein kinase C, blocked the effect of okadaic acid on receptor phosphorylation. Neither genistein nor wortmannin altered the effect of okadaic acid. The intense adrenoceptor phosphorylation induced by okadaic acid altered the adrenoceptor-G protein coupling, as evidenced by a small decreased noradrenaline-stimulated [(35)S]GTPgammaS binding. Okadaic acid did not alter the noradrenaline-stimulated increases in intracellular calcium or the production of inositol trisphosphate. Our data indicate that inhibition of protein phosphatases increases the phosphorylation state of alpha(1b)-adrenoceptors; this effect seems to involve protein kinase C. In spite of inducing an intense receptor phosphorylation, okadaic acid alters alpha(1b)-adrenergic actions to a much lesser extent than the direct activation of protein kinase C by phorbol myristate acetate.

Topics: Animals; Antifungal Agents; Dose-Response Relationship, Drug; Enzyme Inhibitors; Indoles; Kinetics; Marine Toxins; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C; Pyrans; Pyrethrins; Rats; Receptors, Adrenergic, alpha-1; Spiro Compounds; Staurosporine; Tetradecanoylphorbol Acetate

Clathrin-mediated endocytosis includes cycles of assembly and disassembly of the clathrin-coated vesicle constituents. How these cycles are regulated is still not fully known but previous studies have indicated that phosphorylation of coat subunits may play a role. Here we describe that beta2-adaptin undergoes cycles of phosphorylation/de-phosphorylation in intact cells. Thus, beta2-adaptin was constitutively de-phosphorylated by serine/threonine protein phosphatase 2A and phosphorylated by a staurosporine-sensitive kinase in vivo. Confocal laser scanning microscopy demonstrated that phosphorylated AP2 complexes were found more evenly distributed at the plasma membrane compared to non-phosphorylated AP2 complexes which were found in aggregates. Finally, we found that phosphorylation of beta2-adaptin correlated with inhibition of clathrin-mediated endocytosis. Our results support the hypothesis that phosphorylation/de-phosphorylation of coat proteins plays a regulatory role in the assembly/disassembly cycle of clathrin-coated vesicles.

Topics: Adaptor Protein Complex beta Subunits; Antifungal Agents; Cell Membrane; Cells, Cultured; Endocytosis; Enzyme Inhibitors; Humans; Jurkat Cells; Marine Toxins; Membrane Proteins; Microscopy, Confocal; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinases; Protein Phosphatase 2; Pyrans; Spiro Compounds; Staurosporine

In the present study, the role of phosphoprotein phosphatase in the regulation of L-type Ca2+ channel currents in rat pinealocytes was investigated using the whole-cell version of the patch-clamp technique. The effects of three phosphatase inhibitors, calyculin A, tautomycin, and okadaic acid, were compared. Although all three inhibitors were effective in inhibiting the L-type Ca2+ channel current, calyculin A was more potent than either tautomycin or okadaic acid, suggesting the involvement of phosphoprotein phosphatase-1. To determine the kinase involved in the regulation of these channels, cells were pretreated with H7 (a nonspecific kinase inhibitor), H89 (a specific inhibitor of cyclic AMP-dependent kinase), KT5823 (a specific inhibitor of cyclic GMP-dependent kinase), or calphostin C (a specific inhibitor of protein kinase C). Pretreatment with either H7 or calphostin C decreased the inhibitory effect of calyculin A on the L-type Ca2+ channel current. In contrast, pretreatment with H89 or KT5823 had no effect on the inhibition caused by calyculin A. Based on these observations, we conclude that basal phosphatase activity, probably phosphoprotein phosphatase-1, plays an important role in the regulation of L-type Ca2+ channel currents in rat pinealocytes by counteracting protein kinase C-mediated phosphorylation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adrenergic beta-Agonists; Alkaloids; Animals; Antifungal Agents; Calcium Channels; Calcium Channels, L-Type; Carbazoles; Cyclic AMP; Cyclic GMP; Enzyme Inhibitors; Indoles; Isoproterenol; Isoquinolines; Male; Marine Toxins; Membrane Potentials; Nerve Tissue Proteins; Norepinephrine; Okadaic Acid; Oxazoles; Patch-Clamp Techniques; Phosphoric Monoester Hydrolases; Phosphorylation; Pineal Gland; Protein Kinase C; Pyrans; Rats; Rats, Sprague-Dawley; Spiro Compounds; Sulfonamides; Sympathomimetics

Topics: Animals; Antifungal Agents; Bile Acids and Salts; Calcium; Carrier Proteins; Cloning, Molecular; Cyclic AMP; Endosomes; Enzyme Inhibitors; Liver; Okadaic Acid; Organic Anion Transporters, Sodium-Dependent; Phosphoprotein Phosphatases; Pyrans; Signal Transduction; Spiro Compounds; Symporters

The intermediate filament protein vimentin is a major phosphoprotein in mammalian fibroblasts, and reversible phosphorylation plays a key role in its dynamic rearrangement. Selective inhibition of type 2A but not type 1 protein phosphatases led to hyperphosphorylation and concomitant disassembly of vimentin, characterized by a collapse into bundles around the nucleus. We have analyzed the potential role of one of the major protein phosphatase 2A (PP2A) regulatory subunits, B55, in vimentin dephosphorylation. In mammalian fibroblasts, B55 protein was distributed ubiquitously throughout the cytoplasm with a fraction associated to vimentin. Specific depletion of B55 in living cells by antisense B55 RNA was accompanied by disassembly and increased phosphorylation of vimentin, as when type 2A phosphatases were inhibited using okadaic acid. The presence of B55 was a prerequisite for PP2A to efficiently dephosphorylate vimentin in vitro or to induce filament reassembly in situ. Both biochemical fractionation and immunofluorescence analysis of detergent-extracted cells revealed that fractions of PP2Ac, PR65, and B55 were tightly associated with vimentin. Furthermore, vimentin-associated PP2A catalytic subunit was displaced in B55-depleted cells. Taken together these data show that, in mammalian fibroblasts, the intermediate filament protein vimentin is dephosphorylated by PP2A, an event targeted by B55.

Topics: Antifungal Agents; Cytoplasm; Enzyme Inhibitors; Fibroblasts; Humans; Intermediate Filaments; Interphase; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2; Pyrans; Recombinant Proteins; Spiro Compounds; Vimentin

The function of the retinoblastoma protein (pRB) in controlling the G(1) to S transition is regulated by phosphorylation and dephosphorylation on serine and threonine residues. While the roles of cyclin-dependent kinases in phosphorylating and inactivating pRB have been characterized in detail, the roles of protein phosphatases in regulating the G(1)/S transition are not as well understood. We used cell-permeable inhibitors of protein phosphatases 1 and 2A to assess the contributions of these phosphatases in regulating cyclin-dependent kinase activity and pRB phosphorylation. Treating asynchronously growing Balb/c 3T3 cells with PP2A-selective concentrations of either okadaic acid or calyculin A caused a time- and dose-dependent decrease in pRB phosphorylation. Okadaic acid and calyculin A had no effect on pRB phosphatase activity even though PP2A was completely inhibited. The decrease in pRB phosphorylation correlated with inhibitor-induced suppression of G(1) cyclin-dependent kinases including CDK2, CDK4, and CDK6. The inhibitors also caused decreases in the levels of cyclin D2 and cyclin E, and induction of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1). The decrease in cyclin-dependent kinase activities were not dependent on induction of cyclin-dependent kinase inhibitors since CDK inhibition still occurred in the presence of actinomycin D or cycloheximide. In contrast, selective inhibition of protein phosphatase 1 with tautomycin inhibited pRB phosphatase activity and maintained pRB in a highly phosphorylated state. The results show that protein phosphatase 1 and protein phosphatase 2A, or 2A-like phosphatases, play distinct roles in regulating pRB function. Protein phosphatase 1 is associated with the direct dephosphorylation of pRB while protein phosphatase 2A is involved in pathways regulating G(1) cyclin-dependent kinase activity.

