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The phosphorylation state of 20 types of intracellular proteins in the presence of the protein phosphatase 1 (PP1)- and PP2A-specific Ser/Thr phosphatase inhibitor calyculin A or the Tyr phosphatase inhibitor pervanadate was visualized by Phos-tag SDS-PAGE followed by immunoblotting. All blots showed a Phos-tag pattern indicating increased phosphorylation in the presence of one or both phosphatase inhibitors. The increase in phosphorylation stoichiometry per protein tends to be greater for Ser/Thr phosphatase inhibition than for Tyr phosphatase inhibition. This is consistent with the fact that the number of Ser/Thr kinase genes in the human genome is greater than that of Tyr kinases and with the fact that the phospho-Ser/phospho-Thr ratio in the actual human phosphoproteome is far greater than that of phospho-Tyr ratio. This suggests that cellular proteins are routinely and randomly phosphorylated by different kinases with no biological significance, simply depending on the frequency of substrate encounters. Phosphatase is responsible for routinely removing these unwanted phosphate groups systematically and maintaining the dynamic equilibrium of physiological protein phosphorylation. Phos-tag SDS-PAGE visualized that the kinase reaction involves many incidental phosphorylation and that phosphatases play broader roles besides being strict counterparts to kinases. [ABSTRACT FROM AUTHOR]

The present contribution describes an electrochemical system for the detection of alkaline phosphatase (ALP) enzyme inhibitors in seawater medium. Fluorescence spectroscopy and thermal unfolding results suggest that the state of free ALP is affected in this medium, yet the protein remains active. The enzyme activity can be evaluated using hydroquinone diphosphate as the substrate, with hydroquinone as the electrochemically monitored product. It has been demonstrated that encapsulation in conventional and organic modified silica matrices maintains ALP integrity, although diffusion across the formed monoliths hinders the electrochemical response. ALP@Phenyl‐modified silica exhibits the best performance due to higher affinity between substrate molecules and aromatic moieties and, probably, to larger pore size. This electrochemical system can detect and quantify calyculin A in seawater at sub‐nanomolar concentrations and it can also be employed for the development of electrochemical biosensors tailored for the marine environment. [ABSTRACT FROM AUTHOR]

Abstract The present contribution describes an electrochemical system for the detection of alkaline phosphatase (ALP) enzyme inhibitors in seawater medium. Fluorescence spectroscopy and thermal unfolding results suggest that the state of free ALP is affected in this medium, yet the protein remains active. The enzyme activity can be evaluated using hydroquinone diphosphate as the substrate, with hydroquinone as the electrochemically monitored product. It has been demonstrated that encapsulation in conventional and organic modified silica matrices maintains ALP integrity, although diffusion across the formed monoliths hinders the electrochemical response. ALP@Phenyl‐modified silica exhibits the best performance due to higher affinity between substrate molecules and aromatic moieties and, probably, to larger pore size. This electrochemical system can detect and quantify calyculin A in seawater at sub‐nanomolar concentrations and it can also be employed for the development of electrochemical biosensors tailored for the marine environment.

In normal anaphase cells, telomeres of each separating chromosome pair are connected to each other by tethers. Tethers are elastic at the start of anaphase: arm fragments cut from anaphase chromosomes in early anaphase move across the equator to the oppositely-moving chromosome, telomere moving toward telomere. Tethers become inelastic later in anaphase as the tethers become longer: arm fragments no longer move to their partners. When early anaphase cells are treated with Calyculin A (CalA), an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), at the end of anaphase chromosomes move backward from the poles, with telomeres moving toward partner telomeres. Experiments described herein show that in cells treated with CalA, backwards movements are stopped in a variety of ways, by cutting the tethers of backwards moving chromosomes, by severing arms of backwards moving chromosomes, by severing arms before the chromosomes reach the poles, and by cutting the telomere toward which a chromosome is moving backwards. Measurements of arm-fragment velocities show that CalA prevents tethers from becoming inelastic as they lengthen. Since treatment with CalA causes tethers to remain elastic throughout anaphase and since inhibitors of PP2A do not cause the backwards movements, PP1 activity during anaphase causes the tethers to become inelastic.

Elastic tethers, connecting telomeres of all separating anaphase chromosome pairs, lose elasticity when they lengthen during anaphase. Treatment with phosphatase inhibitor CalyculinA causes anaphase chromosomes to move backwards after they reach the poles, suggesting that dephosphorylation causes loss of tether elasticity. We added 50nM CalyculinA to living anaphase crane-fly spermatocytes with different length tethers. When tethers were short, almost all partner chromosomes moved backwards after nearing the poles. When tethers were longer, fewer chromosomes moved backwards. With yet longer tethers none moved backward. This is consistent with tether elasticity being lost by dephosphorylation. 50nM CalyculinA blocks both PP1 and PP2A. To distinguish between PP1 and PP2A we treated cells with short tethers with 50nM okadaic acid which blocks solely PP2A, or with 1µM okadaic acid which blocks both PP1 and PP2A. Only 1µM okadaic acid caused chromosomes to move backward. Thus, tether elasticity is lost because of dephosphorylation by PP1.

In normal anaphase cells, telomeres of each separating chromosome pair are connected to each other by tethers. Tethers are elastic at the start of anaphase: arm fragments cut from anaphase chromosomes in early anaphase move across the equator to the oppositely-moving chromosome, telomere moving toward telomere. Tethers become inelastic later in anaphase as the tethers become longer: arm fragments no longer move to their partners. When early anaphase cells are treated with Calyculin A (CalA), an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), at the end of anaphase chromosomes move backward from the poles, with telomeres moving toward partner telomeres. Experiments described herein show that in cells treated with CalA, backwards movements are stopped in a variety of ways, by cutting the tethers of backwards moving chromosomes, by severing arms of backwards moving chromosomes, by severing arms before the chromosomes reach the poles, and by cutting the telomere toward which a chromosome is moving backwards. Measurements of arm-fragment velocities show that CalA prevents tethers from becoming inelastic as they lengthen. Since treatment with CalA causes tethers to remain elastic throughout anaphase and since inhibitors of PP2A do not cause the backwards movements, PP1 activity during anaphase causes the tethers to become inelastic.

Humanin (HN) is an extensive neuroprotective peptide. This study aims to investigate the neuroprotective effects of HN on Calyculin A (CA)-induced neurotoxicities in cortical neurons and the underlying mechanism. CA was added into the cultured cortical neurons to induce neurotoxicity. Cortical neurons were preincubated with HN which plays a protective role. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and Calcein-AM were applied to evaluate the neural insults. Caspase 3 signal and Tunnel were performed to test neural apoptosis. Western blot analysis was used to detect the expressions of phosphorylated tau. The corresponding kits were used to measure the contents of malondialdehyde (MDA) and superoxide dismutase (SOD), and the activity of PP2A, respectively. HN preincubation preserved cell viability, protected the neurons, alleviated oxidative stress, and reserved PP2A activity. It also blocked tau overphosphorylation at Ser199/202, Ser396, and Thr231 sites and protected neurons against CA-induced insults. These results suggest that HN may serve as a potential therapeutic agent to prevent the pathological changes induced by CA via modulating the activity of PP2A and oxidative stress in neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Elastic tethers, connecting telomeres of all separating anaphase chromosome pairs, lose elasticity when they lengthen during anaphase. Treatment with phosphatase inhibitor CalyculinA causes anaphase chromosomes to move backwards after they reach the poles, suggesting that dephosphorylation causes loss of tether elasticity. We added 50nM CalyculinA to living anaphase crane-fly spermatocytes with different length tethers. When tethers were short, almost all partner chromosomes moved backwards after nearing the poles. When tethers were longer, fewer chromosomes moved backwards. With yet longer tethers none moved backward. This is consistent with tether elasticity being lost by dephosphorylation. 50nM CalyculinA blocks both PP1 and PP2A. To distinguish between PP1 and PP2A we treated cells with short tethers with 50nM okadaic acid which blocks solely PP2A, or with 1µM okadaic acid which blocks both PP1 and PP2A. Only 1µM okadaic acid caused chromosomes to move backward. Thus, tether elasticity is lost because of dephosphorylation by PP1. [ABSTRACT FROM AUTHOR]

