Comparative effects of protein phosphatase inhibitors (okadaic acid and calyculin A) on human leukemia HL60, HL60/ADR and K562 cells.

Inhibitors of protein phosphatases 1/2A (okadaic acid and calyculin A) exhibited differential cytotoxicity toward three human leukemia cell lines, in an increasing order of resistance, HL60 less than HL60/ADR less than K562 cells. Cytotoxicity of the toxins was associated with marked mitotic arrest of the cells, characterized by chromatid scattering/overcondensation and abnormal mitotic spindles. In all cases, calyculin A was more potent than okadaic acid. Protein phosphorylation experiments in intact cells revealed that HL60/ADR, the adriamycin-resistant variant, showed a higher overall phosphorylation of nuclear proteins than the drug-sensitive parental HL60, and that phorbol ester (protein kinase C activator) and calyculin A appeared to more specifically stimulate phosphorylation of p66 and p60, respectively. It was suggested that the toxins might be useful in delineating mechanisms underlying certain properties of cancer cells (such as multidrug resistance, mitosis and differentiation) related to protein phosphorylation/dephosphorylation reactions.     

Effects of the protein phosphatase inhibitors okadaic acid and calyculin A on insulin release from rat pancreatic islets.

The role of protein phosphatases in the regulation of insulin release from rat pancreatic islets was studied with protein phosphatase inhibitors, okadaic acid and calyculin A. Okadaic acid inhibited glucose- and glyceraldehyde-induced insulin release dose-dependently and also inhibited the potentiation of glucose-induced release either by adding forskolin, an activator of adenylate cyclase or by increasing K+ concentration to 25 mM. At a non-stimulatory concentration of 3 mM glucose, a high concentration (2 microM) of okadaic acid inhibited insulin release induced by high K+ or 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, but a low concentration (1 microM) of okadaic acid did not significantly inhibit TPA-induced insulin release. Calyculin A also inhibited glucose-induced insulin release, and the effect was greater than that of okadaic acid. The data suggest that protein phosphatases may play an important role in the regulation of insulin release.     

Effects of the phosphatase inhibitors,okadaic acid,ATPgammaS, and calyculin A on the dividing sand dollar egg

The effects of the phosphatase inhibitors, okadaic acid (OA), adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), and calyculin A (CL-A) on anaphase chromosome movement, cytokinesis, and cytoskeletal structures at cell division were examined by being microinjected into mitotic sand dollar eggs. When OA was injected, chromosome movement was inhibited and, moreover, chromosomes were ejected from the polar regions of the mitotic apparatus. By immunofluorescence, microtubules were observed to be severed in the OA-injected eggs, causing the smooth cell surface to be changed to an irregular surface. When ATPgammaS and CL-A were injected, the effect on cell shape was remarkable: In dividing eggs, furrowing stopped within several seconds after injection, small blebs appeared on the cell surface and became large, spherical or dumbbell cell shapes then changed to irregular forms, and subsequently cytoplasmic flow occurred. Microfilament detection revealed that actin accumulation in the cortex, which was not limited to the furrow cortex, occurred shortly after injection. Cortical accumulation of actin is thought to induce force generation and random cortical contraction, and accordingly to result in bleb extrusion from the cortex. Consequently, the phosphatase inhibitors inhibited the transition from mitosis to interphase by mediating cortical accumulation of actin filaments and/or fragmentation of microtubules.     

Calyculin A and okadaic acid: inhibitors of protein phosphatase activity

Calyculin A and okadaic acid induce contraction in smooth muscle fibers. Okadaic acid is an inhibitor of phosphatase activity and the aims of this study were to determine if calyculin A also inhibits phosphatase and to screen effects of both compounds on various phosphatases. Neither compound inhibited acid or alkaline phosphatases, nor the phosphotyrosine protein phosphatase. Both compounds were potent inhibitors of the catalytic subunit of type-2A phosphatase, with IC50 values of 0.5 to 1 nM. With the catalytic subunit of protein phosphatase type-1, calyculin A was a more effective inhibitor than okadaic acid, IC50 values for calyculin A were about 2 nM and for okadaic acid between 60 and 500 nM. The endogenous phosphatase of smooth muscle myosin B was inhibited by both compounds with IC50 values of 0.3 to 0.7 nM and 15 to 70 nM, for calyculin A and okadaic acid, respectively. The partially purified catalytic subunit from myosin B had IC50 values of 0.7 and 200 nM for calyculin A and okadaic acid, respectively. The pattern of inhibition for the phosphatase in myosin B therefore is similar to that of the type-1 enzyme.     