Topics: 3T3 Cells; Animals; Antifungal Agents; Cyclin G; Cyclin G1; Cyclin-Dependent Kinases; Cyclins; Enzyme Inhibitors; Isoenzymes; Marine Toxins; Mice; Mice, Inbred BALB C; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Protein Phosphatase 2; Pyrans; Retinoblastoma Protein; Spiro Compounds

Dephosphorylation of central photosynthetic proteins regulates their turnover in plant thylakoid membranes. A membrane protein phosphatase from spinach thylakoids was purified 13000-fold using detergent-engaged FPLC. The purified enzyme exhibited characteristics typical of eukaryotic Ser/Thr phosphatases of the PP2A family in that it was inhibited by okadaic acid (IC(50) = 0.4 nM) and tautomycin (IC(50) = 25 nM), irreversibly bound to microcystin-agarose, and recognized by a polyclonal antibody raised against a recombinant catalytic subunit of human PP2A. Furthermore, the anti-PP2A antibody inhibited protein dephosphorylation in isolated thylakoids. The phosphatase copurified with TLP40, a cyclophilin-like peptidyl-prolyl isomerase located in the thylakoid lumen. TLP40 could be released from the phosphatase immobilized on microcystin-agarose by high-salt treatment. Binding of cyclosporin A (CsA) to TLP40 led to thylakoid phosphatase activation, while cyclophilin substrates, prolyl-containing oligopeptides, inhibited protein dephosphorylation. This dephosphorylation could be modulated by CsA or oligopeptides only after the thylakoids had been ruptured to expose the lumenal membrane surface where the TLP40 is located. Regulation of the PP2A-like phosphatase at the outer thylakoid surface is likely to operate via reversible binding of TLP40 to the inner membrane surface. This is a first example of transmembrane regulation in which the activity of phosphatase is altered by the binding of a cyclophilin to a site other than the active one. We propose that signaling from TLP40 to the protein phosphatase coordinates dephosphorylation and protein folding, two processes required for protein turnover during the repair of photoinhibited photosystem II reaction centers.

Topics: Amino Acid Sequence; Antifungal Agents; Catalytic Domain; Chloroplasts; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Humans; Immunophilins; Molecular Sequence Data; Okadaic Acid; Peptidylprolyl Isomerase; Phosphoprotein Phosphatases; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Plant Proteins; Pyrans; Spiro Compounds; Thylakoids

Vascular endothelial growth factor (VEGF) is a specific mitogen for vascular endothelial cells and has been implicated in tumor angiogenesis. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, is a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoter in two-stage carcinogenesis experiments in mouse skin. To elucidate the role of VEGF in the angiogenesis of these experimental tumors, the effect of okadaic acid on VEGF gene expression was examined. In NIH 3T3, Rat1, HeLa, and A431 cells, VEGF mRNA was upregulated by 5- to 10-fold after incubation with okadaic acid. Furthermore, the amount of VEGF protein in the culture medium was significantly increased after stimulation with okadaic acid. Interestingly, okadaic acid-induced upregulation of VEGF mRNA was not suppressed by protein kinase C (PKC) inhibitor or by tumor necrosis factor alpha blocking antibody, although TPA-induced VEGF upregulation was strongly suppressed by PKC inhibitor. Our results indicate that okadaic acid is a new and potent inducer of VEGF, suggesting the involvement of VEGF as an angiogenic factor during multistep carcinogenesis in vivo.

Topics: 3T3 Cells; Animals; Antifungal Agents; Cell Line; Culture Media; Endothelial Growth Factors; Enzyme Inhibitors; HeLa Cells; Humans; Lymphokines; Mice; Neoplasms; Neovascularization, Pathologic; Neutralization Tests; Okadaic Acid; Phosphoprotein Phosphatases; Protein Kinase C; Pyrans; Rats; RNA, Messenger; Spiro Compounds; Tumor Necrosis Factor-alpha; Up-Regulation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The protein phosphatase (PP) inhibitors nodularin and microcystin-LR induced apoptosis with unprecedented rapidity, more than 50% of primary hepatocytes showing extensive surface budding and shrinkage of cytoplasm and nucleoplasm within 2 min. The apoptosis was retarded by the general caspase inhibitor Z-VAD.fmk. To circumvent the inefficient uptake of microcystin and nodularin into nonhepatocytes, toxins were microinjected into 293 cells, Swiss 3T3 fibroblasts, promyelocytic IPC-81 cells, and NRK cells. All cells started to undergo budding typical of apoptosis within 0.5 - 3 min after injection. This was accompanied by cytoplasmic and nuclear shrinkage and externalization of phosphatidylserine. Overexpression of Bcl-2 did not delay apoptosis. Apoptosis induction was slower and Z-VAD.fmk independent in caspase-3 deficient MCF-7 cells. MCF-7 cells stably transfected with caspase-3 showed a more rapid and Z-VAD.fmk dependent apoptotic response to nodularin. Rapid apoptosis induction required inhibition of both PP1 and PP2A, and the apoptosis was preceded by increased phosphorylation of several proteins, including myosin light chain. The protein phosphorylation occurred even in the presence of apoptosis-blocking concentrations of Z-VAD.fmk, indicating that it occurred upstream of caspase activation. It is suggested that phosphatase-inhibiting toxins can induce caspase-3 dependent apoptosis in an ultrarapid manner by altering protein phosphorylation.

Topics: 3T3 Cells; Animals; Antifungal Agents; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Cell Line; Cell Line, Transformed; Enzyme Inhibitors; Gene Expression; Humans; Intracellular Fluid; Marine Toxins; Mice; Microcystins; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Pyrans; Rats; Rats, Wistar; Spiro Compounds; Tumor Cells, Cultured

Previous studies have shown that treating rat cortical neurons in primary culture with apolipoprotein E (apoE) peptide increased cytoplasmic Ca2+ by 2 mechanisms: 1) an influx of extracellular Ca2+ resulting from the activation of a cell surface Ca2+ channel; and 2) release of Ca2+ from internal Ca2+ stores via a G-protein-coupled pathway (Wang and Gruenstein, 1997). These studies employed a biologically active apoE synthetic peptide (apoEdp) derived from the receptor binding domain of apoE. In the present study we examined whether activation of these 2 signal transduction pathways affects phosphorylation of microtubule-associated protein tau. The levels of tau phosphorylation at thr231, ser235, and ser396 were quantified by ELISA employing monoclonal antibodies PHF-6, SMI33, and PHF-1. ApoEdp treatment resulted in a concentration- and time-dependent dephosphorylation of tau at all 3 phosphorylation sites. The apoEdp-induced dephosphorylation of tau at thr231, and ser235 was dependent on the influx of extracellular Ca2+, while dephosphorylation at ser396 was mediated by a pertusis toxin-sensitive G-protein pathway. The involvement of protein phosphatases in mediating the apoEdp-induced dephosphorylation of tau was examined. Pretreatment with the protein phosphatase 2B inhibitor cyclosporin A blocked the apoEdp-induced dephosphorylation of tau at thr231 and ser235 but not at ser396. Pretreatment with the protein phosophatase 2A/1 inhibitor okadaic acid blocked the apoEdp-induced dephosphorylation of tau at all 3 sites, while pretreatment with the protein phosphates 1 inhibitor tautomycin was without effect. The present study suggests that apoE may affect several Ca2+-associated signal transduction pathways that increase the activity of protein phosphatases 2A and 2B, which in turn dephosphorylate tau.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antifungal Agents; Apolipoproteins E; Calcium; Cyclosporine; Cytoplasm; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Immunosuppressive Agents; Molecular Sequence Data; Neurons; Okadaic Acid; Peptide Fragments; Phosphoric Monoester Hydrolases; Phosphorylation; Pyrans; Rats; Rats, Sprague-Dawley; Signal Transduction; Spiro Compounds; tau Proteins; Time Factors; Virulence Factors, Bordetella