Previous researches of our laboratory reported that addition of cAMP analog cBiMPS and protein phosphatase inhibitor calyculin A (stimulators of cAMP signaling cascades) improved the capacity of incubation medium to induce full-type hyperactivation in bovine ejaculated spermatozoa. However, this modified medium was valid only for samples with relatively good survivability for incubation with stimulators of cAMP signaling cascades. Thus, it is necessary to make further modified medium for evaluation of potentials to exhibit full-type hyperactivation in bovine sperm samples with relatively lower survivability. Na+/K+-ATPase is an integral membrane protein and involved with the regulation of rodent sperm motility. To make further modification of the medium, we examined effects of Na+/K+-ATPase inhibition with digoxin on motility, full-type hyperactivation and protein tyrosine phosphorylation in bovine ejaculated spermatozoa with relatively lower survivability for incubation with stimulators of cAMP signaling cascades and also performed the immunodetection of bovine sperm Na+/K+-ATPase. The addition of Na+/K+-ATPase inhibitor digoxin to the incubation medium containing cBiMPS and calyculin A had the tendency to lessen the decreases in the percentages of motile spermatozoa in all of 12 samples after the incubation for 1–3 h and significantly increased the percentages of full-type hyperactivation in one group of 4 samples (Sample-A1) and another group of 4 samples (Sample-A2) after 1 and 2 h respectively, though it had no significant effects on full-type hyperactivation in the other group of 4 samples (Sample-B). In addition, incubation time-related changes in the sperm protein tyrosine phosphorylation (a good marker for sperm capacitation) were correlated with those in the percentages of full-type hyperactivation in Sample-A1 containing digoxin. Immunodetection showed that Na+/K+-ATPase is present in the middle and principal pieces of the flagella, indicating that Na+/K+-ATPase has possible relations with sperm motility. These results obtained with bull ejaculated spermatozoa with relatively lower survivability indicate that incubation method using digoxin is useful to evaluate potentials of sperm samples to exhibit full-type hyperactivation, that digoxin has effects on suppressing reduction of sperm motility, and that prolonged incubation with digoxin induces reduction of capacitation state which may suppress the maintenance of full-type hyperactivation. • Na+/K+-ATPases were present in the middle and principal pieces of bovine sperm. • Temporal inhibition of Na+/K+-ATPases supported sperm hyperactivation (HA). • Prolonged inhibition of Na+/K+-ATPases reduced sperm capacitation state and HA. • Inhibition of Na+/K+-ATPases was effective on maintenance of sperm motility. [ABSTRACT FROM AUTHOR]

Spontaneous AP (action potential) firing of sinoatrial nodal cells (SANC) is critically dependent on protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent protein phosphorylation, which are required for the generation of spontaneous, diastolic local Ca2+ releases (LCRs). Although phosphoprotein phosphatases (PP) regulate protein phosphorylation, the expression level of PPs and phosphatase inhibitors in SANC and the impact of phosphatase inhibition on the spontaneous LCRs and other players of the oscillatory coupled-clock system is unknown. Here, we show that rabbit SANC express both PP1, PP2A, and endogenous PP inhibitors I-1 (PPI-1), dopamine and cyclic adenosine 3′,5′-monophosphate (cAMP)-regulated phosphoprotein (DARPP-32), kinase C-enhanced PP1 inhibitor (KEPI). Application of Calyculin A, (CyA), a PPs inhibitor, to intact, freshly isolated single SANC: (1) significantly increased phospholamban (PLB) phosphorylation (by 2–3-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites, in a time and concentration dependent manner; (2) increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site; (3) substantially increased sarcoplasmic reticulum (SR) Ca2+ load; (4) augmented L-type Ca2+ current amplitude; (5) augmented LCR’s characteristics and decreased LCR period in intact and permeabilized SANC, and (6) increased the spontaneous basal AP firing rate. In contrast, the selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Our numerical model simulations demonstrated that PP inhibition increases AP firing rate via a coupled-clock mechanism, including respective increases in the SR Ca2+ pumping rate, L-type Ca2+ current, and Na+/Ca2+-exchanger current. Thus, PP1 and its endogenous inhibitors modulate the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca2+ cycling protein phosphorylation, the SR Ca2+ load and LCRs, and L-type Ca2+ current.

Abstract In bull spermatozoa, extracellular Ca2+-dependent full-type hyperactivation, which is characterized by the asymmetrical beating in whole parts of the middle/principal pieces, is suppressed by calyculin A-sensitive protein phosphatases. The aim of this study was to identify isoforms of these protein phosphatases. Ejaculated spermatozoa were used for the investigation on effects of protein phosphatase inhibitors (calyculin A with high specificity for both of protein phosphatases 1 and 2A, and okadaic acid with relatively higher specificity for protein phosphatase 2A than protein phosphatase 1) on the induction of extracellular Ca2+-dependent full-type hyperactivation by incubation with CaCl 2 and cAMP analog (cBiMPS). They were also used for the immunodetection of protein phosphatases 1α, 1β, 1γ, 2Aα and 2Aβ. Percentages of full-type hyperactivated spermatozoa significantly increased after incubation with calyculin A (10 nM) in a concentration-dependent manner of CaCl 2 (0–3.42 mM), though only minor increases in the percentages of full-type hyperactivated spermatozoa were observed after incubation with okadaic acid (10 nM). Moreover, the immunodetection of protein phosphatase isoforms showed sperm connecting piece and flagellum included protein phosphatases 1α and 1γ, but did not do the other isoforms. These results suggest that calyculin A-sensitive and okadaic acid-less sensitive protein phosphatases (1α and 1γ) are suppressors for the extracellular Ca2+-dependent full-type hyperactivation in bull ejaculated spermatozoa. Highlights • Calyculin A-sensitive and okadaic acid-less sensitive protein phosphatases (protein phosphatase 1) suppressed full-type hyperactivation and protein phosphorylation of the connecting piece in bull sperm. • Protein phosphatases 1α and γ (PP1α and PP1γ) were detected in the connecting piece of bull sperm by the immunostaining. • PP1α and PP1γ of the connecting piece were identified as specific suppressors for cAMP-protein phosphorylation signaling cascades which regulated full-type hyperactivation in bull sperm. [ABSTRACT FROM AUTHOR]