Differing effects of the protein phosphatase inhibitors okadaic acid and microcystin on translation in reticulocyte lysates.

The effects of the cyanobacterial toxin and protein phosphatase inhibitor, microcystin, on translation in rabbit reticulocyte lysates have been studied. Microcystin inhibited translation with similar potency to the protein phosphatase inhibitor okadaic acid. Unlike low concentrations of okadaic acid, however, it inhibited both the initiation and elongation stages. This was demonstrated using EGTA to inhibit the phosphorylation and inactivation of elongation factor eEF-2. A method for detecting changes in eEF-2 phosphorylation was developed. eEF-2 was found to exist as three different species: eEF-2 was largely monophosphorylated in reticulocyte lysates under control conditions, the remainder being unphosphorylated. Okadaic acid and microcystin increased the level of the bisphosphorylated species. The implications of multiple phosphorylation of eEF-2 for the control of translation is discussed. Microcystin was also found to increase the phosphorylation of eIF-2 alpha (and therefore to inhibit initiation) at lower concentrations than okadaic acid, suggesting that the major eIF-2 alpha phosphatase in the reticulocyte lysate is phosphatase-1.     

Reversible protein phosphorylation regulates the dynamic organization of the pollen tube cytoskeleton: effects of calyculin A and okadaic acid

We investigated the cytoskeleton of Lilium longiflorum pollen tubes and examined the effects of the type 2A protein phosphatase (PP2A) inhibitors calyculin A and okadaic acid. An improved method for actin visualization, the simultaneous fixation and staining with rhodamine-labelled phalloidin during microscopical observation, revealed abundant actin filaments of no preferential orientation in the apical clear zone. Microtubules, visualized by indirect immunofluorescence, were mostly absent from the apices of straight-growing pollen tubes but present in those with irregular shape. Double labelling showed that both actin bundles and microtubules had a similar longitudinal or slightly helical orientation in the pollen tube shaft. In the presence of 30 nM calyculin A or okadaic acid, pollen tubes grew very slowly, branched frequently, and contained isolated, randomly oriented, curved actin bundles and microtubules. Treating pollen tubes with calyculin A or okadaic acid after germination arrested growth immediately, reversibly altered the alignment of actin bundles from axial to transverse, and disassembled microtubules. The changes in actin organization caused by the PP2A inhibitors were similar to those observed upon overexpression of AtRop1 (Y. Fu, G. Wu, Z. Yang, Journal of Cell Biology 152: 1019-1032, 2001), suggesting that hyperphosphorylation interferes with the signalling pathway of small GTPases. The effects of the PP2A inhibitors could be ameliorated with nanomolar concentrations of latrunculin B.     

Inhibition of alkaline phosphatase activity by okadaic acid, a protein phosphatase inhibitor

This paper reports on a potential physiological target of okadaic acid (OA), the toxin metabolite responsible for shellfish poisoning and, consequently, human intoxication. OA is a potent promoter of tumor activity, most likely by inhibiting protein phosphatase 1 and 2A (Adv. Cancer. Res. 61 (1993) 143). However, all of its cellular targets have not yet been characterized. The interaction of OA with alkaline phosphatase (ALP) has been investigated in view of its protein phosphatase inhibition activity. Kinetic analysis of ALP from Escherichia coli, human placental and calf intestinal ALP has shown that OA acts as a non-competitive inhibitor of ALP. The bacterial enzyme displays a higher affinity for OA (K(i) 360 nM) than the eukaryotic proteins (human placental ALP, K(i) 2.05 microM; calf intestinal ALP, K(i) 3.15 microM). The inhibition by OA suggests a putative role of ALP in the phosphorylation status, through regulation of the phosphorylation/dephosphorylation equilibrium of proteins with phosphoseryl or phosphothreonyl residues.     

Activation of p38 mitogen-activated protein kinase and mitochondrial Ca(2+)-mediated oxidative stress are essential for the enhanced expression of grp78 induced by the protein phosphatase inhibitors okadaic acid and calyculin A