The role of Rho GTPase and its downstream targets Rho kinase and myosin light chain phosphatase in thrombin-induced endothelial cell contraction was investigated. The specific Rho inactivator C3-transferase from Clostridium botulinum as well as microinjection of the isolated Rho-binding domain of Rho kinase or active myosin light chain phosphatase abolished thrombin-stimulated endothelial cell contraction. Conversely, microinjection of constitutively active V14Rho, constitutively active catalytic domain of Rho kinase, or treatment with the phosphatase inhibitor tautomycin caused contraction. These data are consistent with the notion that thrombin activates Rho/Rho kinase to inactivate myosin light chain phosphatase in endothelial cells. In fact, we demonstrate that thrombin transiently inactivated myosin light chain phosphatase, and this correlated with a peak in myosin light chain phosphorylation. C3-transferase abolished the decrease in myosin light chain phosphatase activity as well as the subsequent increase in myosin light chain phosphorylation and cell contraction. These data suggest that thrombin activates the Rho/Rho kinase pathway to inactivate myosin light chain phosphatase as part of a signaling network that controls myosin light chain phosphorylation/contraction in human endothelial cells.

Topics: ADP Ribose Transferases; Alkaloids; Antifungal Agents; Botulinum Toxins; Carbazoles; Cells, Cultured; Cytoskeletal Proteins; Endothelium, Vascular; Enzyme Inhibitors; Humans; Indoles; Intracellular Signaling Peptides and Proteins; Myosin-Light-Chain Kinase; Myosin-Light-Chain Phosphatase; Okadaic Acid; Phosphoprotein Phosphatases; Protein Serine-Threonine Kinases; Pyrans; rho-Associated Kinases; Spiro Compounds; Thrombin

Cyclic AMP has been proposed to stimulate Na+/taurocholate (TC) cotransport in hepatocytes by translocating Na+/TC cotransport polypeptide (Ntcp) to the plasma membrane and to induce Ntcp dephosphorylation. Whether protein phosphatases 1 and 2A (PP1/2A) are involved in the regulation of Na+/TC cotransport by cAMP was investigated in the present study. Okadaic acid and tautomycin, inhibitors of PP1/2A, inhibited cAMP-mediated increases in TC uptake and cytosolic [Ca2+], and only tautomycin inhibited basal TC uptake. Removal of cAMP reversed cAMP-mediated increases in TC uptake and plasma membrane Ntcp mass. Okadaic acid alone increased Ntcp phosphorylation without affecting Ntcp mass in plasma membranes and homogenates. In the presence of okadaic acid, cAMP failed to increase plasma membrane Ntcp mass, induce Ntcp dephosphorylation, and decrease endosomal Ntcp mass. Phosphorylated Ntcp was detectable in endosomes isolated from okadaic acid-treated hepatocytes but not in endosomes from control and cAMP-treated hepatocytes. PP1 was found to be enriched in plasma membranes, whereas PP2A was mostly in the cytosol. Cyclic AMP did not activate either PP1 or PP2A, whereas okadaic acid inhibited primarily PP2A. These results suggest that 1) the effect of cAMP on Na+/TC cotransport is not mediated via either PP1 or PP2A; rather, cAMP-mediated signaling pathway is maintained by PP2A and inhibition of PP2A overrides cAMP-mediated effects, and 2) okadaic acid, by inhibiting PP2A, inhibits cAMP-mediated increases in Na+/TC cotransport by decreasing the ability of cAMP to increase cytosolic [Ca2+]. It is proposed that cAMP-mediated dephosphorylation of Ntcp leads to an increased retention of Ntcp in the plasma membrane, and okadaic acid, by inhibiting PP2A, inhibits cAMP-mediated stimulation of Na+/TC cotransport by reversing the ability of cAMP to increase cytosolic [Ca2+] and to induce Ntcp dephosphorylation.

Topics: Animals; Antifungal Agents; Biological Transport; Calcium; Carrier Proteins; Cells, Cultured; Cyclic AMP; Cytosol; Enzyme Inhibitors; Liver; Okadaic Acid; Organic Anion Transporters, Sodium-Dependent; Phosphoprotein Phosphatases; Phosphorylation; Pyrans; Rats; Spiro Compounds; Symporters

Protein phosphatases play significant roles in signal transduction pathways pertaining to cell proliferation, gene expression, and neurotransmission. Serine/threonine phosphatases PP1 and PP2A, which are closely related in primary structure (approximately 50%), are inhibited by a structurally diverse group of natural toxins. As part of our study toward understanding the mechanism of inhibition displayed by these toxins, we have developed research in two directions: (1) The standardization of an assay to be used in acquisition of the structure--activity relationship of inhibition data is reported. This nonradioactive assay affords detection levels of molecular phosphate released from a phosphorylated hexapeptide in subnanomolar quantities. The comparison of our IC50 values of these inhibitors against corresponding literature data provided validation for our method. (2) Computational analysis provided a global model for binding of these inhibitors to PP1. The natural toxins were shown to possess remarkably similar three-dimensional motifs upon superimposition and van der Waals minimization within the PP1 active site.

Topics: Antifungal Agents; Crystallography, X-Ray; Enzyme Inhibitors; Marine Toxins; Microcystins; Models, Molecular; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Binding; Protein Conformation; Pyrans; Signal Transduction; Spiro Compounds; Stereoisomerism; Structure-Activity Relationship; Toxins, Biological

The effects of three protein phosphatase inhibitors, okadaic acid, calyculin A and tautomycin, on the formation of cleavage furrows and the induction of furrow-like dents in the egg of the newt, Cynops pyrrhogaster, were examined. Solutions of the individual compound were injected into the animal hemisphere of one of the two presumptive blastomere regions of the embryo during the first cleavage. Injection of a solution containing any of the chemicals often disturbed the formation of a normal furrow in the injected blastomere at second cleavage. Injection with okadaic acid or calyculin A often induced furrow-like dents on the surface of the injected blastomere at the same time as second cleavage in control embryos, while that with tautomycin usually did not induce them. In an injected blastomere, formation of dents started in the animal half and moved towards the vegetal half as the furrow in its counterpart blastomere extended from the animal half towards the vegetal. Dents gradually became slightly deeper and formed cytoplasmic projections that later degenerated, leaving a surface scar. Cytological observations on blastomeres injected with calyculin A revealed that nuclear division occurred normally.