Triple negative breast cancer cell lines express high levels of β2-adrenergic receptor, which have a significant influence on the activity of extracellular signal-regulated kinase (ERK)1/2. Therefore, it is important to understand the link between β2-adrenergic receptor signaling and ERK1/2 activity in terms of cancer cell regulation and cancer progression. Although the molecular mechanisms are not completely clarified, β2-adrenergic receptor stimulation appears to reduce the basal levels of phosphorylated (p)ERK1/2 in MDA-MB-231 breast cancer cells. The aim of the current study was to determine the mechanism of β2-adrenergic receptor-mediated ERK1/2 dephosphorylation by investigating the role of dual-specificity phosphatase (DUSP)1/6 and protein phosphatase (PP)1/2, which are established regulators of ERK1/2 phosphorylation, in MDA-MB-231 and MDA-MB-468 breast cancer cell lines. (E)-2-benzylidene-3-(cyclohexyl amino)-2,3-dihydro-1H-inden-1-one (BCI) and calyculin A were employed as DUSP1/6 and PP1/PP2 inhibitors, respectively. Subsequently, the protein levels of DUSP1, PP1, pPP1, ERK1/2 and pERK1/2 were measured by western blot analysis. Cells were transfected with DUSP1 small interfering (si)RNA or PP1 siRNA to inhibit their expression. The results demonstrated that β2-adrenergic receptor agonists led to the dephosphorylation of basal pERK1/2 in MDA-MB-231 and MDA-MB-468 cells. The DUSP1/6 inhibitor, BCI, and the PP1/PP2 inhibitor, calyculin A, antagonized the β2-adrenergic receptor-mediated dephosphorylation of ERK1/2. Furthermore, β2-adrenergic receptor stimulation increased the protein expression level of DUSP1, with no effects on DUSP6, PP1 and PP2 expression, and enhanced the expression of the active form of PP1. Downregulation of the expression of DUSP1 or PP1 led to a decline in the β2-adrenergic receptor-mediated dephosphorylation of ERK1/2. The results of the present study indicate that β2-adrenergic receptor-mediated dephosphorylation of ERK1/2 may be associated with the activity of DUSP1 and PP1 in MDA-MB-231 and MDA-MB-468 triple negative breast cancer cell lines. The clinical importance of β2-adrenergic receptor-mediated inactivation of ERK1/2 as well as the activation of DUSP1 and PP1 should be carefully evaluated in future studies, particularly when β2-adrenergic blockers are used in patients with triple negative breast cancer. [ABSTRACT FROM AUTHOR]

Cyclin D1 is a key regulator of the cell cycle that is over expressed in more than half of breast cancer patients. The levels of cyclin D1 are controlled primarily through post-translational mechanisms and phosphorylation of cyclin D1 at T286 induces its proteasomal degradation. To date, no studies have explored the involvement of phosphatases in this process. Here we treated human breast cancer cells with the structurally distinct toxins calyculin A, okadaic acid, and cantharidin, which are known to inhibit Ser/Thr phosphatases of the PPP family. At low nanomolar concentrations calyculin A induced T286 phosphorylation and degradation of cyclin D1 via the proteosome in MDA-MB-468 and MDA-MB-231 cells. Cyclin D1 degradation also was dose-dependently induced by okadaic acid and catharidin, implicating a negative regulatory role for type-2A phosphatases. These effects occurred without increasing phosphorylation of p70S6K, cyclin D3, or myosin light chain that were used as endogenous reporters of cellular PP2A and PP1 activity. A reverse phase phosphoprotein array analysis revealed increased phosphorylation of only 6 out of 33 Ser/Thr phosphosites, indicating selective inhibition of phosphatases by calyculin A. Calyculin A treatment induced cell cycle arrest in MDA-MB-468 and MCF-7 breast cancer cells. These findings suggest that a specific pool of type-2A phosphatase is inhibited by calyculin A leading to the degradation of cyclin D1 in human breast cancer cells. The results highlight the utility of toxins as pharmacological probes and points to the T286 cyclin D1 phosphatase inhibited by calyculin A as a possible target for chemotherapy to treat triple negative breast cancer.

Marine sponges are among the most primitive animals and often contain unique, biologically active compounds. Several of these compounds have played an important roles as pharmaceutical leads for anti-cancer drugs, such as halichondrin B, which led to the development of an anti-breast cancer drug. Some compounds with remarkable biological activities are accumulated in significantly high concentrations in the sponge. How and why the marine sponges produce and accumulate bioactive natural products are long-standing questions with both biochemical and ecological implications, since in sponges, the animal-microbe symbioses are presumed to be responsible for the biosynthetic machinery, consisting of efficient enzymes and regulatory systems for the specific biological activities of medicinally relevant natural products. In this review, I focus on the chemically rich Theonellidae family sponges and discuss the biosynthesis of bioactive peptides and polyketides. In particular, the biosynthetic pathway of calyculin A suggests that crosstalk between the sponge host and bacterial symbiont confers a chemical defense system on the immobile animal-microbe holobiont.

A basic question of cell biology is how DNA folds to chromosome. A number of recently accumulated evidences have suggested that folding of chromosome proceeds tightly coupled with DNA replication progresses. Drug-induced PCC is a useful tool for visualization of the interphase nuclei, in particular, S-phase, as S-phase prematurely condensed chromosomes (S-phase PCC). Active replicating DNA is labeled directly with Cy3-dUTP by bead loading method, and then S-phase nuclei is immediately condensed prematurely by calyculin A to obtain S-phase PCC. Active replicating regions on S-PCC are observed under a scanning confocal microscope. Cy3-dUTP-labeled S-phase PCCs clearly reveal the drastic transitional change of chromosome formation through S-phase, starting from a "cloudy nebula" to numerous numbers of "beads on a string" and finally to "striped arrays of banding structured chromosome" known as G- or R-banding pattern. The number, distribution, and shape of replication foci were also measured in individual subphase of S-phase; maximally ~1400 foci of 0.35 μm average radius size were scored at the beginning of S-phase, and the number is reduced to ~100 at the end of S-phase. Drug-induced PCC clearly provided the new insight that eukaryote DNA replication is tightly coupled with the chromosome condensation/compaction for construction of eukaryote higher-ordered chromosome structure., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background In response to diverse genotoxic stimuli p53 is activated as transcription factor to exert its tumor-suppressor function. P53 activation requires protein stabilization, nuclear localization and posttranslational modifications in key residues that may influence p53 selection of target genes. Among them, serine 46 (Ser46) phosphorylation is considered specific for apoptotic activation. Hyperglicaemia, the high blood glucose condition, may negatively affect tumor response to therapies through several mechanisms, conferring resistance to drug-induced cell death. However, whether high glucose might modify p53Ser46 phosphorylation has never been addressed. Methods and results Here, we performed biochemical and molecular analyses in different cancer cell lines treated with chemotherapy in the presence or absence of high glucose condition. Analyses of p53 posttranslational modifications showed that drug- induced p53Ser46 phosphorylation was reduced by high glucose. Such reduction depended by high glucose-induced calyculin A sensitive phosphatase(s), able to specifically target p53Ser46 phosphorylation. The specific effect on Ser46 phosphorylation was addressed by analysing Ser15 phosphorylation that instead was not modified by high glucose. In agreement, a constitutively phosphorylated Ser46D p53 mutant was resistant to high glucose. As a consequence of phosphoSer46 impairment, high glucose reduced the tumor cell response to drugs, correlating with reduced p53 apoptotic transactivation. The drug-induced apoptotic cell death, reduced by high glucose, was finally restored by the phosphatase inhibitor calyculin A. Conclusions These data indicate that high glucose specifically inhibited Ser46 phosphorylation thus reducing p53 apoptotic activity. These results uncover a new mechanism of p53 inactivation providing an interesting novel molecular link between metabolic diseases such as diabetes or obesity and tumor progression and resistance to therapies. [ABSTRACT FROM AUTHOR]