We have reported that treatment with okadaic acid, a potent protein phosphatase inhibitor, has the ability to enhance the synthesis of the 78-kDa glucose-regulated protein (GRP78). This article reports our investigation of another protein phosphatase inhibitor, calyculin A, demonstrating the signaling pathways elicited by the protein phosphatase inhibitors that lead to the induction of grp78. Our data showed that the induction process is abolished by SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (p38(MAPK)). Phosphorylation-activation of p38(MAPK) in the treated cells was indicated by its own phosphorylation, as shown by double Western blotting analyses and directly confirmed by the in vitro kinase assay using MAPK-activated protein kinase-2, a well-known downstream effector of p38(MAPK), as a substrate. The involvement of p38(MAPK) in this process is further substantiated by using transient transfection assays with a plasmid, pGRP78-Luc, which contains a 0.72-kbp stretch of the grp78 promoter. By exploiting the same transfection assay, we demonstrated that the up-regulation of the grp78 promoter by the protein phosphatase inhibitors is suppressed in the presence of the cytoplasmic calcium chelator bis(aminophenoxy)ethane N,N'-tetraacetic acid, the mitochondria calcium uniporter inhibitor ruthenium red as well as the antioxidants N-acetyl cysteine and pyrrolidinedithiocarbamate. Taken together, our results lead us to conclude that treatment with the protein phosphatase inhibitors would activate the signaling pathways involving p38(MAPK) and mitochondrial calcium-mediated oxidative stress and that these pathways must act in concert in order to confer the induction of grp78 by okadaic acid and calyculin A.     

PP1/PP2A phosphatases inhibitors okadaic acid and calyculin A block ERK5 activation by growth factors and oxidative stress

Okadaic acid is an inhibitor of the protein Ser/Thr phosphatases PP1 and PP2A, which blocks the activation of extracellular signal-regulated protein kinase 5 (ERK5), a member of the MAP kinase family activated by growth factors and several types of stressors. The blocking of ERK5 activation by okadaic acid was observed in HeLa cells exposed to epidermal growth factor and H(2)O(2) as well as in PC12 cells stimulated by nerve growth factor and H(2)O(2). Calyculin A, another PP1 and PP2A inhibitor, behaved similarly although these compounds are not structurally related. This suggests that either PP1 or PP2A or both are necessary for ERK5 activation. Protein kinase C (PKC) acts as a negative regulator of the ERK5 activation pathway, however our data suggest that the effects of PKC and the phosphatase are unrelated.     

A two stage click-based library of protein tyrosine phosphatase inhibitors

Protein tyrosine phosphatases (PTPs) are important regulators of signal transduction pathways. Potent and selective PTP inhibitors are useful for probing these pathways and also may serve as drugs for the treatment of a variety of diseases including type 2 diabetes and infection by the bacterium Yersinia pestis. In this report Cu(I)-catalyzed 'click' cycloaddition reactions between azides and alkynes were employed to generate two sequential libraries of PTP inhibitors. In the first round library methyl 4-azidobenzoylformate was reacted with 56 mono- and diynes. After hydrolysis of the methyl esters, the resulting alpha-ketocarboxylic acids were assayed in crude form against the Yersinia PTP and PTP1B. Four compounds were selected for further evaluation, and one compound was chosen as the lead for generation of the second round library. This lead compound was modified by conversion of an alcohol into an azide group, and the resulting azide was reacted with the same 56 mono- and diynes that were used in the first generation library. After screening the crude inhibitors against the Yersinia PTP and PTP1B, four compounds were selected and evaluated in pure form against the Yersinia PTP, PTP1B, TCPTP, LAR, and CD45. The best bis(alpha-ketocarboxylic acid) inhibitor 34 had an IC(50) value of 550nM against the Yersinia PTP and an IC(50) value of 710nM against TCPTP. The most potent inhibitor containing a single alpha-ketocarboxylic acid group 32 had IC(50) values of 2.1, 5.7, and 2.6 microM against the Yersinia PTP, PTP1B, and TCPTP, respectively.     

The protein phosphatase inhibitor okadaic acid induces morphological changes typical of apoptosis in mammalian cells.

Okadaic acid, a specific and potent inhibitor of protein phosphatases 2A and 1, was tested for its effect on the morphology of a number of cell types: freshly isolated rat hepatocytes in suspension or in primary culture, the human mammary carcinoma cell line MCF-7, the human neuroblastoma cell line SK-N-SH, rat pituitary adenoma GH3 cells, and rat promyelocytic IPC-81 cells. All the cell types reacted within a few hours to okadaic acid in the concentration range 0.1 to 1 microM with profound morphological alterations. Among the changes noted were: condensation of chromatin, shedding of cell contents via surface bleb formation, redistribution and compacting of cytoplasmic organelles, formation of cytoplasmic vacuoles, and hyperconvolution of the nuclear membrane. In some cells nuclear fragmentation was noted. In addition, cells growing as monolayers rounded up and detached from the substratum. The treated cells had no swollen mitochondria and retained the ability to exclude trypan blue until the final stage of dissolution, supporting the hypothesis that the changes were apoptotic rather than necrotic. In hepatocytes the action of okadaic acid was mimicked by another phosphatase inhibitor, microcystin, and was accompanied by shrinkage of the cell volume, as judged by Coulter counter analysis. The action of phosphatase inhibitor was not abolished by protein synthesis inhibitors, Ca(2+)-depleted medium, or phorbol ester. Although hepatocyte DNA replication was very sensitive to inhibition by okadaic acid, DNA fragmentation was less pronounced in response to okadaic acid than other agents inducing the morphological appearance of apoptosis.     