Topics: Animals; Antifungal Agents; Cleavage Stage, Ovum; Enzyme Inhibitors; Marine Toxins; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Pyrans; Salamandridae; Spiro Compounds

Calyculin-A (CA), okadaic acid (OA), and tautomycin (TAU) are potent inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A) and are widely used on cells in culture. Despite their well characterized selectivity in vitro, their exact intracellular effects on PP1 and PP2A cannot be directly deduced from their extracellular concentration because their cell permeation properties are not known. Here we demonstrate that, due to the tight binding of the inhibitors to PP1 and/or PP2A, their cell penetration could be monitored by measuring PP1 and PP2A activities in cell-free extracts. Treatment of MCF7 cells with 10 nM CA for 2 h simultaneously inhibited PP1 and PP2A activities by more than 50%. A concentration of 1 microM OA was required to obtain a similar time course of PP2A inhibition in MCF7 cells to that observed with 10 nM CA, whereas PP1 activity was unaffected. PP1 was predominantly inhibited in MCF7 cells treated with TAU but even at 10 microM TAU PP1 inhibition was much slower than that observed with 10 nM CA. Furthermore, binding of inhibitors to PP2Ac and/or PP1c in MCF7 cells led to differential posttranslational modifications of the carboxyl termini of the proteins as demonstrated by Western blotting. OA and CA, in contrast to TAU, induced demethylation of the carboxyl-terminal Leu309 residue of PP2Ac. On the other hand, CA and TAU, in contrast to OA, elicited a marked decrease in immunoreactivity of the carboxyl terminus of the alpha-isoform of PP1c, probably reflecting proteolysis of the protein. These results suggest that in MCF7 cells OA selectively inhibits PP2A and TAU predominantly affects PP1, a conclusion supported by their differential effects on cytokeratins in this cell line.

Topics: Antifungal Agents; Blotting, Western; Cell Membrane Permeability; Cell-Free System; Down-Regulation; Enzyme Inhibitors; Humans; Intermediate Filaments; Marine Toxins; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Protein Binding; Protein Phosphatase 1; Protein Processing, Post-Translational; Pyrans; Solubility; Spiro Compounds; Tumor Cells, Cultured

The involvement of protein kinases in the signal transduction pathways controlling adrenal steroidogenesis is well established, and the phosphorylation of substrates by cAMP-dependent protein kinase is a major mechanism in ACTH action. However, the possibility that protein phosphatases (PPs) might also be involved in this process has not been investigated. The aim of this study was, therefore, to measure the function, expression and enzymic activity of PPs in zona glomerulosa (ZG) and zona fasciculata/reticularis (ZFR) tissue from the rat adrenal cortex. Immunoblot analysis using specific antisera demonstrated the presence in whole adrenals and capsules of PP type 1 (PP1) migrating with an apparent molecular mass of 37 kDa, and PP type 2A (PP2A) migrating with apparent molecular masses of 38 and 31 kDa. The PP inhibitors, okadaic acid (OA), calyculin A (CA), tautomycin and microcystin RR, caused a reduction in PP activity in vitro, at doses between 1 nM and 1 microM. In addition, treatment of ZG cells with the adenylate cyclase stimulator, forskolin (10 microM) resulted in a significant reduction in PP activity. The effects of CA and OA on steroid secretion by ZG and ZFR cells were also investigated. Neither CA nor OA had any effect on basal steroid secretion or on yields of steroid obtained from 22R-hydroxycholesterol at doses between 1 and 100 nM. However, both OA and CA (10 and 100 nM respectively) significantly reduced ACTH-stimulated aldosterone and corticosterone production by ZG and ZFR cells. CA and OA (10 and 100 nM respectively) also reduced steroid secretion by cells stimulated by forskolin (10 microM) or dibutyryl cAMP (200 microM). These results suggest that PPs may be involved in the intracellular mechanisms through which adrenocortical steroidogenesis is regulated, acting at a point after cAMP generation and action, but proximal to the side-chain cleavage of cholesterol.

Topics: Adrenal Cortex; Adrenal Cortex Hormones; Adrenocorticotropic Hormone; Aldosterone; Animals; Antifungal Agents; Bucladesine; Cells, Cultured; Colforsin; Corticosterone; Enzyme Inhibitors; Immunoblotting; Male; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoric Monoester Hydrolases; Phosphothreonine; Pyrans; Rats; Rats, Sprague-Dawley; Signal Transduction; Spiro Compounds; Stimulation, Chemical

Nucleotide excision repair of DNA in mammalian cells uses more than 20 polypeptides to remove DNA lesions caused by UV light and other mutagens. To investigate whether reversible protein phosphorylation can significantly modulate this repair mechanism we studied the effect of specific inhibitors of Ser/Thr protein phosphatases. The ability of HeLa cell extracts to carry out nucleotide excision repair in vitro was highly sensitive to three toxins (okadaic acid, microcystin-LR and tautomycin), which block PP1- and PP2A-type phosphatases. Repair was more sensitive to okadaic acid than to tautomycin, suggesting the involvement of a PP2A-type enzyme, and was insensitive to inhibitor-2, which exclusively inhibits PP1-type enzymes. In a repair synthesis assay the toxins gave 70% inhibition of activity. Full activity could be restored to toxin-inhibited extracts by addition of purified PP2A, but not PP1. The p34 subunit of replication protein A was hyperphosphorylated in cell extracts in the presence of phosphatase inhibitors, but we found no evidence that this affected repair. In a coupled incision/synthesis repair assay okadaic acid decreased the production of incision intermediates in the repair reaction. The formation of 25-30mer oligonucleotides by dual incision during repair was also inhibited by okadaic acid and inhibition could be reversed with PP2A. Thus Ser/Thr- specific protein phosphorylation plays an important role in the modulation of nucleotide excision repair in vitro.

Topics: Antifungal Agents; Base Sequence; DNA; DNA Damage; DNA Repair; Ethers, Cyclic; HeLa Cells; Humans; Marine Toxins; Microcystins; Molecular Sequence Data; Okadaic Acid; Oligonucleotides; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Proteins; Pyrans; Spiro Compounds

The present work was undertaken to test whether cytoskeletal components are involved in the control of rat-liver carnitine palmitoyltransferase I (CPT-I) activity by cellular effectors. The microtubule stabilizer taxol abolished the changes in CPT-I activity induced by the effectors tested. Taxol also prevented OA-induced shrinkage of hepatocytes as well as the enhanced release of lactate dehydrogenase from digitonin-permeabilized hepatocytes. On the basis of its relative sensitivity to tautomycin and OA, the modulation of CPT-I activity seemed to involve mostly protein phosphatase 1. These data suggest that the short-term control of hepatic CPT-I by cellular effectors may involve modulation of interactions between CPT-I and cytoskeletal components.