The premature chromosome condensation (PCC) assay is considered as complementary bio-dosimetry tool for chromosome aberration assay and the PCC assay can be used to estimate high dose exposure. Though the PCC ring is considered as prospective biomarker, chromosome length ratio (ratio of longest and shortest chromosome length in PCC spreads) of chemically induced PCC is shown to be very good indicator of ionizing radiation. In view of this, an in-vitro study has been performed using PCC assay to suggest chromosome length ratio (LR) as potential bio-dosimeter induced by high dose ionizing radiation. Blood samples were collected from healthy subjects (n = 3) after prior consent and irradiated to ten different doses ranging between 0 and 20 Gy using 6 MV LINAC X-rays with dose rate of 5.6 Gy/min. Irradiated lymphocytes were cultured and calyculin induced PCC spreads were prepared. PCC spreads were captured using image analysis system and chromosome lengths were measured using open-source ImageJ software. For each dose, LR for 50 chromosome spreads were computed and mean LR value was calculated. LR varies between 6.0 ± 0.08 and 23.6 ± 0.55 for the dose range between 2 and 20 Gy. The dose response curve for LR was observed to be linear with y = 1.02x + 3.36, R2 = 0.97. Linear dose response relationship obtained in the present study confirms the prospective use of LR measurement. This study is first of its kind to examine chromosome length ratio as a biomarker of DNA damage in cells exposed to high dose X-ray exposure. • An in-vitro study using chemically induced PCC in X-ray irradiated lymphocytes. • Chromosome length ratio (LR) shown to be good indicator for high dose exposure. • Dose response curve of length ratio (longest/shortest) observed to be linear. • First of its kind to examine LR as a biomarker of DNA damage for high dose X-ray. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study was to investigate the effect of calyculin A on the number of γ-H2AX foci (phosphorylated histone variant 2AX) in lymphocytes after in vitro and in vivo irradiation with rather low doses as they are used in diagnostic and interventional radiology. Materials and methods: For in vitro experiments blood samples of 14 healthy volunteers were irradiated with different doses (10, 50, 100 mGy) and incubated with (0.01, 0.1, 1, 10 nM) or without calyculin A for up to 2 hours. Non-irradiated samples with and without calyculin A served as controls. For in vivo evaluation blood samples were collected from seven patients undergoing computed tomography (CT) both with 1 nM calyculin A containing vials and vials without calyculin A. Foci were quantified in isolated lymphocytes using γ-H2AX immunofluorescence microscopy. Results: 1 nM calyculin A led to a complete inhibition of γ-H2AX foci loss in irradiated samples whereas no inhibition of p53 binding protein 1 (53 BP1) foci was found. Lower concentrations of the phosphatase inhibitor did not have a sufficient effect on foci decrease. Calyculin A did not affect foci levels in non-irradiated samples. If no calyculin A was added into the vial before the blood draws detectable CT-induced foci levels were lower in all patients with a reduction of the medians of 35%. Conclusions: Using γ-H2AX immunofluorescence microscopy calyculin A can be a useful tool to mark the induced γ-H2AX foci after low dose irradiation and to avoid an underestimation of the real deoxyribonucleic acid (DNA) damage in in vitro and in vivo experiments. [ABSTRACT FROM AUTHOR]

Most cell cycle regulation research has been conducted in model organisms representing a very small part of the eukaryotic domain. The highly divergent human pathogen Giardia intestinalis is ideal for studying the conservation of eukaryotic pathways. Although Giardia has many cell cycle regulatory components, its genome lacks all anaphase-promoting complex (APC) components. In the present study, we show that a single mitotic cyclin in Giardia is essential for progression into mitosis. Strikingly, Giardia cyclin B lacks the conserved N-terminal motif required for timely degradation mediated by the APC and ubiquitin conjugation. Expression of Giardia cyclin B in fission yeast is toxic, leading to a prophase arrest, and this toxicity is suppressed by the addition of a fission yeast degradation motif. Cyclin B is degraded during mitosis in Giardia cells, but this degradation appears to be independent of the ubiquitination pathway. Other putative APC substrates, aurora and polo-like kinases, also show no evidence of ubiquitination. This is the first example of mitosis not regulated by the APC and might reflect an evolutionary ancient form of cell cycle regulation. [ABSTRACT FROM AUTHOR]

Abstract: This work deals with the role of myosin phosphorylation in anaphase chromosome movement. Y27632 and ML7 block two different pathways for phosphorylation of the myosin regulatory light chain (MRLC). Both stopped or slowed chromosome movement when added to anaphase crane-fly spermatocytes. To confirm that the effects of the pharmacological agents were on the presumed targets, we studied cells stained with antibodies against mono- or bi-phosphorylated myosin. For all chromosomes whose movements were affected by a drug, the corresponding spindle fibres of the affected chromosomes had reduced levels of 1P- and 2P-myosin. Thus the drugs acted on the presumed target and myosin phosphorylation is involved in anaphase force production. Calyculin A, an inhibitor of MRLC dephosphorylation, reversed and accelerated the altered movements caused by Y27632 and ML-7, suggesting that another phosphorylation pathway is involved in phosphorylation of spindle myosin. Staurosporine, a more general phosphorylation inhibitor, also reduced the levels of MRLC phosphorylation and caused anaphase chromosomes to stop or slow. The effects of staurosporine on chromosome movements were not reversed by Calyculin A, confirming that another phosphorylation pathway is involved in phosphorylation of spindle myosin. [Copyright &y& Elsevier]

Abstract: Potential mechanisms of the reversible temperature-dependent immobilization of fowl sperm were investigated. At 30 °C, motility of demembranated fowl sperm was inhibited by adding 2 mM ethylene glycol-bis (2-aminoethylether)-N,N,N'',N''-tetraacetic acid (EGTA), but restored immediately after the subsequent addition of 2 mM CaCl2, whereas at 40 °C, such additions did not appreciably affect motility (which remained almost negligible). With intact sperm, 10−9 to 10−3 M Ca2+ had no effect on motility at 30 °C, which remained high. In contrast, intact sperm at 40 °C were almost immotile below 10−5 M Ca2+, and then gradually recovered motility at higher Ca2+ concentrations. The negligible motility of demembranated sperm at 40 °C, and at 30 °C in the presence of EGTA, was stimulated by addition of 100 nM of the protein phosphatase inhibitor calyculin A. Dynein-ATPase activities of sperm at 40 °C in the presence of 2 mM EGTA, 2 μM CaCl2, 2 mM CaCl2, or 100 nM calyculin A were higher than those at 30 °C. Therefore, stimulation of fowl sperm motility by temperature, Ca2+, and phosphatase inhibition was not simply associated with an increase of flagellar dynein-ATPase activity. Furthermore, Ca2+ was essential, at the axonemal level, for initiation of the ‘intrinsic’ motility of fowl sperm at 30 °C, but this Ca2+-dependent mechanism might be different from that involved in restoration of motility of intact sperm at 40 °C. In addition, perhaps inhibition of protein phosphatase activity was involved in initiation of sperm motility, but acting at a location different from Ca2+ on the axoneme. [Copyright &y& Elsevier]

Calyculin A (Cal A) is a serine/threonine phosphatase inhibitor that is capable of inducing apoptosis in cancer cells. In this study, we examined whether Cal A could modulate TRAIL-induced apoptosis in human renal carcinoma-derived Caki cells. Our results show that Cal A is capable of sensitizing Caki cells to TRAIL-induced apoptosis, as well as U2OS human osteosarcoma cells and A549 human lung adenocarcinoma epithelial cells. Cal A increases intracellular ROS production and down-regulates c-FLIP(L) expression. Interestingly, the down-regulation of protein phosphatase 1 (PP1) by PP1 siRNA also reduced c-FLIP(L) expression via reactive oxygen species production. Furthermore, Cal A induced death receptor 4 (DR4) mRNA and protein expression by enhancing DR4 mRNA stability. We also found that PP4 siRNA up-regulated DR4 mRNA and protein expression. Collectively, our results suggest that Cal A could enhance TRAIL-mediated apoptosis via the down-regulation of c-FLIP(L) and the up-regulation of DR4 in human renal cell carcinoma cell line Caki. [ABSTRACT FROM AUTHOR]

