calyculin-a and tautomycin

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; 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; 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

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

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

Magnolol stimulates adrenal steroidogenesis and induces the distributional changes of p160 and adipose differentiation-related protein (ADRP) in rat adrenal cells. This study investigated the underlying signaling mechanisms involved in these processes. Magnolol (30 microM) caused a time-dependent increase in the phosphorylation of extracellular signal-related kinase (ERK) in cultured adrenal cells. The following evidence supports a link between ERK activation and p160 translocation. First, the magnolol-induced redistribution of p160 from the lipid droplet surface to the cytosol, resulting in the decrease in the percentages of p160-positive cells, and this decrease in p160-positive cells was completely blocked by pretreatment with either of the MAPK-ERK kinase (MEK) inhibitors PD98059 or U0126. Second, magnolol did not significantly decrease total p160 protein levels but caused an increase in threonine phosphorylation of p160, which reached a maximum after 5 min of magnolol treatment, and this magnolol-induced phosphorylation of p160 was prevented by pretreatment with U0126, suggesting the involvement of ERK. In addition, magnolol decreased both ADRP immunostaining intensity at the lipid droplet surface and the percentage of ADRP-positive cells. This was further confirmed biochemically by the decrease in ADRP levels in total cell homogenates and in lipid droplet fractions. Magnolol-induced decrease in ADRP staining at the lipid droplet surface was not affected by pretreatment with PD98059 or U0126, indicating that ERK signaling was not involved in this event. Furthermore, treatment with 30 microM magnolol for 6 h resulted in about 50% decrease in ADRP protein level. Therefore, decreased protein levels of p160 and ADRP at the lipid droplet surface induced by magnolol were mediated via two different mechanisms: phosphorylation of p160 and downregulation of ADRP expression, respectively.

Topics: Adrenal Glands; Animals; Biphenyl Compounds; Blotting, Western; Butadienes; Cells, Cultured; Cytoplasmic Granules; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Flavonoids; Fluorescent Antibody Technique; Histone Acetyltransferases; Lignans; Lipid Metabolism; Marine Toxins; Membrane Proteins; Microscopy, Fluorescence; Nitriles; Nuclear Receptor Coactivator 1; Oxazoles; Perilipin-2; Phosphoprotein Phosphatases; Phosphorylation; Protein Transport; Pyrans; Rats; Rats, Wistar; Spiro Compounds; Time Factors; Transcription Factors

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

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

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 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

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

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

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

Tautomycin (TT) and calyculin A (CyA) are inhibitors of protein phosphatases type 1 and 2 (PP1, PP2). Inhibitors 1 and 2 are specific for PP1, which is the major phosphatase functionally relevant in heart and able to dephosphorylate phospholamban (PLB). TT and CyA maintain PLB in its phosphorylated state, thereby increasing calcium uptake. Rabbit saponin skinned fibers (SF) are used to assess calcium load of the sarcoplasmic reticulum (SR). The present investigation aimed to examine the effects of PP1 inhibitors on SR calcium load assessed by caffeine-induced tension transient (CITT), and to correlate this activity with the PLB phosphorylation state. TT and CyA (100 nm) applied during the uptake phase increased the amplitude of CITT by 10 and 20%, respectively,P<0.05 without effect on the release phase. Both CyA and TT were devoid of calcium sensitizing effect when studied on Triton X-100 SF. After skinning procedure, SF were grinded for biochemical studies. SDS-PAGE electrophoresis and immunoblots using a monoclonal PLB antibody showed that cAMP or Ca2+/calmodulin-dependent protein kinases phosphorylated PLB in an additive fashion. Inhibition of PP1 by inhibitor 1, CyA and TT maintained PLB in its phosphorylated state in a dose-dependent manner. The results of this study in which functional and biochemical experiments in cardiac SF were combined demonstrate that strong correlation exists between the phosphorylation-dephosphorylation cycle of PLB and calcium uptake.

Topics: Animals; Antifungal Agents; Caffeine; Calcium; Calcium-Binding Proteins; Contractile Proteins; Cyclic AMP-Dependent Protein Kinases; Egtazic Acid; Enzyme Inhibitors; In Vitro Techniques; Macromolecular Substances; Marine Toxins; Muscle Fibers, Skeletal; Myocardial Contraction; Oxazoles; Papillary Muscles; Phosphoprotein Phosphatases; Protein Phosphatase 1; Pyrans; Rabbits; Spiro Compounds; Structure-Activity Relationship

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

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

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

Effects of the protein phosphatase inhibitors, tautomycin and calyculin A on protein phosphorylation and cytoskeleton of human platelets. It has been discovered recently that many cytotoxic compounds isolated from a variety of sources are potent phosphatase inhibitors. Two of these, tautomycin (TM) and calyculin-A (CL-A) were applied to human platelets to investigate the role of protein phosphorylation on cytoskeletal structure and function. Exposure to 10 microM TM or 0.1 microM CL-A induced marked morphological changes. The granules were centralized and surrounded by actin filaments, but there was no evidence of granule release. Myosin became more centralized, was occluded from the granulomere, but was not confined to the microfilament ring. These changes occurred without an increase in cytosolic Ca2+ concentrations, as determined by measurements with fura-2. TM and CL-A induced an overall increase in protein phosphorylation. Phosphorylation of the 20,000 dalton light chain of myosin increased markedly and multiple phosphorylation sites were indicated. Cytoskeletons were prepared from control, thrombin- and TM-treated platelets, the latter prepared in the absence of external calcium. The major difference in protein composition was the increased content of myosin associated with the cytoskeleton from TM-treated platelets where the dominant phosphoprotein was the 20,000 dalton light chain. These results suggest that myosin phosphorylation drives the initial shape changes, and via a contractile process results in the formation of the microfilament ring and centralization of granules.

Topics: Adult; Antifungal Agents; Blood Platelets; Blotting, Western; Calcium; Cytoskeleton; Cytosol; Enzyme Inhibitors; Humans; Marine Toxins; Microscopy, Electron; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Proteins; Pyrans; Spiro Compounds

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

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