Topics: Animals; Antifungal Agents; Bucladesine; Carnitine O-Palmitoyltransferase; Cells, Cultured; Cytoskeleton; Enzyme Inhibitors; Ethers, Cyclic; Glutamine; Liver; Male; Microtubules; Okadaic Acid; Paclitaxel; Phosphoprotein Phosphatases; Protein Phosphatase 1; Pyrans; Rats; Rats, Wistar; Spiro Compounds; Vanadates

Stimulation of insulin secretion is accompanied by changes in the phosphorylation state of several islet polypeptides. Protein (de)phosphorylation is mediated by the action of protein kinases and phosphoprotein phosphatases. In this study we have investigated expression of phospho-serine/threonine phosphatases (PPs) in rat islets of Langerhans and studied the role of these enzymes in the regulation of insulin secretion. PP1, PP2A and PP2B were identified in rat islets and high levels of PP1/2A activities were detected. Inhibition of PP1/2A markedly inhibited glucose-stimulated insulin secretion, whilst glucose increased islet PP1/2A activities in situ. Insulin secretion at basal glucose was unaffected by inhibitors of PP1/2A. Inhibition of PP2B had no effect on either basal or glucose stimulated insulin secretion. These results suggest that PP1/2A are stimulated by glucose in rat islets and the presence of active PP1/2A is required for stimulation of insulin secretion by glucose.

Topics: Animals; Antifungal Agents; Cantharidin; Cyclosporine; Glucose; Humans; Insulin; Insulin Secretion; Islets of Langerhans; Male; Okadaic Acid; Phosphoprotein Phosphatases; Pyrans; Rats; Rats, Sprague-Dawley; Spiro Compounds

Although the role of protein kinases and phosphorylation in steroidogenesis has received much attention, very little is known about the activities of phosphoprotein phosphatases (PP) and dephosphorylation in steroidogenic tissues. The aims of the present study were therefore to identify which of those serine/threonine PPs more commonly involved in intracellular signalling are expressed in rat luteal cells; to quantify, in vitro, the effects of inhibitors on PP activity extracted from purified rat luteal cells; and to measure the effects of PP inhibitors on the phosphorylation of endogenous luteal cell proteins. Polyclonal antibodies raised against the catalytic subunits of PP types 1 and 2A, and a monoclonal antibody raised against the Ca(2+)-binding subunit of PP2B, were used to identify immunoreactive proteins that migrated on SDS-PAGE with approximate molecular masses of 37, 34 and 16 kDa, corresponding well with the reported molecular mass of PP1, PP2A and PP2B respectively. Five selective inhibitors of PP1/PP2A: okadaic acid, calyculin A, cantharidin, tautomycin and microcystin-RR, each caused a dose-dependent decrease in the activity of PPs in luteal cell homogenates, and also enhanced 32P incorporation into numerous luteal cell proteins; most notably, proteins with approximate molecular masses of 20 and 22 kDa. The results of this study suggest that PPs may play an important role in the regulation of rat luteal cell functions.

Topics: Animals; Antifungal Agents; Calcineurin; Calmodulin-Binding Proteins; Cantharidin; Cells, Cultured; Corpus Luteum; Dose-Response Relationship, Drug; Female; Marine Toxins; Microcystins; Molecular Weight; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Pyrans; Rats; Rats, Sprague-Dawley; Signal Transduction; Spiro Compounds

We examined the effect of okadaic acid, an inhibitor of protein phosphatase type 1 and 2A, on prostaglandin E1 (PGE1)-induced alkaline phosphatase (ALP) activity in osteoblast-like MC3T3-E1 cells. PGE1 increased ALP activity dose dependently in the range between 10 nM and 0.3 microM in these cells. The pretreatment with okadaic acid enhanced the PGE1-induced ALP activity in a dose-dependent manner in the range between 0.1 and 5 nM. On the other hand, 1-norokadaone, a less potent analogue of okadaic acid, had no effect on the PGE1-induced ALP activity. Tautomycin, an another inhibitor of protein phosphatase type 1 and 2A, also enhanced the PGE1-induced ALP activity. PGE1 stimulated cAMP accumulation dose dependently in the range between 10 nM and 0.3 microM. However, PGE1 had no effect on the formation of inositol phosphates. Okadaic acid did not affect the PGE1-induced cAMP accumulation. Okadaic acid dose dependently enhanced the dibutyryl cAMP-induced ALP activity. These results strongly suggest that protein phosphatase type 1 and/or 2A act as a regulator of ALP activity at a point downstream from protein kinase A in osteoblast-like cells.

Topics: Alkaline Phosphatase; Alprostadil; Animals; Antifungal Agents; Bucladesine; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ethers, Cyclic; Inositol Phosphates; Mice; Okadaic Acid; Osteoblasts; Oxazoles; Pyrans; Spiro Compounds

Lactacystin (1.3 microM), a metabolite from an actinomycete, induced the formation of bipolar projections at both sides of the cell body of Neuro 2a cells 1 day after treatment and networks at and after 3 days and enhanced acetylcholinesterase activity (a marker of neuronal differentiation). Thus, the neuronal differentiation was characterized both morphologically and functionally. The experiments with various inhibitors of protein kinases and phosphatases revealed that the protein phosphatase inhibitors calyculin A (0.5 nM) and okadaic acid (0.6 nM) inhibit the formation of bipolar projections at 1 day, but does not inhibit the network formation at and after 3 days.

Topics: Acetylcholinesterase; Acetylcysteine; Animals; Antifungal Agents; Biomarkers; Bucladesine; Cell Differentiation; Cell Line; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Ethers, Cyclic; Hydroquinones; Kinetics; Marine Toxins; Neurites; Neurons; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Protein Kinase Inhibitors; Pyrans; Spiro Compounds; Time Factors

The role of phosphoprotein phosphatase in the regulation of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) accumulation in rat pinealocytes was investigated using the three phosphatase inhibitors calyculin A, tautomycin, and okadaic acid. Calyculin A (0.1 microM) was found to enhance the isoproterenol- and norepinephrine-stimulated cAMP accumulation six- and threefold, respectively, whereas tautomycin and okadaic acid were less effective. The effect of calyculin A was rapid (within 5 min) and persisted in the presence of phosphodiesterase inhibition. However, in contrast to protein kinase C activation or intracellular calcium elevation, the phosphatase inhibitors were less effective in potentiating the cAMP response stimulated by forskolin or cholera toxin, and their effects were not blocked by calphostin C or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. The adrenergic-stimulated cGMP response was also less sensitive to the phosphatase inhibition. Therefore, our results suggest that 1) the adrenergic-stimulated cAMP signal is subjected to the tonic inhibition by phosphoprotein phosphatase; 2) phosphatase inhibitors enhance cAMP synthesis through their actions at the receptor level; and 3) the cAMP signal is more sensitive to the regulation by phosphorylation than cGMP in rat pinealocytes.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Antifungal Agents; Cyclic AMP; Cyclic GMP; Drug Synergism; Ethers, Cyclic; Isoproterenol; Kinetics; Male; Marine Toxins; Norepinephrine; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Pineal Gland; Protein Phosphatase 2; Pyrans; Rats; Rats, Sprague-Dawley; Spiro Compounds

Protein phosphatases regulate the activity of signal transduction mechanisms by dephosphorylating activated components. By utilizing selective inhibitors of these phosphatases, we investigated their role in regulating cAMP accumulation in the UMR 106 osteoblast-like tumor cell line. PTHrP, PTH and PGE2 stimulated cAMP accumulation up to 100-fold. Calyculin A, a potent inhibitor of protein phosphatase type 1 (PP1) and type 2A (PP2A), did not affect basal levels of cAMP, but concentrations of 10(-11) M to 10(-8) M increased PTHrP-, PTH-, and PGE2-stimulated cAMP accumulation up to 1.7-fold, and this increase was concentration-dependent. Similar results were obtained with tautomycin, another potent inhibitor of PP1 and PP2A. In contrast, okadaic acid, a potent inhibitor of PP2A which inhibited PP1 less potently, did not enhance PTHrP-, PTH-, or PGE2-stimulated cAMP accumulation. The effect of calyculin A on agonist-stimulated cAMP accumulation persisted in cells treated with isobutyl methylxanthine, a phosphodiesterase inhibitor. When the effect of calyculin A was compared with that of 4 beta-phorbol 12-myristate 13-acetate (PMA), it was found that while PMA enhanced both the receptor and forskolin-stimulated cAMP accumulation, calyculin A had no effect on the forskolin-stimulated cAMP accumulation. The effect of calyculin A on PTHrP- and PTH-stimulated cAMP accumulation persisted in cells treated with PMA. These results suggest that protein phosphatases play an important role in agonist-stimulated cAMP accumulation in osteoblast-like cells, and that PP1 but not PP2A may be the major phosphatase involved. In contrast to activation by protein kinase C, the site of action for the phosphatase appears to be predominantly at a step prior to the activation of adenylyl cyclase in the cAMP signal transduction pathway.