Aims The role of Ca2+ sensitization induced by a Ca2+-independent myosin light chain kinase (MLCK) in hypertension has not been determined. The aim of this study was to clarify the role of possible Ca2+-independent MLCK activity in hypertension. Methods and results We compared increases in contractile force and phosphorylation of myosin light chain (MLC) evoked by calyculin A, a phosphatase inhibitor, in β-escin-permeabilized mesenteric arteries at pCa 9.0 between spontaneously hypertensive rat (SHR) and Wistar Kyoto rat (WKY). We found that there was no detectable phosphorylation of MLC at pCa 9.0, but that the administration of 1 μM calyculin A gradually increased force and mono- and di-phosphorylation of MLC. This contraction was inhibited by staurosporine but not by wortmannin, Y-27632, or calphostin-C. The calyculin A-induced contraction was significantly greater in the SHR than in the WKY and was associated with an increase in mono- and di-phosphorylation of MLC. SM-1, a zipper-interacting protein kinase (ZIPK)-inhibiting peptide, significantly inhibited the amplitude of the calyculin A-induced contraction and di-phosphorylation. Total ZIPK expression (54 + 32 kDa) was greater in the SHR than in the WKY. Phosphorylation of myosin phosphatase target subunit at Thr697, but not at Thr855, was consistently stronger in the SHR than in the WKY in calyculin A-treated tissues at pCa 9.0. Conclusions Our results suggest that Ca2+-independent MLCK activity is enhanced in the SHR, and that ZIPK plays, at least in part, an important role as a candidate for this kinase in rat mesenteric arteries. [ABSTRACT FROM PUBLISHER]

Microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) often induce rises in cytosolic free Ca concentration ([Ca]) and protein phosphorylation. Though they are postulated to play pivotal roles in plant innate immunity, their molecular links and the regulatory mechanisms remain largely unknown. To investigate the regulatory mechanisms for MAMP-induced Ca mobilization, we have established a transgenic rice ( Oryza sativa) cell line stably expressing apoaequorin, and characterized the interrelationship among MAMP-induced changes in [Ca], production of reactive oxygen species (ROS) and protein phosphorylation. Oligosaccharide and sphingolipid MAMPs induced Ca transients mainly due to plasma membrane Ca influx, which were dramatically suppressed by a protein phosphatase inhibitor, calyculin A (CA). Hydrogen peroxide and hypo-osmotic shock triggered similar [Ca] elevations, which were not affected by CA. MAMP-induced protein phosphorylation, which is promoted by CA, has been shown to be required for ROS production and MAPK activation, while it negatively regulates MAMPs-induced Ca mobilization and may play a crucial role in temporal regulation of [Ca] signature. [ABSTRACT FROM AUTHOR]

Cyclin D1 is a key regulator of the cell cycle that is over expressed in more than half of breast cancer patients. The levels of cyclin D1 are controlled primarily through post-translational mechanisms and phosphorylation of cyclin D1 at T286 induces its proteasomal degradation. To date, no studies have explored the involvement of phosphatases in this process. Here we treated human breast cancer cells with the structurally distinct toxins calyculin A, okadaic acid, and cantharidin, which are known to inhibit Ser/Thr phosphatases of the PPP family. At low nanomolar concentrations calyculin A induced T286 phosphorylation and degradation of cyclin D1 via the proteosome in MDA-MB-468 and MDA-MB-231 cells. Cyclin D1 degradation also was dose-dependently induced by okadaic acid and catharidin, implicating a negative regulatory role for type-2A phosphatases. These effects occurred without increasing phosphorylation of p70S6K, cyclin D3, or myosin light chain that were used as endogenous reporters of cellular PP2A and PP1 activity. A reverse phase phosphoprotein array analysis revealed increased phosphorylation of only 6 out of 33 Ser/Thr phosphosites, indicating selective inhibition of phosphatases by calyculin A. Calyculin A treatment induced cell cycle arrest in MDA-MB-468 and MCF-7 breast cancer cells. These findings suggest that a specific pool of type-2A phosphatase is inhibited by calyculin A leading to the degradation of cyclin D1 in human breast cancer cells. The results highlight the utility of toxins as pharmacological probes and points to the T286 cyclin D1 phosphatase inhibited by calyculin A as a possible target for chemotherapy to treat triple negative breast cancer. [ABSTRACT FROM AUTHOR]

The small GTPase RhoA increases the Ca2+ sensitivity of smooth muscle contraction and myosin light chain (MLC) phosphorylation by inhibiting the activity of MLC phosphatase. RhoA is also a known regulator of cytoskeletal dynamics and actin polymerization in many cell types. In airway smooth muscle (ASM), contractile stimulation induces MLC phosphorylation and actin polymerization, which are both required for active tension generation. The objective of this study was to evaluate the primary mechanism by which RhoA regulates active tension generation in intact ASM during stimulation with acetylcholine (ACh). RhoA activity was inhibited in canine tracheal smooth muscle tissues by expressing the inactive RhoA mutant, RhoA T19N, in the intact tissues or by treating them with the cell-permeant RhoA inhibitor, exoenzyme C3 transferase. RhoA inactivation reduced ACh-induced contractile force by ~60% and completely inhibited ACh-induced actin polymerization but inhibited ACh-induced MLC phosphorylation by only ~20%. Inactivation of MLC phosphatase with calyculin A reversed the reduction in MLC phosphorylation caused by RhoA inactivation, but calyculin A did not reverse the depression of active tension and actin polymerization caused by RhoA inactivation. The MLC kinase inhibitor, ML-7, inhibited ACh-induced MLC phosphorylation by ~80% and depressed active force by ~70% but did not affect ACh-induced actin polymerization, demonstrating that Ach-stimulated actin polymerization occurs independently of MLC phosphorylation. We conclude that the RhoA-mediated regulation of ACh-induced contractile tension in ASM results from its role in mediating actin polymerization rather than from effects on MLC phosphatase or MLC phosphorylation. [ABSTRACT FROM AUTHOR]

Abstract: This study provides a useful biodosimetry protocol for radiation accidents that involve high doses of heavy particle radiation. Human peripheral blood lymphocytes (PBLs) were irradiated in vitro with high doses (5–50Gy) of charged heavy-ion particles (carbon ions, at an effective linear-energy-transfer (LET) of 34.6keV/μm), and were then stimulated to obtain dividing cells. PBLs were treated with 100nM calyculin A to force chromosomes to condense prematurely, and chromosome spreads were obtained and stained with Giemsa. The G2 prematurely condensed chromosome (G2-PCC) index and the number of G2-PCC including fragments (G2-PCC-Fs) per cell for each radiation dose point were scored. Dose-effect relationships were obtained by plotting the G2-PCC indices or G2-PCC-Fs numbers against radiation doses. The G2-PCC index was greater than 5% up to doses of 15Gy; even after a 30Gy radiation dose, the index was 1 to 2%. At doses higher than 30Gy, however, the G2-PCC indices were close to zero. The number of G2-PCC-Fs increased steeply for radiation doses up to 30Gy at a rate of 1.07Gy−1. At doses higher than 30Gy, the numbers of G2-PCC-Fs could not be accurately indexed because of the limited numbers of cells for analysis. Therefore, the number of G2-PCC-Fs could be used to estimate radiation doses up to 30Gy. In addition, a G2-PCC index close to zero could be used as an indicator for radiation doses greater than 40Gy. [Copyright &y& Elsevier]