The protein phosphatase inhibitor, okadaic acid, inhibits phosphatidylcholine biosynthesis in isolated rat hepatocytes.

There is evidence that phosphatidylcholine (PC) biosynthesis in hepatocytes is regulated by a phosphorylation-dephosphorylation mechanism. The phosphatases involved have not been identified. We, therefore, investigated the effect of okadaic acid, a potent protein phosphatase inhibitor, on PC biosynthesis via the CDP-choline pathway in suspension cultures of isolated rat hepatocytes. Okadaic acid caused a 15% decrease (P less than 0.05) in [Me-3H]choline uptake in continuous-pulse labeling experiments. After 120 min of treatment, the labeling of PC was decreased 46% (P less than 0.05) with a corresponding 20% increase (P less than 0.05) in labeling of phosphocholine. Cells were pulsed with [Me-3H]choline for 30 min and subsequently chased for up to 120 min with choline in the absence or presence of okadaic acid. The labeling of phosphocholine was increased 86% (P less than 0.05) and labeling of PC decreased 29% (P less than 0.05) by 120 min of chase in okadaic acid-treated hepatocytes. The decrease of label in PC was quantitatively accounted for in the phosphocholine fraction. Incubation of hepatocytes with both okadaic acid and CPT-cAMP did not produce an additive inhibition in labeling of PC. Choline kinase and cholinephosphotransferase activities were unaltered by treatment with okadaic acid. Hepatocytes were incubated with digitonin to cause release of cytosolic components. Cell ghost membrane cytidylyltransferase (CT) activity was decreased 37% (P less than 0.005) with a concomitant 33% increase (P less than 0.05) in released cytosolic cytidylyltransferase activity in okadaic acid-treated hepatocytes. We postulate that CT activity and PC biosynthesis are regulated by protein phosphatase activity in isolated rat hepatocytes.     

Essential role of protein phosphatase 2A in metaphase II arrest and activation of mouse eggs shown by okadaic acid, dominant negative protein phosphatase 2A, and FTY720

Vertebrate eggs arrest at second meiotic metaphase. The fertilizing sperm causes meiotic exit through Ca(2+)-mediated activation of the anaphase-promoting complex/cyclosome (APC/C). Although the loss in activity of the M-phase kinase CDK1 is known to be an essential downstream event of this process, the contribution of phosphatases to arrest and meiotic resumption is less apparent, especially in mammals. Therefore, we explored the role of protein phosphatase 2A (PP2A) in mouse eggs using pharmacological inhibition and activation as well as a functionally dominant-negative catalytic PP2A subunit (dn-PP2Ac-L199P) coupled with live cell imaging. We observed that PP2A inhibition using okadaic acid induced events normally observed at fertilization: degradation of the APC/C substrates cyclin B1 and securin resulting from loss of the APC/C inhibitor Emi2. Although sister chromatids separated, chromatin remained condensed, and polar body extrusion was blocked as a result of a rapid spindle disruption, which could be ameliorated by non-degradable cyclin B1, suggesting that spindle integrity was affected by CDK1 loss. Similar cell cycle effects to okadaic acid were also observed using dominant-negative PP2Ac. Preincubation of eggs with the PP2A activator FTY720 could block many of the actions of okadaic acid, including Emi2, cyclin B1, and securin degradation and sister chromatid separation. Therefore, in conclusion, we used okadaic acid, dn-PP2Ac-L199P, and FTY720 on mouse eggs to demonstrate that PP2A is needed to for both continued metaphase arrest and successful exit from meiosis.     

Mutation induction by okadaic acid, a protein phosphatase inhibitor, in CHL cells, but not in S. typhimurium.