Topics: 1-Methyl-3-isobutylxanthine; Antifungal Agents; Colforsin; Cyclic AMP; Dinoprostone; Ethers, Cyclic; Humans; Kinetics; Marine Toxins; Okadaic Acid; Osteoblasts; Oxazoles; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Protein Serine-Threonine Kinases; Proteins; Pyrans; Spiro Compounds; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

A specific inhibitor of protein tyrosine phosphatase (PTPase), RK-682 (3-hexadecanoyl-5-hydroxymethyl-tetronic acid) was isolated from microbial metabolites. In vitro, RK-682 inhibited dephosphorylation activity of CD45 and VHR with IC50 54 and 2.0 microM, respectively. In situ, sodium orthovanadate and RK-682 enhanced the phosphotyrosine level of Ball-1 cells, a human B cell leukemia, but not the phosphoserine/threonine level. The PTPase inhibitors, however, had the different arrest point on the cell cycle progression. Sodium orthovanadate inhibited the cell cycle progression at G2/M boundary phase, on the other hand, RK-682 inhibited the G1/S transition.

Topics: Aniline Compounds; Antifungal Agents; Blotting, Western; cdc25 Phosphatases; Cell Cycle; Cell Cycle Proteins; Dual Specificity Phosphatase 3; Enzyme Inhibitors; Ethers, Cyclic; G1 Phase; Humans; Kinetics; Leukemia, B-Cell; Okadaic Acid; Organophosphorus Compounds; Phosphoprotein Phosphatases; Phosphoproteins; Phosphotyrosine; Protein Tyrosine Phosphatases; Pyrans; Spiro Compounds; Streptomyces; Tumor Cells, Cultured; Vanadates

Tautomycin isolated from Streptomyces spiroverticillatus is an inhibitor of protein phosphatases 1 and 2A. Tautomycin induced hyperphosphorylation of cytokeratin peptides in human keratinocytes (PHK 16-I cells) 30 times less strongly than did okadaic acid. Repeated applications of tautomycin (30 micrograms, 40 nmol/application) did not induce tumor promotion in a two-stage carcinogenesis experiment on mouse skin initiated with 7,12-dimethylbenz[a]anthracene, whereas okadaic acid (1 microgram, 1.2 nmol/application) as a control induced tumor promotion strongly. As for mucosa of rat glandular stomach, tautomycin induced ornithine decarboxylase 4 h after intubation into the stomach. The tumor-promoting activity of tautomycin was next studied in the glandular stomach initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Administration of tautomycin in the diet (1 mg rat-1 day-1), from week 9 to week 52 of the experiment, inhibited rather than enhanced tumor development in the glandular stomach initiated with MNNG. The percentages of tumor-bearing rats of the groups treated with MNNG plus tautomycin, MNNG alone, and tautomycin alone were 20.0%, 40.6%, and 0% respectively in week 52. The reason for the absence of tumor-promoting activity of tautomycin was studied in relation to tumor necrosis factor alpha (TNF alpha), an endogenous tumor promoter. We found that tautomycin neither enhanced TNF alpha mRNA expression in mouse skin nor induced TNF alpha release in a human stomach cancer cell line (KATO III cells), whereas okadaic acid did both. These results indicate that not all inhibitors of protein phosphatases are tumor promoters, and suggest that tumor promotion of the okadaic acid class of compounds is mediated by TNF alpha.

Topics: Animals; Antifungal Agents; Carcinogens; Enzyme Induction; Enzyme Inhibitors; Ethers, Cyclic; Female; Gene Expression; Humans; Keratins; Male; Mice; Okadaic Acid; Ornithine Decarboxylase; Phosphoprotein Phosphatases; Phosphorylation; Pyrans; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Spiro Compounds; Stomach Neoplasms; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Several groups have reported that okadaic acid (OA) and some other tight-binding protein phosphatase inhibitors including microcystin-LR (MCLR), calyculin-A and tautomycin prevent each other from binding to protein phosphatase 2A (PP2A). In this paper, we have introduced an improved procedure for examining to what extent the affinity of an enzyme for a labelled tight-binding ligand is reduced by binding of an unlabelled tight-binding, ligand to the enzyme. Using this procedure, we have analysed the dose-dependent reduction of PP2A binding of [24-3H]OA by addition of OA, MCLR, calyculin-A and tautomycin. The results indicate that the binding of the unlabelled inhibitors to the PP2A molecule causes a dramatic (10(6)-10(8)-fold) increase in the dissociation constant associated with the interaction of [24-3H]OA and PP2A. This suggests that OA and the other inhibitors bind to PP2A in a mutually exclusive manner. The protein phosphatase inhibitors may share the same binding site on the PP2A molecule. We have also measured values of the dissociation constant (Ki) for the interaction of these toxins with protein phosphatase 1 (PP1). For MCLR and calyculin-A, the ratio of the Ki value obtained for PP1 to that for PP2A was in the range 4-9, whereas it was 0.01-0.02 for tautomycin. The value of tautomycin is considerably smaller than that (0.4) calculated from previously reported Ki values.

Topics: Antifungal Agents; Ethers, Cyclic; Marine Toxins; Microcystins; Okadaic Acid; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Phosphatase 1; Protein Phosphatase 2; Pyrans; Radioligand Assay; Spiro Compounds

The effects of three serine/threonine protein phosphatase inhibitors, calyculin-A, tautomycin and okadaic acid, on the Ca2+ entry across the plasma membrane was studied in Fura-2-loaded rat parotid acinar cells. These protein phosphatase inhibitors did not affect the peak elevation of cytosolic free Ca2+ concentration ([Ca2+]i) just after stimulation with the muscarinic agonist carbachol (CCh), but they suppressed the sustained increase in [Ca2+]i. In the absence of extracellular Ca2+, CCh produced a transient increase in [Ca2+]i due to Ca2+ release from intracellular Ca2+ stores, and this increase in [Ca2+]i was unaffected by the phosphatase inhibitors. When Ca2+ was added to the external medium after the transient [Ca2+]i response, the increase in [Ca2+]i in the cells treated with the phosphatase inhibitors was significantly smaller than that in the control cells, indicating that the Ca2+ entry was reduced. Similar suppression of Ca2+ entry by the phosphatase inhibitors was observed when intracellular Ca2+ stores were previously depleted by the microsomal Ca(2+)-ATPase inhibitor thapsigargin (TG). In addition, the phosphatase inhibitors reduced the Mn2+ (Ca2+ surrogate) influx following the addition of CCh or TG. The enhancement of Ca2+ entry by the protein kinase inhibitor staurosporine was significantly attenuated by the phosphatase inhibitors. These results suggest that the phosphatase inhibitors suppressed the Ca2+ entry mechanism activated by depletion of intracellular Ca2+ stores in rat parotid acinar cells. The capacitative Ca2+ entry may be regulated by protein phosphorylation/dephosphorylation.