AbstractAim:KCNQ4 channels play an important part in adjusting the function of cochlear outer hair cells. The aim of this study was to investigate the effects of ser/thr phosphatase inhibitors on human KCNQ4 channels expressed in Xenopuslaevis oocytes.Methods:Synthetic cRNA encoding human KCNQ4 channels was injected into Xenopus oocytes. We used a two-electrode voltage clamp to measure the ion currents in the oocytes.Results:Wild-type KCNQ4 expressed in Xenopus oocytes showed the typical properties of slow activation kinetics and low threshold activation. The outward K+ current was almost completely blocked by a KCNQ4 blocker, linopirdine (0.25 mmol/L). BIMI (a PKC inhibitor) prevented the effects of PMA (a PKC activator) on the KCNQ4 current, indicating that PKC may be involved in the regulation of KCNQ4 expressed in the Xenopus oocyte system. Treatment with the ser/thr phosphatase inhibitors, cyclosporine (2 μmol/L), calyculin A (2 μmol/L) or okadaic acid (1 μmol/L), caused a significant positive shift in V1/2 and a decrease in the conductance of KCNQ4 channels. The V1/2 was shifted from −14.6±0.5 to −6.4±0.4 mV by cyclosporine, −18.8±0.5 to −9.2±0.4 mV by calyculin A, and −14.1±0.5 to −0.7±0.6 mV by okadaic acid. Moreover, the effects of these phosphatase inhibitors (okadaic acid or calyculin A) on the induction of a positive shift of V1/2 were augmented by further addition of PMA.Conclusion:These results indicate that ser/thr phosphatase inhibitors can induce a shift to more positive potentials of the activation curve of the KCNQ4 current. It is highly likely that the phosphatase functions to balance the phosphorylated state of substrate protein and thus has an important role in the regulation of human KCNQ4 channels expressed in Xenopus oocytes. [ABSTRACT FROM AUTHOR]

The analysis of chromosomal aberrations by premature chromosome condensation (PCC) induced by Calyculin A (Cal) is feasible in tumor biopsies from patients and has the potential to predict sensitivity to radiotherapy. As hyperthermia (HT) improves radiotherapy outcome in certain tumor sites, it was investigated whether PCC induction is still possible after temperatures reached in the clinic. Human cervical carcinoma (CaSki) and lung carcinoma (SW-1573) cells were incubated with Cal to induce PCC immediately after 1 h treatment at temperatures ranging from 41°C to 43°C and after recovery for up to 24 h after treatment with 43°C. Levels of phosphorylated Cdc2 (at the Tyr15 residue), histone H3 (at the Ser10 residue) and Cyclin B1 were investigated by immunoblotting. The amount of cells positive for phosphorylated histone H3 was determined by flow cytometry. Temperatures ≥42.5°C inhibited the induction of PCC by Cal, while recovery of PCC-induction was observed at >20 h after treatment in both cell lines. The phosphorylation status of Cdc2 as well as of histone H3 in cells treated with Cal directly after HT at 43°C was similar to that of cells treated with Cal alone or treated with Cal 24 h after HT at 43°C. HT alone did not affect the levels of phosphorylated Cdc2, while phosphorylation levels of histone H3 were increased as compared with control status of these two proteins. Phosphorylated and total Cyclin B1 levels were not influenced by any of the treatments. Flow cytometric analysis confirmed that HT at 43°C did not interfere with phosphorylation of histone H3. Our data indicate that HT transiently inhibits PCC induction by Cal in a temperature-dependent manner. Therefore, an interval of at least 24 h after HT should be applied before taking tumor biopsies for karyogram analysis of patients treated with temperatures above 42.5°C. [ABSTRACT FROM AUTHOR]

Objective: The effect of calyculin A (CA), a serine/threonine protein phosphatase inhibitor, on tumor necrosis factor-α (TNF-α) in primary osteoblasts was investigated to determine whether protein phosphatases could affect primary osteoblasts and if so which signaling pathways would be involved. Materials and methods: Primary osteoblasts were prepared from newborn rat calvaria. Cells were treated with 1 nM CA for different time periods. The expressions of TNF-α and GAPDH mRNA were determined by RT-PCR. Cell extracts were subjected to SDS-PAGE and the activation of Akt and NF-κB were analyzed by western blotting. Results: Calyculin A-treatment markedly increased the expression of TNF-α mRNA and enhanced the phosphorylation level of Akt (Ser473) in these cells. Pretreatment with the PI3K inhibitor LY294002 suppressed the increase in TNF-α mRNA expression and the phosphorylation of Akt in response to CA. Western blot analysis showed that CA stimulated the phosphorylation and nuclear translocation of NF-κB in primary osteoblasts, and these responses were blocked by pretreatment with LY294002. Conclusion: Calyculin A elicits activation of PI3K/Akt pathway which leads to expression of TNF-α mRNA and activation of NF-κB. This NF-κB activation involves both phosphorylation and nuclear translocation of NF-κB. [ABSTRACT FROM AUTHOR]

Abstract: The present study investigated the role of inhibitor of protein phosphatases 1 and 2A on the modulation of the phosphorylation of the spinal N-methyl-d-aspartate receptor (NMDAR) NR1 and NR2B subunits following electroacupuncture (EA) stimulation in rats. Bilateral 2Hz EA stimulations with 1.0mA were delivered at those acupoints corresponding to Zusanli and Sanyinjiao to men via needles for 30min. EA analgesia was slightly reduced by the intrathecal injection of calyculin A during EA stimulation. At 60min after the termination of EA stimulation, the levels of c-fos, serine phosphorylation of NR1 and NR2B by Western analysis had increased in the L4–5 segments of the spinal cord after EA treatment. These expressions were enhanced by the intrathecal injection of calyculin A and immunohistochemical analyses confirmed the significant increase of these proteins. As for the regional reaction of NMDAR subunits, a mean integrated optical density of phosphorylated NR1 and NR2B subunits was potentiated by calyculin A injections in the superficial laminae and neck region and superficial laminae and nucleus proprius, respectively. It can be concluded that protein phosphatase may play an important role in EA analgesia by modulating the phosphorylation state of spinal NMDAR subunits. [Copyright &y& Elsevier]

Abstract: Platelets are produced by megakaryocytes (MKs) through proplatelet formation (PPF), or cytoplasmic extensions, in vitro. Through the use of video-enhanced light microscopy, as well as localization of cytoskeletal proteins by confocal microscopy, the reaction of fully mature MK proplatelets, derived from murine embryonic stem cells, to various agents was studied. Calyculin A (protein phosphatase 1/2A inhibitor) treatment induced proplatelet retraction. In MKs with PPF, the expression of actin, myosin IIA, monophosphorylated myosin light chain (MLC-P1), and diphosphorylated myosin light chain (MLC-P2) was diffusely located. Following calyculin A treatment, actin was diffusely localized in retracted MKs and was expressed particularly in the periphery. MLC-P1 was also localized primarily in the periphery; however, MLC-P2 was expressed mostly in the inner area of proplatelets. Protein phosphatase inhibitors may result in increased hyperphosphorylation of localized MLC, which could alter the balance of actomyosin force in a cell, and therefore induce proplatelets retraction. [Copyright &y& Elsevier]