Okadaic acid (OA) is a specific and strong inhibitor of protein phosphatase 1 and 2A present in eukaryotes, and a potent promoter of carcinogenesis in mouse skin. In this study, we examined the mutagenicity of OA. OA did not induce mutations in S. typhimurium TA100 and TA98, with or without a microsomal metabolic activation system. However, it was strongly mutagenic to Chinese hamster lung (CHL) cells without a microsomal activation system, as shown using diphtheria toxin (DT) resistance (DT^r) as a selective marker. Treatment of CHL cells with OA at 17.5 ng/ml induced 164 DT^r mutants per 10⁶ survivors. A plot of the mutation frequency against the OA concentration gave a concave curve, and the mutant frequency was calculated to be 5500/10⁶ survivors/μg, with OA in the dose range of 10–15 ng/ml. This value was about 680 times that of ethyl methanesulfonate (EMS), and comparable to that of 2-amino-N⁶-hydroxyadenine, one of the strongest knowon mutgens. Elongation factor 2 (EF-2) obtained from 4 DT^r clones was not ADP-ribosylated by DT fragment A. PCR-direct sequencing revealed that the hot spot of EF-2 for EMS mutagenesis in CHO-K1 cells, the first letter of codon 717, was not a t spot for OA mutagenesis in CHL cells.     

Differentiation induction in human breast tumor cells by okadaic acid and related inhibitors of protein phosphatases 1 and 2A.

Okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, induces differentiation in human MCF-7, AU-565, and MB-231 breast tumor cells. In MCF-7 cells, OA elicited within 5 min an increase in the levels of a set of phosphorylated cellular proteins, within hours expression of the early response genes junB, c-jun, and c-fos, and within days manifestation of differentiation. Differentiation was also induced by two related protein phosphatase inhibitors, but not by an inactive OA derivative or by an inhibitor that penetrates epithelial cells poorly. These results indicate that OA and related agents can induce tumor breast cell differentiation, and this induction is correlated with their ability to inhibit PPH 1 and 2A.     

Apoptosis and mitotic arrest are two independent effects of the protein phosphatases inhibitor okadaic acid in K562 leukemia cells.

Treatment of human myeloid leukemia K562 cells with the serine/threonine protein phosphatases inhibitor okadaic acid induced mitotic arrest followed by apoptosis in a synchronized manner. The effect was observed at drug concentrations that inhibited the protein phosphatase type 2A but not type 1. We investigated whether apoptosis was a consequence of the preceding mitosis arrest or was induced independently by okadaic acid. We found that (1) apoptosis, but not mitotic arrest, was inhibited in cells with constitutive expression of Bcl-2; (2) pretreatment of cells with the DNA synthesis inhibitor hydroxyurea blocked the mitotic arrest but not the apoptosis mediated by okadaic acid; (3) down-regulation of c-myc gene was associated with apoptosis, but not with mitotic arrest; and (4) inhibition of protein synthesis abrogated mitotic arrest, but not apoptosis. The results suggest that inhibition of protein phosphatase 2A by okadaic acid provokes mitotic arrest and apoptosis of leukemia cells by independent mechanisms.     

Sucrose-phosphate synthase is dephosphorylated by protein phosphatase 2A in spinach leaves: Evidence from the effects of okadaic acid and microcystin

Sucrose-phosphate synthase (SPS) purified from spinach leaves harvested in the dark, was activated by mammalian protein phosphatase 2A (PP2A). Activation of SPS in a fraction from darkened spinach leaves was largely prevented by either okadaic acid or microcystin-LR (specific inhibitors of PP1 and PP2A), while inhibitor-2 (a PP1 inhibitor) or Mg²⁺ (essential for PP2C) were ineffective. In vivo, okadaic add and microcystin-LR prevented the light-induced activation of SPS and decreased sucrose biosynthesis and CO₂ fixation. It is concluded that PP2A is the major SPS phosphatase in spinach. This study is the first to employ microcystin-LR for modulating protein phosphorylation in vivo.     

Mitotic arrest and enhanced nuclear protein phosphorylation in human leukemia K562 cells by okadaic acid, a potent protein phosphatase inhibitor and tumor promoter.

We investigated the effects of the non-phorbol tumor promoter okadaic acid on human leukemia K562 cells. It was found that okadaic acid potently and reversibly inhibited cell growth, with a nearly complete inhibition of thymidine uptake seen at about 10 nM. The cytotoxicity of okadaic acid was characterized by a marked mitotic arrest of the cells exhibiting scattered chromosomes and abnormal anaphase-like structures, a phenomenon distinct from the typical metaphase arrest caused by colchicine. Okadaic acid (10-1,000 nM) greatly stimulated phosphorylation of a number of nuclear proteins in K562 cells. Phosphorylation of many of the same proteins was also stimulated by 12-O-tetradecanoylphorbol-13-O-acetate, a protein kinase C activator. The present findings, consistent with recent reports that okadaic acid is a potent inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A) shown to be essential for normal mitosis, provided evidence for the first time that okadaic acid inhibition of PP1/PP2A resulted in enhanced nuclear protein phosphorylation and subsequent mitotic arrest.