Topics: Alkaloids; Animals; Antifungal Agents; Calcium; Carbachol; Enzyme Inhibitors; Ethers, Cyclic; Male; Okadaic Acid; Parotid Gland; Phosphoric Monoester Hydrolases; Pyrans; Rats; Rats, Wistar; Spiro Compounds; Staurosporine

Recent studies suggest that the ability to inhibit the activity of certain serine/threonine protein phosphatases underlies the toxicity of several natural compounds including: okadaic acid, microcystin-LR, nodularin, calyculin A and tautomycin. To characterize further the actions of these toxins, this study compares the inhibitory effects of okadaic acid, chemical derivatives of okadaic acid, microcystin-LR, microcystin-LA, nodularin, calyculin A and tautomycin on the activity of serine/threonine protein phosphatases types 1 (PP1), 2A (PP2A) and a recently identified protein phosphatase purified from bovine brain (PP3). This study shows that, like PP1 and PP2A, the activity of PP3 is potently inhibited by okadaic acid, both microcystins, nodularin, calyculin A and tautomycin. Further characterization of the toxins employing the purified catalytic subunits of PP1, PP2A and PP3 under identical experimental conditions indicates that: (a) okadaic acid, microcystin-LR, and microcystin-LA inhibit PP2A and PP3 more potently than PP1 (order of potency PP2A > PP3 > PP1); (b) nodularin inhibits PP1 and PP3 at a similar concentration that is slightly higher than that which affects PP2A, and (c) both calyculin A and tautomycin show little selectivity among the phosphatases tested. This study also shows that the chemical modification of the (C1) carboxyl group of okadaic acid can have a profound influence on the inhibitory activity of this toxin. Esterification of okadaic acid, producing methyl okadaate, or reduction, producing okadaol, greatly decreases the inhibitory effects against all three enzymes tested. Further reduction, producing 1-nor-okadaone, or acetylation, producing okadaic acid tetraacetate, results in compounds with no inhibitory activity. In contrast, the substitution of alanine (-LA) for arginine (-LR) in microcystin has no apparent effect on the inhibitory activity against PP1, PP2A or PP3.

Topics: Animals; Antifungal Agents; Cattle; Cyclic AMP-Dependent Protein Kinases; Esterification; Ethers, Cyclic; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Oxidation-Reduction; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylase b; Phosphorylase Kinase; Pyrans; Rabbits; Spiro Compounds

The effects of three phosphatase inhibitors including okadaic acid, calyculin A and tautomycin were evaluated on platelet Ca2+ mobilization. Calyculin A and tautomycin at appropriate concentrations appeared to have a selective inhibitory effect on thrombin-induced Ca2+ influx, but not on [Ca2+]i release from intracellular Ca2+ storage sites. In contrast, pretreatment with okadaic acid at concentrations that effectively lowered Ca2+ influx also suppressed Ca2+ release from intracellular Ca2+ stores. In a system that specifically evaluates the effects of agents on Ca2+ influx induced by the Ca(2+)-depleted state of intracellular Ca2+ storage sites, the three phosphatase inhibitors attenuated Ca2+ influx in a dose dependent manner and showed complete inhibition at appropriate concentrations. These findings suggest that protein phosphorylation/dephosphorylation plays an important role in mediating signals to open Ca2+ channels when Ca2+ depletion in intracellular Ca2+ stores is caused by thrombin. In contrast, Ca2+ influx induced by thapsigargin, a Ca(2+)-ATPase inhibitor, was only partially suppressed by pretreatment with each of the three phosphatase inhibitors. Based on these findings, we suggest that the Ca(2+)-depleted state of intracellular Ca2+ stores by thapsigargin induces the opening of Ca2+ channels via phosphatase inhibitor-insensitive pathways. All the phosphatase inhibitors, at the highest concentrations tested in the present study, only partially inhibited Mn2+ entry induced by thrombin. These findings suggest that there are at least two types of divalent ion channels on platelet plasma membranes and that one of them, that preferentially allows Mn2+ entry, is resistant to the inhibitory effects of phosphatase inhibitors.

Topics: Antifungal Agents; Blood Platelets; Calcium; Ethers, Cyclic; Humans; In Vitro Techniques; Magnesium; Marine Toxins; Okadaic Acid; Oxazoles; Phosphoric Monoester Hydrolases; Platelet Aggregation Inhibitors; Pyrans; Spiro Compounds; Terpenes; Thapsigargin; Thrombin

Effects of compounds of the okadaic acid type (okadaic acid, dinophysistoxin-1, calyculin A and tautomycin) on proliferation by digestive-tract epithelial cells were investigated in mice and rats. In mice, a single oral administration of these agents caused significant enhancement of BrdU labeling indices in a dose/response manner. Exceptions showing no response were limited to the pyloric mucosa for okadaic acid, the pyloric and fundic mucosa for calyculin A and the pyloric mucosa for tautomycin. Sequential analysis of labeling indices after a single oral administration of dinophysistoxin-1 revealed two peaks of cell proliferation at 18 h and 36 h in the esophagus, ileum and colon. The labeling indices of the forestomach, fundus, pylorus and jejunum, on the other hand, continuously increased from 6 h after the administration. Elevated proliferation was also observed in the skin after 30 h or after, but no effects on the liver or kidney were evident. A single oral administration of the okadaic acid type of compounds also dose-dependently enhanced cell proliferation of the rat digestive tract. These results strongly suggest that the okadaic acid class of compounds may exert promoting potential for the gastrointestinal mucosa when administered orally.

Topics: Administration, Oral; Animals; Antifungal Agents; Bromodeoxyuridine; Carcinogens; Cell Division; Dose-Response Relationship, Drug; Ethers, Cyclic; Intestinal Mucosa; Male; Marine Toxins; Mice; Mice, Inbred ICR; Okadaic Acid; Oxazoles; Pyrans; Rats; Rats, Sprague-Dawley; Spiro Compounds; Time Factors

Protein phosphatase 1 is considered to be involved in thrombin-induced platelet activation (Murata et al., Biochem Int 26:327-334, 1992). To clarify the mechanism, we examined the effects of protein phosphatase 1 and 2A inhibitors (calyculin A, tautomycin, okadaic acid) on Ca2+ influx. In the presence of 1 mM Ca2+, thrombin- (0.1 U/ml) induced platelet aggregation and ATP release were inhibited by calyculin A, while this inhibitory effect was abolished in the absence of Ca2+ (EGTA 1 mM). Furthermore, thrombin-induced Mn2+ influx but not intracellular Ca2+ mobilization was inhibited by calyculin A in a dose-related manner. Calyculin A also blocked the ongoing Ca2+ influx when added 3 min after thrombin stimulation. Similar inhibitory effects were observed with okadaic acid and tautomycin in the same potency sequence as the reported one for protein phosphatase 1 (calyculin A > tautomycin > okadaic acid). These results suggest that the anti-platelet effects of phosphatase inhibitors are due to the inhibition of Ca2+ influx and that protein phosphatase 1 plays a key role in the regulation of receptor operated Ca2+ channel of human platelets.