The availability of the eukaryotic polypeptide chain initiation factor 4E (eIF4E) for protein synthesis is regulated by the 4E-binding proteins (4E-BPs), which act as inhibitors of cap-dependent mRNA translation. The ability of the 4E-BPs to sequester eIF4E is regulated by reversible phosphorylation at multiple sites. We show here that, in addition, 4E-BP1 is a substrate for polyubiquitination and that some forms of 4E-BP1 are simultaneously polyubiquitinated and phosphorylated. In Jurkat cells inhibition of proteasomal activity by MG132 enhances the level of hypophosphorylated, unmodified 4E-BP1 but only modestly increases the accumulation of high-molecular-weight, phosphorylated forms of 4E-BP1. In contrast, inhibition of protein phosphatase activity with calyculin A reduces the level of unmodified 4E-BP1 but strongly enhances the amount of phosphorylated, high-molecular-weight 4E-BP1. Turnover measurements in the presence of cycloheximide show that, whereas 4E-BP1 is normally a very stable protein, calyculin A decreases the apparent half-life of the normal-sized protein. Affinity chromatography on m7GTP-Sepharose indicates that the larger forms of 4E-BP1 bind very poorly to eIF4E. We suggest that the phosphorylation of 4E-BP1 may play a dual role in the regulation of protein synthesis, both reducing the affinity of 4E-BP1 for eIF4E and promoting the conversion of 4E-BP1 to alternative, polyubiquitinated forms.Oncogene (2008) 27, 811–822; doi:10.1038/sj.onc.1210678; published online 23 July 2007 [ABSTRACT FROM AUTHOR]

Caffeic acid phenethyl ester (CAPE) is one of the most effective components of propolis which is collected by honey bees. The aim of this study was to investigate the cytotoxic and apoptotic effects of CAPE in the CCRF-CEM cell line and to clarify the role of serine/threonine protein phosphatase 2A (PP2A) and human telomerase reverse transcriptase (hTERT) activity as an underlining mechanism of CAPE-induced apoptosis. Trypan blue dye exclusion test and XTT methods were used to evaluate the cytotoxicity and ELISA based oligonucleotide detection, which can be seen during apoptosis, was used to determine apoptosis. Acridine orange/ethidium bromide dye technique was also used to evaluate apoptosis. The cytotoxic effect of CAPE was detected in a dose and time dependent manner with the IC50 of 1 μM. ELISA and acridine orange/ethidium bromide methods have shown remarkable apoptosis at 48th hour in CAPE treated cells. To investigate the role of PP2A in CAPE-induced apoptosis of CCRF-CEM cells, we performed combination studies with CAPE and, Calyculin A and Okadaic acid, which are very well known inhibitors of PP2A, in IC20 of inhibitors and IC50 of CAPE. Combination studies revealed synergistic effect of both drugs by concomitant use. Western blot analyses of PP2A catalytic and regulatory subunits showed down-regulation of expression of PP2A catalytic subunit in CAPE treated cells at 48th hour. Since, PP2A is important in hTERT (telomerase catalytic subunit) activation and deactivation, we also performed hTERT activity in CAPE treated cells simultaneously. Treating cells with IC50 of CAPE for 96 h with the intervals of 24 h showed marked reduction of hTERT activity. The reduction of hTERT activity in CAPE treated CCRF-CEM cells was more prominent in the initial 48 h. The variation of hTERT activity in CAPE treated CCRF-CEM cells may be the reason for the protein phosphatase interaction that occurred after treatment with CAPE. [ABSTRACT FROM AUTHOR]

Aim: This study was to investigate the effect of dehydroevodiamine (DHED) on Alzheimer's disease (AD)-like tau hyperphosphorylation induced by calyculin A (CA), an inhibitor of protein phosphatase (PP)-2A and PP-1, and the involvement of PP-2A in metabolically competent rat brain slices. Methods: Rat brain slices were pre-incubated at 33°C in the presence (10, 100, and 200 μmol/L, respectively) or absence of DHED for 1 h. Then, CA 0.1 μmol/L was added and the slices were treated for another 2 h. Western blotting and/or immunohistochemistry were used to measure the phosphorylation level of tau and PP-2A. Results: CA treatment could remarkably increase the immunoreactivity of pS262 and decrease the staining of Tau-1, representing tau hyperphosphorylation at Ser262 (pS262) and Ser198/ 199/202 (Tau-1, as the antibody reacts with unphosphorylated tau, therefore, decreased staining represents increased phosphorylation). Pre-incubation of the brain slices with DHED could efficiently attenuate the CA-induced tau hyperphosphorylation at the above AD-related sites. Additionally, DHED also decreased the basal phosphorylation level of tau at Ser396, although CA failed to induce tau hyperphosphorylation at this site. Furthermore, CA treatment induced an increased level of Tyr307-phosphorylated PP-2A, which represents inactivation of the phosphatase, whereas DHED arrested the elevation of the inhibitory modification of PP-2A. Conclusion: DHED can attenuate CA-induced tau hyperphosphorylation at multiple AD-related sites in metabolically active rat brain slices. The underlying mechanism may involve a decreased inhibitory phosphorylation of PP-2A at Tyr307. [ABSTRACT FROM AUTHOR]

The activity of protein phosphatase (PP)-2A and PP-1 decreased in the brains of Alzheimer’s disease and inhibition of the phosphatases led to spatial memory deficit in rats. However, the molecular basis underlying memory impairment of the phosphatase inhibition is elusive. In the present study, we observed a selective inhibition of PP-2A and PP-1 with Calyculin A (CA) not only caused hyperphosphorylation of cytoskeletal proteins, but also impaired the transport of pEGFP-labeled neurofilament-M subunit in the axon-like processes of neuroblastoma N2a cells and resulted in accumulation of neurofilament in the cell bodies. To analyze the morphological alteration of the cells during inhibition of the phosphatases, we established a cell model showing steady outgrowth of axon-like cell processes and employed a stereological system to analyze the retraction of the processes. We found CA treatment inhibited outgrowth of the cell processes and prolonged treatment with CA caused retraction of the processes and meanwhile, the early neurodegenerative varicosities were also obvious in the CA-treated cells. We conclude suppression of PP-2A and PP-1 by CA not only damages intracellular transport but also leads to cell degeneration, which may serve as the functional and structural elements for the memory deficits induced by suppression of the phosphatases. [ABSTRACT FROM AUTHOR]

Abstract: Intracellular phosphatase activity has been recognized to play a central role in signal transduction. In the present study, we investigated the effects of calyculin A, an inhibitor of protein phosphatases, on the expression of TNF-α mRNA and the possible signaling pathways in mouse osteoblastic MC3T3-E1 cells. The result of semiquantitative RT-PCR showed that calyculin A increased the expression of TNF-α mRNA in MC3T3-E1 cells. Pre-treatment of LY294002 and Wortmannin, inhibitors of PI3K, inhibited the calyculin A-stimulated TNF-α mRNA expression. Western blot result disclosed that calyculin A increased the phosphorylation status of Akt at Ser473. However, U0126 and SB203580, specific inhibitor of MEK1/2 and p38MAPK, respectively, had no effect on calyculin A-stimulated expression of TNF-α mRNA. BAY11-7085 and CAPE, inhibitors of NF-κB activity, did not alter the calyculin A-stimulated TNF-α mRNA expression. Indirect immunofluorescent study confirmed that NF-κB was not translocated to the nucleus by calyculin A treatment. Our present results suggest that inhibition of phosphatase activity by calyculin A stimulate the phosphorylation of Akt at Ser473 by PI3K/Akt signaling pathway, resulting in the expression TNF-α mRNA. [Copyright &y& Elsevier]