Topics: Antifungal Agents; Blood Platelets; Calcium; Calcium Channels; Ethers, Cyclic; Humans; Manganese; Marine Toxins; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Platelet Aggregation; Protein Phosphatase 1; Pyrans; Spiro Compounds; Thrombin

Phosphatase inhibitors microcystin-LR, tautomycin, and okadaic acid caused contraction and increased 20-kDa myosin light chain (MLC20) phosphorylation in Ca(2+)-free solutions in both phasic and tonic smooth muscle permeabilized with beta-escin, and inhibited the heavy meromyosin (HMM) phosphatase activity of smooth muscle homogenates with the same potency sequence: microcystin-LR greater than tautomycin greater than okadaic acid. The sensitivity to all three inhibitors was significantly higher, the half-times of relaxation and dephosphorylation were 4-6 times longer, and the HMM phosphatase and MLC20 kinase activity/smooth muscle cell wet weight was 2.0- and 1.9-fold lower in the tonic, femoral artery, than in the phasic, ileum or portal vein, smooth muscle. Preincubation with 0.2 microM inhibitor-2 decreased the HMM phosphatase activity by 35% in the ileum and by 60% in the femoral artery. The results suggest that the HMM phosphatases of smooth muscle have properties common to type 1 protein phosphatases, but are inhibited only partially by high concentrations of inhibitor-2, and that the lower HMM phosphatase activity of tonic smooth muscle may contribute to its greater sensitivity to phosphatase inhibitors and its slower rate of relaxation.

Topics: Animals; Antifungal Agents; Calcium; Cations, Divalent; Escin; Ethers, Cyclic; Guinea Pigs; In Vitro Techniques; Marine Toxins; Microcystins; Microscopy, Electron; Muscle Contraction; Muscle, Smooth; Muscle, Smooth, Vascular; Myosin-Light-Chain Phosphatase; Okadaic Acid; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Pyrans; Rabbits; Spiro Compounds

Phorbol dibutyrate induced a nitroblue tetrazolium-reducing reaction in differentiated HL-60 cells, which was inhibited by protein kinase inhibitors such as staurosporine and H-7. ID50 of staurosporine and H-7 were 1.4 ng/ml and 0.19 mM, respectively. When tautomycin, an inhibitor of protein phosphatases, was added with the kinase inhibitors, the nitroblue tetrazolium-reducing reaction again appeared. ID50 of staurosporine was 510 ng/ml in the presence of tautomycin. Tautomycin itself weakly induced the reaction, which was inhibited by kinase inhibitors. Such a competitive effect between tautomycin and staurosporine was not observed in a cell-free system of protein kinase C. Okadaic acid had the same effect as tautomycin. The similar results were obtained when respiratory burst was quantitated by measuring H2O2 produced by canine peripheral neutrophils. The mechanism of competitive effect of tautomycin and staurosporine on respiratory burst is discussed.

Topics: Alkaloids; Animals; Antifungal Agents; Cells, Cultured; Dogs; Echinomycin; Ethers, Cyclic; Hydrogen Peroxide; Nitroblue Tetrazolium; Okadaic Acid; Phorbol 12,13-Dibutyrate; Pyrans; Respiratory Burst; Spiro Compounds; Staurosporine

The effects of tautomycin, a protein phosphatase inhibitor, on recycling of cell surface molecules were studied with transferrin receptor (TFR) of human myeloid leukemia K562 cells and with CD4 of murine thymocytes. Tautomycin increased expression of TFR of K562 cells whereas phorbol dibutylate (PDBu) decreased it. Tautomycin inhibited PDBu-induced down-regulation of CD4 although it did not induce up-regulation. Okadaic acid also inhibited down-regulation of CD4 which was induced by PDBu. The results suggest that certain inhibitors of protein phosphatases preferentially inhibit endocytosis of cell surface molecules.

Topics: Alkaloids; Antifungal Agents; Carbazoles; CD4 Antigens; Down-Regulation; Ethers, Cyclic; Indole Alkaloids; Okadaic Acid; Phorbol 12,13-Dibutyrate; Protein Kinase C; Pyrans; Receptors, Transferrin; Spiro Compounds; Staurosporine

The antibiotic, tautomycin, was found to be a potent inhibitor of protein phosphatases and equally effective for the type-1 and type-2A enzymes. For the catalytic subunits of the type-1 and type-2A phosphatases the IC50 value was 22 to 32 nM. For the phosphatase activity present in chicken gizzard actomyosin the IC50 value was 6 nM. Tautomycin had no effect on myosin light chain kinase activity. Tautomycin induced a Ca(2+)-independent contraction of intact and permeabilized smooth muscle fibers and this was accompanied by an increase in the level of myosin phosphorylation. Thus, tautomycin by virtue of its ability to inhibit phosphatase activity is a valuable addition for studying the role of protein phosphorylation.

Topics: Animals; Antifungal Agents; Aorta; Calcium; Enzyme Inhibitors; Ethers, Cyclic; Guinea Pigs; Kinetics; Macromolecular Substances; Marine Toxins; Muscle Contraction; Muscle, Smooth, Vascular; Myosin-Light-Chain Kinase; Myosins; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Pyrans; Rabbits; Rats; Spiro Compounds; Verapamil

Okadaic acid, a specific inhibitor of protein phosphatase 1 in Paramecium causes sustained backward swimming in response to depolarising stimuli (S. Klumpp et al. (1990) EMBO J. 9, 685). Here, we employ okadaic acid, tautomycin, microcystin LR and inhibitor 1 as phosphatase inhibitors to identify a 42 kDa protein in the excitable ciliary membrane that is dephosphorylated by protein phosphatase 1. Identification of the 42 kDa protein was facilitated by the finding that the protein kinase responsible for its phosphorylation uses Ca-ATP as a substrate just as effectively as Mg-ATP. Notably, dephosphorylation of the 42 kDa protein is specifically inhibited by cyclic AMP; cyclic GMP has no effect.

Topics: Animals; Antifungal Agents; Calcium Channels; Calcium-Transporting ATPases; Cilia; Cyclic AMP; Ethers, Cyclic; Marine Toxins; Microcystins; Okadaic Acid; Paramecium; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Phosphatase 1; Protozoan Proteins; Pyrans; Spiro Compounds

Tautomycin inhibited the catalytic subunits of protein phosphatase-1 (Kiapp = 0.16 nM) more potently than protein phosphatase 2A (Kiapp = 0.4 nM), and the native forms of these enzymes in mammalian, protozoan and plant extracts were inhibited in a similar manner. Protein phosphatase 2B was inhibited 10,000-fold less potently, while two other phosphatases and six protein kinases were unaffected at 10 microM. Okadaic acid prevented the binding of tautomycin to protein phosphatase 2A, indicating a common binding site for both inhibitors. The different relative potencies of tautomycin and okadaic acid for protein phosphatases 1 and 2A suggest that parallel use of both inhibitors may help to identify physiological substrates for each enzyme.

Topics: Antifungal Agents; Ethers, Cyclic; In Vitro Techniques; Kinetics; Marine Toxins; Microcystins; Okadaic Acid; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylases; Protein Phosphatase 1; Protein Phosphatase 2; Pyrans; Spiro Compounds; Streptomyces