Abstract: In this study, we used calyculin A to induce premature condensed chromosomes (PCC). S-phase PCC is as “pulverized” appearance when viewed by light microscopy. Then, we applied atomic force microscopy (AFM) to investigate the ultrastructual organization of S-phase PCC. S-phase PCC shows ridges and grooves as observed by AFM. After trypsin treatment, chromosome surface roughness is increased and chromosome thickness is decreased. At high magnification, the ridges are composed of densely packed 30nm chromatin fibers which form chromosome axis. Around the ridges, many 30nm chromatin fibers radiate from center. Some of the 30nm chromatin fibers are free ends. The grooves are not real “gap”, but several 30nm chromatin fibers which connect two ridges and form “grid” structure. There are four chromatin fibers detached from chromosome: two free straight 30nm chromatin fibers, one loop chromatin fiber and one straight combining with loop chromatin fiber. These results suggested that the S-phase PCC was high-order organization of 30nm chromatin fibers and the 30nm chromatin fibers could exist as loops and free ends. [Copyright &y& Elsevier]

Abstract: Exposure of platelets to toxins (calyculin A or okadaic acid) that inhibit protein serine/threonine phosphatases types 1 and 2A, at concentrations that block aggregatory and secretory responses, results in the phosphorylation of several platelet proteins including integrin β3. Since protein phosphorylation represents a balance between kinase and phosphatase activities, this increase in phosphorylation reflects either the removal of phosphatases that oppose constitutively active kinases known to reside in the platelet (e.g., casein kinase 2) or the activation of endogenous kinases. In this study, we demonstrate that the addition of calyculin A promotes the activation of several endogenous platelet protein kinases, including p42/44mapk, p38mapk, Akt/PKB, and LKB1. Using a pharmacologic approach, we assessed whether inhibition of these and other enzymes block phosphorylation of β3. Inhibitors of p38mapk, casein kinase, AMP kinase, protein kinase C, and calcium–calmodulin-dependent kinases did not block phosphorylation of β3 on thr753. In contrast, 5′-iodotubercidin, at 50 μM, blocks β3 phosphorylation without affecting the efficacy of calyculin A to inhibit platelet aggregation and spreading. These data dissociate threonine phosphorylation of β3 molecules and inhibition of platelet responses by protein phosphatase inhibitors. [Copyright &y& Elsevier]

We describe here the chemical induction of premature condensed chromosomes in human peripheral lymphocytes after culture for 6 h. Many have attempted this induction without culture or with short-term culture, because this technique permits prompt cytogenetic biodosimetry of radiation accidents. Lymphocytes were separated from blood, incubated in the presence of phytohemagglutinin, ATP, and p34cdc2/cyclin B kinase, then treated with calyculin A during the last hour. The culture medium was supplemented with a lower concentration of fetal calf serum than conventionally used to minimize its possible interference with the effects of these drugs. We obtained, rarely, a suitable morphology of premature chromosome condensation in short-term cultured lymphocytes for conventional chromosome aberration analysis. [ABSTRACT FROM AUTHOR]

Three human malignancy cell lines were irradiated with 60Co γ-rays. Initial chromatid breaks were measured by using the chemically induced premature chromosome condensation technique. Survival curves of cells exposed to gamma rays was linear-quadratic while the efficiency of Calyculin A in inducing PCC of G2 PCC was about five times more than G1 PCC. A dose-dependent increase in radiation-induced chromatid/isochromatid breaks was observed in G1 and G2 phase PCC and a nearly positive linear correlation was found between cell survival and chromatin breaks. This study implies that low LET radiation-induced chromatid/isochromatid breaks can potentially be used to predict the radiosensitivity of tumor cells either in in vitro experimentation or in in vivo clinical radiotherapy. [ABSTRACT FROM AUTHOR]

Pharmacological inhibitors of protein kinase A (PKA) and protein phosphatases 1/2A were used to determine whether basal L-type Ca2+ current (ICa) observed in the absence of exogenous β-adrenergic receptor stimulation is sustained by PKA-mediated phosphorylation. Amphotericin B was used to record whole-cell ICa in the perforated patch-clamp configuration.Calyculin A and isoprenaline (both 1 μmol l−1) increased basal ICa (P<0.05), whereas H-89 inhibited ICa in a concentration-dependent manner with an IC50 ∼5 μmol l−1. H-89 also inhibited the response to 1.0 μmol l−1 isoprenaline, although relatively high concentrations (30 μmol l−1) were required to achieve complete suppression of the response.Double-pulse protocols were used to study the effects of 10 μmol l−1 H-89 on time-dependent recovery of ICa from voltage-dependent inactivation as well as the steady-state gating of ICa. T0.5 (time for ICa to recover to 50% of the preinactivation amplitude) increased in the presence of H-89 (P<0.05) but was unaffected by calyculin A or isoprenaline.Steady-state activation/inactivation properties of ICa were unaffected by 10 μmol l−1 H-89 or 1 μmol l−1 calyculin A, whereas isoprenaline caused a leftward shift in both curves so that V0.5 for activation and inactivation became more negative.Data show that basal ICa is regulated by cAMP-PKA-mediated phosphorylation in the absence of externally applied β-receptor agonists and that relatively high concentrations of H-89 are required to fully suppress the response to β-adrenergic receptor stimulation, thereby limiting the value of H-89 as a useful tool in dissecting signalling pathways in intact myocytes.British Journal of Pharmacology (2006) 148, 1108–1115. doi:10.1038/sj.bjp.0706809; published online 26 June 2006 [ABSTRACT FROM AUTHOR]

It is well known from various mammalian cells that anoxia has a major impact on the mitogen-activated protein kinase ERK, but a possible similar effect in fish cells has not been investigated. Here we characterise a p44ERK-like protein in the rainbow trout cell line RTHDF and study the effect of (i) serum stimulation, (ii) sodium azide (chemical anoxia) and removal of azide (recovery) and (iii) anoxia (PO2<0.1%) and recovery. During both chemical and true anoxia p44ERK was inhibited and recovery resulted in robust reactivation of p44ERK activity, far above the initial level. The inhibition was secondary to activation of p38MAPk and the increase was MEK dependent, as SB203580 inhibited the dephosphorylation during anoxia and the presence of PD98059 inhibited phosphorylation of p44ERK during recovery. In addition, we demonstrated that the reactivation of p44ERK during recovery also was dependent on reactive oxygen species and a PP1/PP2A-like phosphatase. [ABSTRACT FROM AUTHOR]

Various studies suggest a tight relationship between chromosome rearrangements driving tumor progression and breaks at loci called common fragile sites. Most of these sites are induced after perturbation of the replication dynamics, notably by aphidicolin treatment. We have mapped the majority of these sites to the interlace of R and G bands, which calls into question the previous assignment of aphidicolin-sensitive sites to R bands. This observation suggests that most of them correspond to loci that ensure the transition between early and late replicating domains. We show that calyculin A, which triggers chromosome condensation at any phase of the cell cycle but does not markedly impair replication, induces damage in the chromosomes of human lymphocytes treated in G2 but not in G1 phase. We demonstrate that these lesions colocalize with those induced by aphidicolin treatment. Hence, common fragile site stability is compromised, whether aphidicolin delays replication or calyculin A advances condensation. We also show that, in cells that go through an unperturbed S phase, completion of their replication and/or replication-associated chromatin reorganization occur all along the G2 phase, which may explain their inability to condense properly after calyculin A treatment during this phase of the cell cycle. [ABSTRACT FROM AUTHOR]